Leave a comment

Biosynthesis, and ,Cytoplasmic Trafficking, Membranes, Vesicles, and, Intracellular Transport

11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
11. Biosynthesis and Cytoplasmic
Membranes, Vesicles and Intracellular Transport
I.  Transport Vesicles
A.  Assembly of Coats, Budding and Formation of Vesicles
Adaptors and receptors
Involvement of phosphatides
B.  Targeting of Proteins and Vesicles; SNAREs
C.  Fusion
D.  The GTPases
E.  ER and Golgi Transport
ER to Golgi Transport
Transport from Golgi Stacks
II.  Recognition of Targets
A.  Cell Polarity
Maintenance of polarity
Components of tight junctions
B.  Targeting of Plasma Membrane Proteins
C.  Targeting in Secretion and Transcytosis
D.  Transport of the Vesicles
E.  Recycling of the Plasma Membrane
F.  Formation of Lysosomes and Storage Vesicles
Secretory storage granules
G.  Synaptic Vesicles
http://www.albany.edu/~abio304/text/chapter_11.html (1 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
Suggested Readings
Web Resources
Back to List of Chapters
Understanding the intricate details of the interactions between membranes and membrane bound
compartments is a challenging task. Electronmicroscopy using immunological methods together
with genetic approaches and molecular studies of reconstituted systems are beginning to provide the
needed framework of information. This chapter focuses on some of these processes.
As we saw in Chapter 10, most intracellular transport is thought to be mediated by small vesicles,
although an alternative mechanism for the transfer from the VTCs to the cis-Golgi (see Chapter 10,
Section III) is likely and, in addition, transport between Golgi stacks may involve alternative
mechanisms (see Chapter 10, Section IIIC). The transport can occur in either direction: from the ER
to the periphery of the cell or in the opposite direction. The transport processes are discussed in
relation to endocytosis in Chapter 9, in relation to the motility of cell components in Chapter 23 and
the discussion of neuronal transport can be found in that Chapter and in Chapter 24.
Chapter 9 discussed the involvement of clathrin in the formation of endocytotic vesicles. Some other
transport vesicles are also clathrin-coated. They carry proteins originating from the trans-Golgi
system (see Brodsky, 1988). They include transport vesicles destined to the lysosomes (e.g., Schulze-Lohoff et al., 1985) containing acid hydrolases and mannose-6-phosphate receptors, and those
destined to storage vesicles containing densely packed secretory products (see Section II F, below).
Vesicles coated with proteins other than clathrin (non-clathrin coated) represent another set of
transport vesicles. These are involved in the translocations within the Golgi stack, between the ER
and the Golgi, from the TGN in the constitutive secretory pathway and in retrograde transport (e.g.,
from Golgi to ER)
The present conventional view maintains that non-clathrin coated vesicles are involved in the
transport between ER and Golgi and between Golgi stacks. The transport from the ER to the cis-Golgi is carried out by COPII vesicles (see Barlowe, 1998). In contrast, retrograde transport from
Golgi to ER takes place in coatomer protein (COPI) coated vesicles (Cosson and Letourneur, 1997;
Gaynor et al., 1998) for proteins either with KKXX, KKXX-like (see below) or the KDEL amino
acid motif (see Chapter 10) but not by glycosylated Golgi enzymes or Shiga toxin (Girod et al.,
1999). Forward and retrograde transport between Golgi stacks is also thought to be mediated by
COPI (Orci et al., 1997). In this latter case, electron microscope immunocytochemistry shows that
both anterograde-cargo and retrograde-cargo are present in separate COPI-coated vesicles budding
from all stacks of the Golgi. Packaging of anterograde and retrograde cargo into separate vesicles
can also be demonstrated in vitro even when budding is driven by highly purified coatomer and a
recombinant, small GTPase, ARF.
http://www.albany.edu/~abio304/text/chapter_11.html (2 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
All in all, at this time four different coat proteins are clearly defined, including two clathrin proteins
and COPI and COPII (listed in Table 1, Rothman and Wieland, 1996). In Table 1, the two different
clathrin coats are shown to function in conjunction with different adaptor molecules (see below). All
these coats are associated with GTP-binding proteins (or GTPases) (see below). Their origin and
initial destination are listed in the fourth column. Besides the four coat proteins listed in the Table,
more coat proteins have been demonstrated and others are likely to be revealed by further studies. A
lace-like coat has been described in the TGN (Ladinsky et al., 1994; Narula et al., 1995), a neuronal
variant of COPI has been found (Newman et al., 1995) and another is thought to be associated with
endosomal vesicle traffic (Whitney et al., 1995). A distinct coating for vesicles involved in
endosome-to-Golgi retrograde transport of sorting receptors is operative in yeast (Seaman et al.,
1998). The vesicles are slightly larger than the COPI vesicles. In Saccharomyces cerevisiae, a novel
vesicle 30-40 nm in diameter transferring fructose-1,6-bisphosphatase to the vacuole for
degradation, has been described (Huang and Chiang, 1997) . There is growing evidence for other
coat proteins similar to clathrin. A gene (HC22) has been identified with the potential of coding for
a protein similar to the clathrin heavy chain. mRNA corresponding to HC22 is expressed
predominantly in skeletal muscle and alternative transcripts of H22 are expressed in a tissue specific
fashion (see Brodsky, 1997).
Table 1. Coat proteins or vesicle shuttles (from Rothman and Wieland 1996, reproduced by
Type of coated
Subunits of coat  GTPase  Origin-destination
AP-1 clathrin Clathrin, AP1
ARF  TGN-prelysosomes
AP-2 clathrin Clathrin, AP2
ARF?  Plasma membrane-endosomes
COPI Coatomer (COPI
ARF  ER-Golgi;
bidirectional within
Golgi; Golgi-ER
COPII COPII proteins SAR  ER-Golgi
It is now recognized that the process of targeting and interaction between components occurs in
many steps starting from the translocation of vesicles to their target and ending with their fusion to
their target. The translocation of the vesicles (see Section IID and Chapter 24, section on
cytoplasmic dynein and kinesin) has not been elucidated in detail. Upon arrival processes involved
in membrane recognition (tethering) leaves the two sets of membranes at some distance from each
http://www.albany.edu/~abio304/text/chapter_11.html (3 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
other. The binding of SNAREs (docking) leaves the membranes in close proximity and is followed
by fusion. One kind of SNARE, SNAREv,resides on the surface of the vesicle being transported and
SNAREt resides on its membrane target (see Section C). Tethering is thought to be the primary step
in determining specificity. Rab GTP-binding proteins have been implicated as well (see Pfeffer,
1999; Novick and Zerial, 1997; Zerial and McBride, 2001). However, a variety of proteins and
protein complexes have been found to play essential roles in targeting and to be specific for
individual steps. Before fusion, priming events must take place (Klenchin and Martin, 2000) and a
fusion trigger, most frequently Ca2+, is needed (e.g., Heidelberger et al., 1994; Peters and Mayer,
1998; Beckers and Bach, 1989; Colombo et al., 1997). The priming includes ATP-dependent steps,
such as the NSF-mediated priming of SNARE protein complexes, the ATP-dependent synthesis of
phosphoinositides, and protein kinase-mediated protein phosphorylation. The protein munc 13 is
also involved in the priming.
The intracellular transport of proteins in vesicles raises a number of issues: (a) how the appropriate
proteins are selected in the formation of specific vesicles, (b) the nature of the mechanism of
budding, (c) how the vesicles are targeted unidirectionally and in an orderly fashion, and (d) the
mechanism of fusion of the targeted vesicles. Unfortunately, only partial answers are known.
The first issue, (a), was discussed in Chapter 10. The formation of vesicles (Section A), their
targeting (Section B) and fusion to their target membranes (Section C) and the special role of the
GTPases (Section D) will be discussed first, followed by an examination of some of the details of
the various pathway segments (Section E).
A. Assembly of Coats, Budding and Formation of Vesicles
This and the following sections will be referring to several components: NSF, SNAP and SNARE.
As in many other cases in this book, these acronyms stand for certain appellations that are only of
historical importance.  NSF stands for N-ethyl maleimide sensitive factor, SNAP for soluble NSF-attachment protein and SNARE  for SNAP receptors. The acronysms will be used  in the text that
The process of vesicle formation and their delivery to a target are summarized in the diagram of Fig.
1 which will serve as the basis of this discussion. This representation reflects the bare bones, so-called SNARE hypothesis. As discussed below some of the present evidence indicates that SNAREs
are responsible for docking and act downstream from tethering. The assembly described in Fig. 1A
typified for COPI vesicles, occurs stepwise with (i) the binding of the small GTP-GTPase first (see
Section E) (in this case ARF; open spheres: GDP-GTPase; closed spheres: GTP-GTPase) where
GDP has been replaced by GTP (i-ii), followed by activation by GTP and formation of coated bud
by recruitment from the cytoplasmic pool. The pinching off of the vesicle requires acyl-CoA binding
(not shown, Ostermann, et al., 1993) and the GTPase, dynamin (not shown, e.g., Takei et al., 1995;
Hinshaw and Schmid, 1995). Dynamin is thought to assemble around the neck of endocytotic
invagination and participates in pinching off the vesicles (see below and Chapter 9). Subsequently,
the hydrolysis of GTP induces the detachment of the GTP-binding protein, now bound to GDP
(Melançon et al., 1987; Orci et al., 1989) (iii-iv). Finally, dissociation of the coat follows (v) . The
http://www.albany.edu/~abio304/text/chapter_11.html (4 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
steps diagrammed in Fig. 1B and C are discussed in more detail below. Basically, the uncoated
vesicles bind to the target protein on the target membrane (part B of the diagram). The binding is
mediated by the v-SNARE and its cognate, t-SNARE, in the target membrane (see Section C). The
fusion (part C of the diagram) requires ATP, GTP, AcylCoA, the NEM-sensitive-factor (NSF),
SNAPs and other factors. NSF catalyzes the hydrolysis of ATP which disrupts the SNARE complex
(see Section C), and initiates fusion.
This model is supported by experimental observations: (a) the presence of coated vesicles coincides
with biochemically defined transport (Orci et al., 1986); (b) coated vesicles containing cargo
proteins, in this case G-VSV-protein, transfer from donor to acceptor stacks of the Golgi and can be
trapped at this stage by the presence of GTPγS (Orci et al., 1989); (c) after a GTPγS block is
reversed the vesicles disappear; (d) budding requires fatty acylCoA; (e) addition of NEM (or AlF4-,
which acts as a phosphate analog) causes a buildup of uncoated 75 nm vesicles at the Golgi complex
in intact cells; and (f) the accumulation of uncoated vesicles produced by NEM is reversed by the
addition of a soluble cytoplasmic factor (NSF) (Malhotra et al., 1988).

Fig. 1 Model of the vesicle shuttle (see text) (A) Vesicle budding. (B) Targeting of vesicles. (C)
Fusion. Reproduced with permission from Rothman, J.E. and Wieland, F.T. (1996) Protein sorting
by transport vesicles, Science 272:227-234, copyright &copy1996, American Association for the
Advancement of Science.
http://www.albany.edu/~abio304/text/chapter_11.html (5 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
As implied by Fig. 1A, several features in the assembly of coated vesicles are common to all three
systems that have been studied. They all first require the recruitment of small GTP-GTPases. Later
binding of coat proteins induces a deformation of the membrane to form a bud. Transmembrane
proteins of the donor membranes recruit the proteins that will form the coat (see Fig. 12A of Chapter
10). In addition, they can function in the selection of cargo. However, there are significant
differences. The assembly of COPI-coated vesicles recruits a pre-assembled coat present as a
soluble complex ( e.g., Zhao et al., 1997, 1999), whereas COPII proceeds stepwise. The recruitment
of clathrin is also nucleotide dependent and requires ARF1. However, unlike COPI vesicles, in
clathrin vesicle assembly, ARF1 only recruits AP-1 (an adaptor protein, see below) (e.g. Zhu et al,
1998) followed by formation of the coat. In addition, clathrin disassembly in neurons requires the
heat shock protein Hsc70 together with the cofactor auxilin and proceeds with the hydrolysis of
ATP. In other cells cyclin G-associated protein kinase (GAK) acts similarly to auxilin. The
disassembly of the clathrin coat (e.g., Ungewickell et al., 1995; Umeda et al., 2000) differs from the
dissociation of the other two coats.
The ability of clathrin to self assemble into polyhedral cages (Woodward and Roth, 1978) has
suggested that budding occurs by a stepwise assembly of coat structure from subunits in the
cytoplasm. In this model, each addition progressively deforms the membrane to eventually form the
bud and then a detached vesicle (see Le Borgne and Hoflack, 1998).
However, the process is best understood for the cases of budding from the ER (Barlowe et al., 1994)
and the Golgi cisternae that do not involve clathrin. As already mentioned, the budding process is
regulated by GTP-binding proteins (see Table 1, Fig. 1A), which initiate the process (Donaldson et
al., 1992; Helms and Rothman, 1992). Then the coat proteins can begin to be assembled. In contrast,
the hydrolysis of the bound GTP to form bound GDP, initiates the release of the coat (Tanigawa et
al., 1993). An enzyme in the donor compartment catalyzes the exchange of GTP for GDP (Barlowe
and Shekman, 1993).
The components of the COPII coat needed for vesicle assembly can be shown by genetic approaches
in yeast. In addition, ER membranes can be used in an in vitro assay after extraction of peripheral
proteins. The stripped membranes produce vesicles by budding after the addition of cytosol extracts
and GTP. The active ingredients of the cytosol extracts correspond to a 700 kDa complex of
Sec31p/Sec13p, a 400 kDa complex of Sec23p/Sec24p and the small GTPase, Sarp1 (Barlowe and
Sheckman, 1993). Sar1p-GDP is normally in the cytoplasm. It is recruited to the ER membrane by
Sec12p, an integral membrane protein of the ER. Sec12p also functions as a guanine exchange
factor (GEF) (Barlowe and Sheckman, 1993), thereby facilitating the exchage of GTP for GDP. In
contrast, the disassembly, required for fusion, involves GTP hydrolysis activated by Sec23p (see
Kaiser and Ferro-Novick, 1998).
Another protein of 240 kDa, Sec16p, is tightly bound to the ER and acts as a scaffold for the
assembly. Sec23p, Sec24p, Sec31p and Sed4p (a homolog of Sec12p) are bound to different sites in
the Sec16p molecule (e.g., Shaywitz et al., 1997).
The minimum system to function in the formation of COPII vesicles and buds has also been
http://www.albany.edu/~abio304/text/chapter_11.html (6 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
determined by reconstituting purified proteins in entirely synthetic liposomes (Matsuoka et al.,
1998). By adding 5’guanylyl imidodiphosphate (GMP-PNP), a non-hydrolyzable analog of GTP,
Sec12p (which functions as a nucleotide exchange protein) is not required. After Sar1p-GMP-PNP
is bound to the membrane, the Sec23p/Sec24p and Sec31p/Sec13p complexes are sequentially
attached in that order. The process differs somewhat from the native process suggesting that other
factors (e.g., the presence of cargo) may facilitate the process. Apparently a minimum of 10% acidic
phospholipids is required (Matsuoka et al., 1998).
Sorting of integral membrane proteins is thought to require binding of the cytoplasmic domain of
the protein to the Sec23p-Sec24p complex (Aridor, et al., 1998). Sorting of soluble proteins requires
transmembrane receptors. These receptors would require at least one transmembrane domain, a
lumenal domain that can bind to the cargo and a cytoplasmic exposed domain that would bind to
coat subunits. One of these is the KDEL receptor that binds to a carboxy-terminal KDEL peptide
and retrieves the proteins that have escaped from the ER (Lewis and Pelham, 1992). Two proteins of
the p24 family are receptors that have been found in COPII-coated vesicles (Schimoller et al., 1995)
and are needed for the secretion of certain proteins. Their cytoplasmic domain contains a diphenyl
and a dibasic motif at the carboxy terminals.
In order to fuse to their target the vesicles must contain v-SNAREs (see Section B). vSNAREs have
been demonstrated in COPI and COPII coats. Two ER to Golgi v-SNAREs, Bet1p and Bos1p,
interact specifically with Sar1p, Sec23p, and Sec24p in a guanine nucleotide-dependent fashion
(Springer and Sheckman, 1998).
The assembly of COPI-coated vesicles proceeds as follows. First the GTPase ARF1 (ADP-ribosylation factor 1) binds to the membrane. This requires binding to its GEF. ARF1 is
myristoylated. The myristoyl moiety is exposed when the GTPase binds GTP, allowing ARF1 to
bind to lipids. The hydrolysis of GTP leads to the retraction of the meristoyl-moiety into a pocket of
the ARF1 molecule, so that the GTPase is no longer able to attach to lipids. The assembly of the
coat takes place by recruitment of a preassembled coat via its β and γ-COP subunits, resulting into a
deformation of the membrane to form the bud (Zhao et al., 1997; 1999).
Adaptors and receptors
The assembly of coated vesicles probably involves many more interactions than those considered so
far. The adaptor molecules AP-1 (present in the clathrin coated vesicles that originated from the
TGN) and AP-2 (from the clathrin coated vesicles originating during endocytosis) appear to have a
key role in the processes involved in vesicle formation. Their structure and function have been
recently reviewed (Traub, 1997; Robinson, 1997). In addition to AP-1 and AP-2, a third adaptor
protein has been identified, AP-3 (Dell’Angelica et al., 1997). AP-3 is likely to function in transport
to the lysosomes (in yeast the vacuole). Deletion of any of the subunits, leads to mistargeting of
some of the vacuolar proteins but not others (Cowles et al., 1997; Stepp et al., 1997; Vowels and
Payne, 1998). Similarly, in mammalian cells, antisense oligonucleotides for the AP-3 gene (see
Chapter 1) also send lysosomal glycoproteins to the cell surface without affecting AP-1 mediated
transport, such as that of the mannose 6-phosphate receptors (Le Borgne et al., 1998). An additional
http://www.albany.edu/~abio304/text/chapter_11.html (7 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
function is suggested by the observation that in vitro synaptic vesicle can be formed from
endosomes in the presence of AP-3 (Faundez et al., 1998).
AP-1, AP-2 and AP3 are heterotetramers [two large δ subunits of 160 kDa, and two β3A (120 kDa)
subunits associated with µ3 (47 kDa) and σ3 (22 kDa)].The β-subunits of these adaptors promote
clathrin-cage formation (e.g., Gallusser and Kirchhausen, 1993). AP-1 and -2 also bind in vitro to
the cytoplasmic domains of membrane receptors (e.g., Pearse, 1988, Glickman et al., 1989) and
clathrin (e.g., Ahle and Ungewickell, 1989; Shih et al., 1995; Traub et al., 1995, Schröder and
Ungewickell, 1995). In addition, they bind the tyrosine or di-leucine sorting motifs (Heilker et al.,
1996) important for endocytosis and lysosomal targeting (see Sandoval and Bakke, 1994). The µ-1
and µ-2 subunits of AP-1 and AP-2, bind to the tyrosine endocytotic motif (Ohno et al., 1995). The
adaptors are therefore responsible for the selectivity of the vesicle as well as their assembly.
The receptor molecules, such as the mannose 6-phosphate receptors (MPRs) sorted out in the TGN,
were found to be needed for the recruitment of AP-1 to the clathrin vesicles in an in vitro system (Le
Borgne et al., 1996). The amount of AP-1 recruited by clathrin coated vesicles of the TGN was
found to depend on the presence of the MPRs and the integrity of their cytoplasmic domains (Le
Borgne and Hoflack, 1997).
Many homologues of adaptor subunits AP-1, AP-2 and AP-3 have been found. In one case, clathrin
coated vesicles associated with endosomes have been shown to have neither AP-1 nor AP-2,
suggesting the presence of another adaptor complex (see Stoorvogel et al., 1996). Some of the
adaptors function independently of clathrin (e.g., Stepp et al., 1995; Simpson et al., 1996). These
adaptors could function in conjunction with clathrin-like molecules or other unknown coat proteins,
possibly interacting with COPI.
In summary, adaptors, receptors and clathrin appear to act in concert to produce coated vesicles.
The subunits of COPI, coatomer, (β, δ and ζ-COP) have some sequence homology with the β, µ and
σ-adaptor subunits of AP-1 and AP-2. Interestingly, the B subcomplex of COPI binds the di-lysine
motif (e.g., Fiedler et al., 1996). Therefore, in contrast to clathrin, coatomer subunits have a role
similar to the adaptor molecules. Coatomer coat subunits have been shown to interact with KKXX
motif (Letourneur et al., 1994) needed for retrieval to the ER. Therefore they do function as
receptors for retrograde transport.
The interaction of coatomer with a domain of the peptide p23 (a p24 protein thought to act as a
receptor for cargo) has been studied in vitro (Reinhard et al., 1999). The binding of the two kinds of
protein results in a conformational change and polymerization of the complex in vitro with a
stoichiometry of 1:4, COPI:peptide. This conformation is also seen on the surface of isolated COPI
vesicles. These results suggest a mechanism by which the induced conformational change of
coatomer accompanying its polymerization is responsible for the formation of the bud on the Golgi
membrane during biogenesis of a COPI vesicle. In vitro experiments using liposomes reveal the
formation of coatomer vesicles requires ARF, GTP and the cytoplasmic tails of the p24 proteins
receptors, or cargo proteins with the KKXX retrieval signal (Bremser et al., 1999).
http://www.albany.edu/~abio304/text/chapter_11.html (8 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
Knowledge of the possible role of adaptor-like molecules in COPII is still lacking. However, COPII-coated vesicles are selective, in some cases, as shown by the fact that they concentrate proteins
exported from the ER (Balch et al., 1994; Bednarek et al., 1995). We saw that only the COPII
proteins are required for vesicle formation (see above) (Matsuoka et al, 1998). However,
phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate are needed to bind them
to liposomes and the GTP-bound form of Sar1p is needed to recruit the proteins to either liposomes
or the ER membranes.
Heteromeric complexes of proteins of the p24 family are found in both COPI and COPII vesicles as
well as ER and Golgi membranes (e.g., Füllekrug et al., 1999; Marzioch et al., 1999) suggesting that
p24 cycles between ER and Golgi as would be expected for receptor proteins. In mammals the p24
proteins are thought to be involved in exit from the ER (Lavoie et al., 1999). In yeast there are eight
genes encoding these family members. Mutations of several of these genes exhibit selective protein
transport defects or secretion of the ER lumenal protein Kar2p (e.g., Marzioch et al., 1999). For
example, deletion of one p24 gene slows down the transport of Gas1p from ER to Golgi (e.g.,
Marzioch et al., 1999). Gas1p is a GPI-anchored protein.
Mutations of the genes coding for p24 proteins do not inhibit anterograde passage from the ER
completely and the export of some of the proteins from the ER do not require p24-proteins at all.
This suggests that only some proteins require this receptor or any receptor at the ER exit step. In
agreement with this notion, many secreted proteins have been shown not to be concentrated in
COPII-coated vesicles (Martínez-Menárguez et al., 1999). The incomplete inhibition by some of the
mutants that seem to be involved in the p24 system may be explained by the occurrence of bulk flow
which would be of significance in the absence of receptors. However, in some cases, the role of p24-proteins is clearly that of receptors. The Emp24 complex is needed for for packing Gas1p into
vesicles of the ER (Muñiz et al., 2000). In agreement with the notion that Emp23 acts a receptor,
Gap1p was shown to become chemically cross-linked to two of the Emp24 proteins in experiments
using cross-linking reagents.
Cargo proteins have been found to have a role in the assembly of coat proteins (see Springer et al.,
1999; Bremser et al., 1999). In addition, in view of the central role of the GTPases in coat assembly,
it would seem likely that the GTPases have a role in regulating cargo recognition. A special role of
GTPases is suggested by experiments using a cell-free system. During COPI vesicle formation,
competing sorting signals were shown to act through a GTPase switch thereby providing specificity
for the process (Goldberg, 2000). The signal sequence of hp24a (a p24 protein, supposedly a
receptor) inhibits coatomer-dependent GTP hydrolysis. In contrast, the di-lysine retrieval signal (see
above) that binds the same coatomer site has no effect on the GTPase. The results suggest that the
activity of the GTPase can select or discard a cargo protein.
Involvement of phosphatides
Several phospholipid modifying enzymes are involved in the formation of vesicles from buds (e.g.,
endophilin, synaptojanin and phospholipase D). Endophilin is an acyltransferase that binds to the
small GTPase, dynamin (see below), and transfers fatty-acyl from arachidonic and palmitic acid to
http://www.albany.edu/~abio304/text/chapter_11.html (9 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
lysophosphatidic acid to produce phosphatidic acid (Schmidt et al., 1999). This reaction may induce
negative membrane curvature by converting an inverted-cone-shaped lipid into a cone-shaped lipid
in the cytoplasmic layer of the bilayer. Phospholipase D produces phosphatidic acid from
phospholipids, possibly affecting membrane curvature (Zimmerberg, 2000). Synaptojanin is a
polyphosphoinositide phosphatase. See also Chapter 4 for a discussion of membrane curvature.
Although their role is not well understood, the phosphoinositides (PIs) have been shown to be part
of the machinery responsible for the formation of vesicles, for example, in the the TGN, the plasma
membrane and the endosomes (see Martin, 1997; De Camilli, 1996; Ohashi et al., 1995). The
phosphoinositides are thought to be part of the discrete membrane sites (see Chapter 4) required for
the recruitment of cytoplasmic proteins needed for vesicle formation and budding.
An involvement of the lipid system is shown by the dependence of secretion on phosphatitylinositol
transfer proteins (PITPs). PITPs are enzymes reponsible for the transfer of phospholipids between
membrane structures or serum lipoproteins (see Wirtz et al., 1991). These enzymes have a distinct
preference for phosphatidylinositol over phosphatidylcholine. The β isoform, present in the Golgi
system, has a high transfer activity in relation to sphingomyelin (see deVries et al., 1995). In yeast,
mutations in the SEC14 gene which codes for a PITP, block post-Golgi secretory traffic (e.g.,
Bankaitis et al., 1990). In agreement with these results, experiments carried out on a cell free system
from a neuroendocrine cell line (Ohashi et al., 1995) found that the α and β isoforms of PITPs
stimulate formation of vesicles from the TGN.
Phosphatidylinositol kinases (PIKs) which phosphorylate PIs have also been implicated. In yeast,
the Vps15 protein kinase and the Vps34 PI-3-kinase have been shown to function as a membrane-associated complex which facilitates the delivery of proteins to the yeast vacuole (e.g., Stack and
Emr, 1994) which has a similar function than lysosomes in a mammal. Subsequent work has
indicated that polyphosphoinosides phosphorylated at the 4′ and 5′ or alternatively the 3′ positions of
the inositol ring determine the location of events involved in membrane traffic. It is now generally
recognized that phosphorylation-dephosphorylation of the polar heads of phosphoinositides in
specific locations coincides with the recruitment or the activation of proteins essential for vesicular
transport (see De Camilli et al., 1996).
The compartmentation of PPIs is probably the result of synthesis at specific locations. The targeting
of PI 4-kinase would permit a segregated synthesis. PI 4-kinase catalyzes the phosphorylation of
phosphatidylinositol to form PI 4-phosphate. PI 4-kinases have been found in plasma membranes
and intracellular organelles including Golgi, lysosomes, ER, nuclear envelope, coated vesicles,
exocytotic vesicles and secretory granules (see De Camilli et al., 1996; Carpenter and Cantley,
1996). The immunoreactivity for this PI 4-kinase molecule was found mostly in close association
with the membranes of the Golgi vesicles and vacuoles (Nakagawa et al., 1996).
The association between PI-kinases and membranes occurs despite the absence of transmembrane
sectors. However, these proteins could bind directly to lipids or indirectly by anchoring to integral
proteins. Some of the mammalian kinases have a pleckstrin-homology (PH) domain (Nakagawa et
al., 1996) that would allow them to interact with lipids. Alternatively, the kinase could be anchored
http://www.albany.edu/~abio304/text/chapter_11.html (10 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
by binding to membrane receptors, as is the case of PI 4-kinase that links to transmembrane proteins
and integrins (Berditchevski et al., 1997). A mammalian adaptor protein p150 (homolog of the yeast
VPS15) that recruits the PI 3-kinase to the Golgi membrane, has been identified (Panaretou et al.,
1997). Recombinant p150, PI 3-kinase and PITP form a complex also present in the cytoplasm of
human cells.
Not surprisingly, a polyphosphoinositide phosphatase (synatpojanin 1) has been found to be
involved in presynaptic endocytotic vesicle recycling in neurons. Synaptojanin 1 was found at high
levels at nerve terminals (McPherson et al., 1996), which are involved in the exocytosis of synaptic
vesicles. Synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth,
phosphatidylinositol 4,5-biphosphate (PIP2) levels are increased and clathrin-coated vesicles
accumulate at the nerve endings (Cremona et al., 1999)
Undoubtedly, the binding of phosphoinositides to coat components has a role in trafficking. They
bind AP-2 (e.g., Gaidarov et al., 1996), AP-3 (AP180) (e.g., Hao et al., 1997) and COPI coatomer
(e.g., Chaudhary et al., 1998), dynamin, synaptojanin (Cremona et al., 1999) and in addition
arrestins (Gaidarov et al., 1999). Dynamin is involved in vesicle formation (see Chapter 9).
Arrestins are involved in the inhibition of Gs proteins (e.g., see Chapter 7) and also act as clathrin
adaptors (see Goodman et al., 1996). Mutants of arrestin3 expressed in cells in culture fail to
participate in β2-adrenergic receptor internalization and fail to be recruited to the coated pits
although they are recruited to the plasma membrane (Gudrov et al., 1999). Similarly, mutation of the
α subunit of the AP-2 adaptor protein at high expression levels results in failure to localize at
clathrin coated pits (Gaidarov and Keen, 1999).
In addition to their separate roles in vesicle formation and secretion, the phosphoinositide system
and the GTP-binding proteins are functionally and intimately linked. ARF activates phospholipase
D (PLD) (Brown et al., 1993). PLD catalyzes the conversion of phosphatidyl choline to phosphatidic
acid. The latter activates PI-4 kinase to produce PIP2 (see Martin, 1997; DeCamilli et al., 1996)
which has been shown to be involved in vesicle formation (Tüscher et al., 1997). In addition, ARF
has a direct effect in increasing the level of PIP2 (Godi et al., 1998) and PI-4 kinase-β and, by
recruitment, another unidentified kinase in the Golgi (Godi et al., 1999). This increases the synthesis
of PI-4-phosphate and PI2 levels independently of PLD activation. The PI-4 kinase is required to
maintain the integrity of the Golgi. Mutants that lack the kinase exhibit a disorganized Golgi (Godi
et al., 1999).
Other effects of ARF on the Golgi and vesicle pathway are less clearly understood. The actin
binding proteins ankyrin and spectrin, are at the cytoplasmic surface of the Golgi (see Beck et al.,
1998; De Matteis et al., 1998). ARF recruits a specific spectrin to Golgi membranes (Godi et al.,
1998) in a mechanism involving the PIP2 binding domain of spectrin [the pleckstrin-homology (PH)
domain]. Spectrin is needed for maintaining the structural integrity of the Golgi. Agents that block
the binding of spectrin inhibit the transport of vesicular stomatitis virus G protein from the ER to the
medial compartment of the Golgi complex.
http://www.albany.edu/~abio304/text/chapter_11.html (11 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
B. Targeting of Proteins and Vesicles; SNAREs
As we saw in Chapter 10, the sorting signals of cargo proteins are responsible for the targeting of
proteins. Similarly, retention signals are likely to be required for retention by the appropriate
compartment. The sorting signal is recognized directly or indirectly by the proteins that form the
vesicle coat to insure packaging of the cargo protein in the corresponding vesicle. Once assembled,
specific tethering and docking of the transport vesicle are needed to deliver the vesicle to the
acceptor compartment. At the target site, the docking depends on the complementary SNAREs.
SNAREs are integral proteins of intracellular membranes with a large domain on the cytoplasmic
phase. SNAREs present in a transport vesicle (v-SNARE; e.g. synaptobrevin in neurons) (Söllner et
al., 1993a) are recognized by another similar protein on the surface of the target compartment (t-SNARE; e.g. syntaxin in neurons) (Rothman, 1994; Søgaard et al., 1994) (see Fig. 1B). The two
usually correspond to R-and Q-SNAREs respectively (i.e., named after the glutamine and arginine
residues of the their cytoplasmic domains) (see Jahn and Südhof, 1999).The specific interaction is
apparently mediated by the extended cytoplasmic domain of the two different kinds of SNAREs
(Chapman et al., 1994). The role of SNAREs has been delineated most completely for the shuttles
between the ER and the Golgi (see Rothman; 1994, Paek et al., 1997) and from the Golgi to the
plasma membrane (Brennwald et al, 1994). A SNARE protein that functions within the Golgi
system has been isolated from mammalian cells in culture (Hay et al., 1997; Nagahama et al., 1997).
This protein of 28 kDa is associated with Golgi membranes. The transport from the ER to the trans-Golgi and TGN is blocked by the cytoplasmic domain of this protein or an antibody to it. These
agents are thought to block transport between the medial-Golgi and the trans-Golgi and TGN (Hay
et al., 1997). A yeast v-SNARE protein has been implicated in retrograde transport to the cis-Golgi
(Lupashin et al., 1997) and probably the anterograde transport from late Golgi and a prevacuolar
compartment (von Mollard, et al., 1997).
The two kinds of SNARES, v-SNARES and t-SNARES, must be capable of binding each other
specifically. Complementary pairs have been identified in yeast for the ER-Golgi step (Lian and
Ferro-Novick, 1993; Søgaard et al., 1994) and the Golgi-plasma membrane step (Aalto et al., 1993;
Propopov et al., 1993; Brennwald et al., 1994). In addition, SNARE-pairs have been identified in
regulated exocytosis of neuronal synapses (see Südhof, 1995). A study of Nichols et al. (1997) has
shown, in yeast vacuoles, the need for t-SNARE in one membrane and v-SNARE in another for
fusion to occur. In a more systematic approach, McNew et al. (2000) tested all of the potential v-SNAREs encoded in the yeast genome to examine whether they can partner t-SNAREs of the Golgi,
the vacuole and the plasma membrane. Vesicle and target SNAREs were reconstituted into two
separate sets of liposomes and tested for fusion (see Weber et al., 1998). In these experiments, the
phospholipids in the vesicles reconstiuted with v-SNAREs were labeled with a mixture of probes
whose fluorescence is quenched. When fusion to the vesicles containing t-SNAREs occurs, the
fluorescent probes are diluted and become fluorescent as the quenching decreases. The SNARE
proteins reconstituted in liposomes were found to function as predicted by the SNARE hypothesis
by exhibiting the appropriate specificity. However, other factors seem to play an important role as
well (see below). These are proteins involved in the localization of vesicles, such as tethering
factors, or those involved in the activation of the SNARE complexes (see Waters et al., 1999;
http://www.albany.edu/~abio304/text/chapter_11.html (12 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
Mellman and Warren, 2000).
The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices (Parlati et al.,
2000; Fukuda et al., 2000). For the transition between ER and Golgi, one SNARE contains three of
the helices (t-SNARE) and the other one (v-SNARE). Fusion does not take place with any other
combination. For t-SNAREs on the plasma membrane, the protein syntaxin, supplies one helix and a
SNAP-25 protein contributes the other two. The assembly of SNARE complexes involves Rab (see
Rothman, 1994; Søgaard et al., 1994) and Sec1. Rab proteins are GTP-binding proteins (Simons and
Zerial, 1993, see below). Sec1 proteins (Aalto et al., 1992) bind specifically to t-SNARE subunits
(Pevner et al., 1994).
The need for separate and complementary v-SNAREs and t-SNARES, one in the cargo carrying
vesicle and the other in the target membrane, explains many of the available data. However, in
COPII vesicles the situation is more complex. The vesicles originating from the ER must first
cluster to form VTCs (see Chapter 10). The large vesicles of the VTC are then targeted to the cis-Golgi with which they fuse. Consistent with this view, yeast vesicles can be isolated in clusters
(Lian and Ferro-Novick, 1993). In addition, the t-SNARE syntaxin 5 is present in the vesicles, as we
might expect if they fuse and not in the Golgi membranes (Rowe et al., 1998) and is essential for the
assembly of vesicular-tubular-preGolgi intermediates as well as for the delivery of the cargo to
We saw that the SNARE hypothesis postulates that the specificity of membrane fusion events
resides on the SNARE receptors, SNAREv and SNAREt joining with each other. A good deal of
data just reviewed supports this view (e.g., see Weber et al., 1998). However, recent studies suggest
that the process is more complex. It has been argued that the specificity of targeting may not depend
on SNAREs (e.g., Kaiser and Ferro-Novick, 1998). t-SNAREs are not localized at specific sites of
the target membrane (e.g., Garcia et al., 1995) and v-SNARE can bind to more than one t-SNARE
(von Mollard, et al., 1997; Holthuis et al., 1998). In some cases the system may be able to bypass
SNAREs entirely. The disruption of SNAREs [e.g., caused by microinjection of the cytoplasmic
domain of synaptobrevin (Hunt et al., 1994) or cell mutants lacking synaptobrevin or syntaxin
(Broadie et al., 1995)] does not prevent vesicle docking. A v-SNARE can reside in both anterograde
and retrograde-directed vesicles and a single v-SNARE can bind to several t-SNARES and a single t-SNARE can bind to several different v-SNAREs (see Götte and Fischer von Mollard, 1998; Pfeffer
et al., 1999). In the case of the ER-to-Golgi transport, another entity, the 800 kDa complex of 8
subunits, the transport protein particle (TRAPP) may account for the specificity. TRAPP, located in
the cis-Golgi, has a role in targeting and fusion (Sacher et al., 1998) and may have a role in
providing specificity since it is unique to this step, whereas all SNARE molecules are very similar.
As reviewed above, t-SNAREs and v-SNAREs present in separate vesicles interact. However, other
components are active in this process (Ungermann et al., 1998; Peters and Mayer, 1998). The small
GTP-binding protein Ypt7p holds together the vacuoles in a reversible reaction (Ungermann et al.,
1998). This stage requires the presence of various factors (see next section) but not SNAREs. A
similar SNARE independent attachment was first described in the attachment of the ER vesicles to
the Golgi (Cao et al., 1998a).
http://www.albany.edu/~abio304/text/chapter_11.html (13 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
In Saccharomyces cerevisiae, Vam7 (a t-SNARE) is targeted to vacuoles in a manner dependent on
the presence of phosphatidylinositol 3-phosphate in the vacuolar membrane. This interaction
involves the phox homology (PX) domain of Vam7 (Cheever et al., 2001). The PX domain is an 80-125 amino acid residue region of proteins involved in binding phosphoinositides (Kanai et al.,
C. Fusion
The machinery responsible for fusion is composed of three distinct proteins (Söllner et al., 1993):
NSF (Sec18p in yeast), SNAP (Sec17p in yeast) family members, and the SNAREs. Once
assembled, the complex forms a 20S particle (where 20S refers to the sedimentation coefficient).
Models of the structures of the proteins in the complex derived from crystallography give us some
understanding of the assembly of the 20S fusion particle (May et al. 1999; Yu et al., 1999; Rice and
Brunger, 1999). These studies provide us with a detailed view of the very similar structures seen
with the EM of the 20S particles (Hohl et al., 1998). The SNARE complex is rod shaped (2.5 x 15
nm). SNAP binds laterally, whereas NSF binds to one end of the complex to form a particle 22 nm
in length.
The association of SNAP and NSF with SNARE to form the 20S particle is sequential. The v-SNARE-t-SNARE complex binds 3 to 6 SNAP proteins and subsequently NSF (Söllner et al.,
1993b; Hayashi et al, 1995). The release of SNAP, which requires NSF, is accomplished before
vesicle docking (Mayer et al., 1996). Subsequently, v-SNARE from t-SNARE dissociate (Söllner et
al., 1993) and the fusion takes place. The SNAP and NSF proteins are common to most, if not all,
different transport steps. It might be expected the SNAREs are specialized since their interaction
determines the final fate of the cargo (Fig. 1B) (see Ferro-Novick and Jahn, 1994). However, this
was found not to be the case. In vitro testing showed very promiscuous binding of SNAREs derived
from different sources (Yang et al., 1999).
The SNAP proteins (Clary et al., 1990) and an ATPase, the NSF (Block et al., 1988; Malhotra et al,
1988; Wilson et al, 1989) assemble after docking. The v-SNARE-t-SNARE complex binds 3 to 6
SNAP proteins and subsequently NSF (Söllner et al., 1993b; Hayashi et al, 1995). The hydrolysis of
ATP dissociates v-SNARE from t-SNARE and releases the SNAP molecules (Söllner et al.,
1993).The fusion process is still not well understood.
In one of the current models of fusion, SNAREs have a role in bringing the two bilayers together in
a zipper like process (see Chen and Scheller, 2001). When the two bilayers become fused a pore
opens and then pore expands producing one continuous bilayer. At least in exocytosis, freeze
fracture EM (Chandler and Heuser, 1980) and patch clamping ( Breckenridge and Almers, 1987)
confirm the formation of a pore. Present thinking suggests that the pore is in the lipid components
with protein scaffolding, possibly SNAREs (see Monck and Fernandez, 1994, 1996;Lee and Lentz,
In mammals, the fusion of early endosomal vesicles and more mature endosomal vesicles requires
http://www.albany.edu/~abio304/text/chapter_11.html (14 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
the early endosome-associated protein (EEA1) and Rabaptin-5. EEA1 binds to phosphatidylinositol
3-phosphate in the endosomal membrane through its FYVE domain (e.g., Simonsen et al., 1998)
NSF was discovered in isolated systems. In low concentrations, the sulfhydryl alkylating agent
NEM, inactivated the acceptor membrane fraction of the intra-Golgi transport system so that it
would no longer be able to fuse to the incoming vesicles (Glick and Rothman, 1987). Untreated
peripheral proteins of the Golgi membranes restored function. A 76 kDa polypeptide, forming a
homotetramer in its native form, NSF, was found to be responsible for the activity (Block et al.,
1988). EM examination of the Golgi membranes of blocked cells (Diaz et al., 1989) indicated an
accumulation of uncoated intermediate transport vesicles associated with membranes. This
observation suggested that NSF acts as part of the complex needed to fuse the vesicle membrane
with the acceptor membrane. NSF was subsequently found to be needed for the in vitro fusion of
vesicles involved in the transport from ER to Golgi and for the fusion of endocytotic vesicles.
NSF shows 48% sequence identity to Sec18p of yeast. Furthermore, Sec18p can replace NSF in the
mammalian cell free system. NSF is a hydrophilic molecule not likely to interact with hydrophobic
domains such as the hydrocarbon leaflets of the bilayer membrane. However, it has two ATP-binding domains and displays ATPase activity, which determines its attachment to the membrane. In
addition to NSF, five other proteins have been found to be necessary in vesicle mediated transport.
Many others might also be required. The in vivo requirement for Sec18p was demonstrated using a
temperature sensitive mutant (Graham and Emr, 1991). The proteins of the cells were labelled by
exposure for a short period to radioactive amino acids at the permissive temperature (20oC),
followed by a chase at the nonpermissive temperature (37oC). The fate of two proteins, F and CPY,
was examined. These two were selected because the proteins undergo stepwise modifications in the
Golgi complex that can be easily recognized using SDS gel electrophoresis and
immunoprecipitation. In the case of the wild type cells, the F precursor proteins were rapidly chased
to the mature form of the protein (mF). In contrast, in the mutant cells the inactivation of the sec18
protein left the F protein in all intermediate compartments. These results indicate that each
sequential step must occur in a separate compartment, as we saw for other systems, and Sec18p is
involved in each step.
NSF is a member of the ATPases responsible for varied cellular activities, the AAA (ATPases
associated with a variety of cellular activities) superfamily (see Patel and Latterich, 1998;
Confalonieri and Duguet, 1995, see Frölich on the Web) which also includes proteasomal
components (see also Chapter 15). Some members of this family perform chaperone tasks (i.e.,
folding tasks) (see Chapter 15), others are associated with assembly, remodeling and disassembly.
They act in a variety of cellular functions (see Neuwald et al., 1999), including cell-cycle regulation,
protein degradation, organelle biogenesis and vesicle-mediated protein transport, and the initiation
of transcription. The AAA motif corresponds to a 230-amino-acid domain that contains Walker ATP-binding homology sequences and imparts ATPase activity.
NSF is involved in eukaryotic fusion events which also require SNAREs. The SNAREs are
associated at their cytoplasmic domains forming stable complexes (Hay and Scheller, 1997; Weber
http://www.albany.edu/~abio304/text/chapter_11.html (15 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
et al., 1998). SNAREs (v and t) constitute the minimum requirement for fusion of membranes for
one round of fusion. NSF and SNAPs being required for separation of the two SNAREs to allow for
the next round of fusions (see Weber et al., 1998). This activity depends on ATP hydrolysis. The
dissociation events require the soluble NSF attachment proteins (SNAPs) to bind to SNAREs
providing a binding site for NSF (Whiteheart et al., 1992; Wilson et al., 1992). The binding to
SNARE and SNAP increases the ATPase activity of NSF and at the same time the complex is
disassembled (Morgan et al., 1994, Barnard et al., 1997). NSF is made up of three domains: the
amino-terminal domain which is capable of interacting with SNAPs and SNAREs, and two similar
ATPase domains (D1 and D2). D1 is thought to be involved in remodeling the 20S particle; D2 is
thought to be responsible for the formation of NSF-hexamers (Neuwald, 1999). The complex is
thought to function similarly to chaperones in driving the remodeling of effector molecules using the
free energy generated by the hydrolysis of ATP (see Patel and Latterich, 1998; Neuwald, 1999)
SNAPs constitute a family of soluble proteins required for NSF to bind to Golgi membranes
(Weidman et al., 1989, Clary et al., 1990). α, β and γ SNAPs are 35, 36 and 39 kDa in molecular
weight, respectively. α-SNAP has also been shown to be required in vivo. In yeast, α-SNAP is
encoded by the SEC17 gene. In the absence of this protein, vesicles are accumulated (Kaiser and
Shekman, 1990). SNAPs bind to specific sites on the membranes the SNAREs) and this binding is
required for interaction with NSF. The binding site can be identified by crosslinking with
crosslinking reagents (Whiteheart et al., 1992). These show that α-SNAP attaches to a 30-40 kDa
protein. The various SNAPs bind at different sites of a receptor complex.
D. The GTPases
The GTP-binding proteins or GTPases discussed above (also referred to as G-proteins, not to be
confused with the VSV-G glycoproteins) have been shown to have a key role in intracellular
transport. Mutations in the genes known to code for GTP-binding proteins block several steps in
secretion. Furthermore, in mammalian cells, the Rab or Ras proteins which correspond to GTPases
are localized in specific membranes associated with the secretory pathway and intracellular transport
is blocked by treatments that inhibit small GTP-binding proteins.
There are seven major groups of GTPases. They share domains needed for guanine binding and
GTP hydrolysis. These domains are so similar that the three-dimensional crystal structure of the
proteins are superimposable. They diverge significantly in amino acid sequences in other regions of
the molecules. Presumably, these are the regions which determine functional specificity.
The role of heterotrimeric GTP-binding proteins in signal transduction was discussed in Chapter 7.
In this complex, the α-subunit of 40-50 kDa binds to the guanidine nucleotide. There are also two
additional subunits (β and γ of 35-36 and 8 kDa respectively). The present section will discuss both
the heterotrimeric and the lower molecular weight GTPases in relation to intracellular trafficking.
In contrast to the heterotrimeric GTPase, those of the Ras superfamily (also called Rab when
http://www.albany.edu/~abio304/text/chapter_11.html (16 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
derived from cDNA libraries from rat brain) are monomers (Sar1, ARF, Rab/YPT, Rac/CDC42 and
Rho families) 20 to 30 kDa in molecular weight. Those of the dynamin family are 60-80 kDa. With
the exception of ARF, all form complexes with cytoplasmic or membrane proteins which may have
a function similar to the β and γ subunits of the trimeric GTPase. Fatty acid residues are added
posttranscriptionally to several GTPases. In some cases, these have been shown to permit direct
interaction with membranes (Rossi et al., 1991). Rab, Rho, Rac and the γ-subunit of the
heterotrimeric GTP-binding protein have one or two prenyl moieties at the carboxyl terminal or at
cysteine residues. Members of the ARF and Gα family are myristylated at an amino-terminal
glycine residue. Palmitylation of cysteine residues may occur in the amino or carboxyl terminal of
Ras and G.
GTPases were originally shown to have a role in all the steps of intracellular transport through the
effects of guanosine-5-O-thiotriphosphate (GTPγS) and AlF4-. GTPγS, a non-hydrolyzable analog of
GTP blocks all GTPases. It inhibits almost all of the steps in the transport.
The heterotrimeric GTPase appears to function as an inhibitor of transport. Two specific α-subunits
are associated with the Golgi. Overexpression (by transfection, see Chapter 1) of one of these Gαs
leads to slowing of the transport of proteoglycan through the Golgi (Stow et al., 1991). Conversely,
inhibition of Gα speeds up transport. A similar role is suspected for ER to Golgi transport at the
budding stage (Schwaninger et al., 1992).
The well-defined morphology of the mammalian system provides a clear picture of the role of the
small GTP-binding proteins. A minimum of 12 mammalian rab genes have been isolated from
cDNA libraries using probes recognizing ras sequences. The proteins of six rab genes (rab1 to
rab6) have been shown to bind and hydrolyze GTP. Immunofluorescence,
immunoeletronmicroscopy and the immunological reactions after cell fractionation have provided
information on the localization of these proteins, as summarized in Table 2 (Rothman and Orci,
1992). This association of each Rab protein with a specific structure in the intracellular transport
pathway suggests that each GTP protein acts at a different step. However, direct evidence has been
provided by genetic approaches in yeast. These findings were extended to other systems using
cDNA technology and in vitro reconstitution systems. Numerous additional rab110,111 and ARF65
genes have been cloned and sequenced but the proteins have not yet been localized. In most cases,
there is a substantial pool of soluble GTPases in addition to the membrane-bound forms.
The transport of VSV G-protein was followed in vitro by measuring the incorporation of [3H]-N-acetylglucosamine (GlcNAc) from donor membranes (from a cell line lacking GlcNAc-transferase)
to acceptor membranes isolated from wild type cells (containing GlcNAc transferase) (Balch et al.,
1984a,b). The use of synthetic polypeptides representing partial amino acid sequences of specific
GTPases, or the use of antibodies to the individual GTPases, identified the individual steps in which
they are involved. A polypeptide will block the action of a GTPase when it competes with it, for
example, for a receptor site. Similarly, a specific antibody can block individual steps by depleting
the cells of the Rab-protein.
Table 2 Partial list of GTPases and their location.
http://www.albany.edu/~abio304/text/chapter_11.html (17 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
Small GTP-binding
Location(s) when
membrane bound
Rab family rab1Ap/rab1Bp/ypt
ER and Golgi
Neurosecretory vesicles
Early endosomes, plasma
Early endosomes, plasma
Golgi stack
Late endosomes
Post-Golgi vesicles, plasma
ARF family ARF1p
Golgi stack
Reproduced with permission from Nature (1992) Rothman, J.E. and Orci, L. 355:409-415.
Research efforts are now directed toward understanding how the small GTP-binding proteins
interact with specific membranes. This is likely to require the identification and study of multiple
binding proteins in the various membranes.
A summary of some of the functions of GTPases is presented in Table 3. In this table, an important
function of the GTPase is listed in the first column, however, some of them have multiple functions.
The GTPase responsible is listed in the second column and the evidence available is listed in the
The precise mechanism of the action of small GTPases is not known. However, their properties
provide hints that they can function as switches, initiating or terminating biological processes. We
have seen this kind of role in the docking of ribosomes and nascent polypeptide chains in the ER.
http://www.albany.edu/~abio304/text/chapter_11.html (18 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
The GTPases change in conformation in response to the phosphorylative state of the bound
nucleotide. The active conformation occurs when the bound GDP exchanges with GTP. Hydrolysis
of the GTP returns the protein to its inactive conformation. The reactions of the GTPases require the
presence of other proteins. Two proteins regulate the conformational changes. The exchange of GDP
for GTP is facilitated by the guanine nucleotide dissociation proteins (GEPs) and inhibited by
guanine nucleotide dissociation inhibitors (GDIs). In addition, the GTPase activity of the Ras
superfamily is slow in the absence of GTPase activating proteins (GAPs).
Table 3 Role of GTPases in Intracellular Transport
vesicle budding from ER  Sar 1  overcoming mutant block
location in ER, trnaslational
elements and Golgi (2)
formation of COP vesicles
from Golgi
ARF (ADP ribosylation
in vitro assay (3)
endocytotic role dynamins accumulation of coated pits
in defective mutants (4)
transport from ER and cis to
trans Golgi
Rab1 in vitro assay (5)
early endosome fjunction Rab2, 5 and 7 Rab domains target to
endosomes (6)
TGN vesicle budding  Rab6 antibodies to Rab6 block
TGN budding (7)
exocytosis: synaptic vesicle Rab3A required for transfer from
vesicle to cell surface (8)
exocytosis: mast cells   discharge of granules when
Rab delivered into cells (9)
Ref. (1) Nakano, A. and Maramatsu, M. (1969), J. Cell Biol. 109:2677-2691.
(2) Kuge, O., Dascher, C., Orid, L., Amherdt, M., Plutner, H., Ravazzola, M., Tanigawa, G.
Rothman, J.E. and Balch, W.E. (1994) J. Cell Biol.. 125:51-65
http://www.albany.edu/~abio304/text/chapter_11.html (19 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
(3) Orci, L., Palmer, D.J.,, Perrelet, A., Amerdt, M.., Palmer, D.J. and Rothman, J.E.. Nature
362:648-652; 364:732-734.
(4) Kosaka, T. and Ikeda, L. (1983) J. Neurobiol.. 14:207-225.
(5) Plutner, H., Cox, A.D., Pind, S., Khosravi-Far, R., Bourne, J.R. et al., (1991) J. Cell Biol. 115:31-43.
(6) Chavier, P., Gorvel, J.P., Steizer, E., Simons, K., Greenberg, J. and Zerila, M. (1991) Nature
(7) Jones, S.M., Crosby, J.R., Salamero, J. and Howell, K.E. (1993) J. Cell Biol . 122:775-788.
(8) Matteoli, M., Takel, K., Cameron. R., Hurbult, P., Johnston, P.A. et al. (1991) J. Cell Biol .
(9) Oberhauser, A.F., Monck, J.R., Balch, W.E. and Fernandez, J.M.. (1992) Nature 360:270- 273.
Dynamins (see McNiven et al., 2000) constitute a family of large (100-kDa) GTPases that seem to
fulfill several roles in membrane trafficking in eukaryotic organisms. Different dynamin isoforms
are present in different tissues and even in the same tissue. At least 25 different mRNAs are
produced by the 3 dynamin genes by alternative splicing ( Cao et al., 1998b).
Dynamins have been shown to play important roles in endocytosis and vesicle formation (see
Chapter 9 and above). Dynamin is needed for clathrin-mediated endocytosis (e.g., Herskovits et al.,
1993; van der Bliek, 1993) and the formation of vesicles from caveolae (see Chapter 9) (Henley et
al, 1998; Oh et al., 1998). Inhibition of dynein function, inhibits the scission of caveolae both in
vivo and in vitro. Dynamin has also been implicated in other kinds of endocytosis (see Sandvig and
van Deurs, 1996), including the intake of fluid in cultured mammalian cells (Henley et al., 1999)
and phagocytosis in macrophages (Gold et al., 1999).
Dynamins have a role in vesicle formation in steps of the endocytotic pathway other than vesicle
formation from the plasma membrane. In HeLa cells, the expression of a mutant of dynamin does
not affect clathrin-independent endocytosis. However, the pathway from endosomes to Golgi is
blocked (Llorente et al., 1998). Similarly, disruption of a dynamin-family proteins in Dictyostelium
discoideum has a very broad effect (including a defective fluid-phase uptake) (Wienke et al., 1999).
In addition, dynamin was found to colocalize with vacuolin, a marker of a postlysosomal
compartment. In mammalian cells, confocal imaging (see Chapter 1) showed that dynamin is
associated not only with the plasma membrane but also the trans-Golgi network, and a perinuclear
cluster of structures containing cation-independent mannose 6-phosphate receptor. Electron
microscopy showed that the structures correspond to late endosomes with a localization of dynamin
preferentially in tubulo-vesicular processes of these endosomes (Nicoziani et al., 2000). In other
studies, dynamin was found associated with the TGN (Henley et al., 1996; Maier et al., 1996). GFP-dynamin chimeras (see Chapter 1) expressed in cultured rat hepatocytes appeared in the clathrin
coated vesicles at the cell surface and the TGN (Jones et al., 1998). An in vitro system produced
dynamin in clathrin coated and non-clathrin coated vesicles. Cells expressing a mutant of dynamin
were found to accumulate GFP-protein chimeras in the TGN (Kreizer et al., 2000). In
Saccharomyces cerevisiae, a dynamin, dnm1p, has been shown to be involved in transport to the
vacuole from the TGN (Gammie et al., 1995).
http://www.albany.edu/~abio304/text/chapter_11.html (20 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
The dynamins are capable of interacting with several components. Their pleckstrin homology (PH)
domain allows them to bind to phosphoinositides and their proline-rich domain (PRD) allows to
bind to the SH3-domains of variety of effector molecules. A coiled-coil region (CC domain) f
dynamins is a GTPase effector domain (Sever et al., 1999).
In vivo, dynamin interacts with other proteins that may alter the geometry of lipid structures to
produce vesicles. An interaction with endophilin 1 (Schmidt et al., 1999) (that transfers arachidonate
to lysophosphatidic acid) has been demonstrated. This reaction is likely to favor the formation of
highly curved lipid structures. There is at least one example of this mechanism: BARS (Weigert et
al, 1999) (that acylates lysophosphatidic acid) was shown to induce the formation of vesicles in
Golgi membranes of rat brain. However, in this case the system seems to operate independently of
Several studies have indicated the dynamin is associated with actin or actin-binding or actin-depolymerizing proteins, suggesting a role of these interactions in the formation of vesicles (e.g.,
Witke et al., 1998; Qualmann et al., 1999)
E. ER and Golgi Transport
ER to Golgi Transport
Present information indicates that COPII vesicles mediate the transfer of cargo from the ER to the
Golgi. However, the process may be more complex. We saw that larger vesicular components,
formed by vesicle clustering, carry cargo to the cis-Golgi (see Chapter 10, Section III). Therefore,
the vesicular tubular clusters (VTC) and not the COPII vesicles are targeted to the cis-Golgi.
Consistent with this view, the t-SNARE, syntaxin 5, is present in the vesicles as well as in the cis-Golgi (Rowe et al., 1998) and is essential for the assembly of VTCs. What this suggests is that the
VTCs are formed by an aggregation of these vesicles. Unfortunately, since retrograde transport also
takes place, it is difficult to consider this argument decisive.
The role of COPII in anterograde transport from the ER is well established. However, much of the
present evidence also supports a role of COPI for anterograde transport between ER and Golgi and
between Golgi stacks (Chapter 10, Section 3C; see also Kreis et al., 1995). However, studies with
yeast COPI mutants demonstrate ER to Golgi transport in COPI-impaired cells for some proteins but
a complete block for others (Gaynor and Emr, 1997). In contrast, the remaining ER to Golgi
transport required COPII.
A possible explanation for the complex role of COPI in anterograde transport from the ER, might
rest on a sequential role of the two coat complexes. In in vitro experiments using isolated ER
fragments, showed that vesicles released by the ER-derived system were COPII coated and
contained VSV-G protein and p58. p58 is an endogenous recycling protein. However, preparations
from ARF1 mutants that prevent COPI recruitment blocked subsequent movement to the isolated
Golgi membranes (Rowe et al., 1996). These observations suggest that COPII drives the export from
the ER and then COPI replaces COPII in the vesicles.
http://www.albany.edu/~abio304/text/chapter_11.html (21 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
In yeast, the transfer of cargo from the ER to the cis-Golgi involves the transport protein particle
(TRAPP), a 1100 kDa complex (Sacher et al., 1998). TRAPP mediates vesicle docking and fusion.
Proteins analogous to the subunits of TRAPP have been found in mammalian species (see Guo et
al., 2000). Subunits of TRAPP have been found in early Golgi membranes, in X-100 insoluble
membrane components suggesting a presence in rafts.The protein p115 (also known as TAP) is
needed for the transport from the VTC to the cis-Golgi (Nelson et al., 1998). It plays a more general
role in intra-Golgi transport (see next section). In yeast, two other factors have been implicated in
ER-to-Golgi transfer of cargo. Uso1p acts before SNAREs and is required for tethering (Cao et al.,
1998). Sec34p and Sec35p also function in tethering (VanRheenen et al., 1998).
Transport from the Golgi stacks
Isolated Golgi stacks incubated with cytosol and ATP generate 75 nm vesicles from the cisternae
(Balch et al., 1984a) (now recognized as COPI-coated vesicles). They correspond to transport
vesicles because they can be shown to contain transported proteins (in these experiments the G-protein of vesicular stomatatis virus, VSV). The transport between cisternae, also mediated by
vesicles, can be followed biochemically through the progression in the glycosylation of proteins.
Like other transfers studied so far in vitro, these also require cytosol and ATP (e.g., Braell et al.,
The 75 nm vesicles can be either coated or uncoated (Orci et al., 1986). The relationship between
these two kinds of vesicles is revealed by the effect of two inhibitors. GTPγS, a non-hydrolyzable
analog of GTP, produces an accumulation of buds and coated vesicles (Melançon et al., 1987). GTP
hydrolysis is needed for the fusion of vesicles onto their target compartment and GTPγS blocks this
process. N-ethylmaleimide (NEM) block, on the other hand, produces an accumulation of uncoated
vesicles (Melançon et al., 1987). As discussed later, NEM blocks the docking of vesicles to the
target membrane (see below). Simultaneous treatment with GTPγS and NEM produces an
accumulation of coated vesicles (Orci et al., 1989); therefore, the coated vesicles are the precursors
of the uncoated vesicles. Both kinds of vesicles should accumulate if the two were produced
independently. Alternatively, if the uncoated vesicles were the precursor, only the uncoated vesicles
would accumulate.
The accumulation of vesicles derived from the Golgi stacks in a cell free system in the presence of
GTPγS, permitted the isolation and direct biochemical study of the COPI (coatomer) coated vesicles
(Malhotra et al., 1989; Serafini and Rothman et al., 1992).
The COPI coatomer is composed of seven proteins (α, β, β’, γ, ε and ζ) ranging in molecular weight
between 60 and 160 kDa. β-COP has been localized with immunofluorescence and immunoelectron
microscopy in the CGN and TGN and has also been found in soluble cytoplasmic complexes
(Duden et al., 1991b, Waters et al., 1991). Presumably, the cytosolic β-COP is the source of this
subunit of the vesicle’s coat.
Members of the p24 protein family bind to COPI coatomeres and may have a role in the recruitment
http://www.albany.edu/~abio304/text/chapter_11.html (22 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
of COPI to the Golgi membranes (Dominguez et al., 1998). p24 and p23 are cargo receptors found
in both COPI and COPII coated vesicles. In COPI coated vesicles, they are present in stoichiometric
amounts relative to coatomer and the GTPase, ARF. The cytoplasmic domains of these proteins bind
to coatomer (e.g., Sohn et al., 1996) and are needed for cycling in the early secretory pathway (e.g.,
Nickel et al., 1997). COPI coatomer vesicles also bind to KKXX (di-lysine motif) and KXKXX
containing proteins. The KDEL receptor that binds to proteins containing the KDEL motif is also
transported in these vesicles. The γ subunit recognized the lysine motifs (Harter et al., 1996). These
motifs are retrieval signals (see Table 2, Chapter 10) in line with the role of COPI coated vesicles in
retrograde transport.
COPI is required for intra-Golgi retrograde transport in vitro (see Lin et al., 1999). COPI vesicles or
transport intermediates can be isolated or produced in vitro and have a high level of Golgi-resident
enzymes (Lanoix et al., 1999; Love et al., 1998) and KDEL receptors (Sönnichsen et al., 1996)
suggesting that they are recycling intermediates.
Although there is evidence for the involvement of COPI in anterograde transport (see previous
section), the evidence for a role of COPI in retrograde transport is less ambiguous. Subunit of the
coatomer bind directly to proteins with the di-lysine retrieval motif (Cosson and Letourneur, 1994).
Furthermore, mutation in some of these subunits prevents recovery to the ER of proteins with the di-lysine retrieval motif (Cosson et al., 1996). In view of these findings, some investigators are
postulating that the role of COPI in anterograde transport is indirect, recycling components needed
for COPII transport by retrograde transport. However, results of several experiments continue to
support a direct role of COPI in both anterograde and retrograde transport. Immunocytochemistry
with the electron microscope using colloidal gold and antibodies (to COPI subunits, KDEL
receptors and proinsulin) have demonstrated that both anterograde transport of proinsulin and VSV
G protein and retrograde transport of a KDEL receptor occur in COPI vesicles. These vesicles
constitute two distinct populations that together account for at least 80% of the vesicles present. The
COPI vesicles bud from every level of Golgi cisternae. Similar results were obtained in in vitro
experiments (Orci et al., 1997).
In addition to a retrograde pathway which involves COPI coat proteins, a transport pathway from
Golgi to ER has been found which functions independently from COPI coat proteins (see Storrie et
al., 2000). This pathway returns Golgi resident proteins (as well as protein toxins) to the ER and
may have a primary role in the recycling of lipids. Microinjection of antibodies to coatomer, block
recycling of KDEL receptor and a lectin-like molecule, ERGIC-53, from Golgi to ER. Proteins
containing sequences recognized by the KDEL receptor are also inhibited (Girod et al., 1999). In
contrast, microinjection of anti-COPI antibodies or an Arf-1 mutant (Arf is required for COPI-coated vesicle assembly) does not interfere with the transport to the ER of Golgi-resident
glycosylation enzymes or Shiga toxin/Shiga-like toxin-1. However, overexpression of a Rab6 (a
small GTPase, see discussion below) mutant blocks retrieval of Golgi-resident glycosylation
enzymes and Shiga toxin/Shiga-like toxin-1. However, it has no effect on KDEL receptor, KDEL
containing proteins or ERGIC-53. These observations indicate that there are two pathways for the
retrieval of proteins from the Golgi to the ER.
The protein p115 (TAP), which has a role in the transport from the VTC to the cis-Golgi (see
http://www.albany.edu/~abio304/text/chapter_11.html (23 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
previous section) is required for intra-Golgi transport (Waters et al., 1992) and is likely to function
in tethering several steps. It has been implicated in the binding to the plasma membrane during
transcytosis (Barroso et al., 1995).
A. Cell Polarity
In an organized tissue, many cells have regions of the cytoplasm and the cell surface that differ in
composition and function. They are said to be polar. This polarity can be preserved when the cells
are cultured on a solid medium. Epithelial and endothelial cells form sheets in which they are held
together by junctional complexes which prevent exchange between the two domains and prevent
exchanges between compartments. A diagrammatic representation of polar cells in Fig. 2 shows
apical and basolateral surfaces. The adherens junction is responsible for adhesion between the cells.
In vertebrates the tight junction and in other animals the septate junction, prevent the exchanges.
Vertebrate epithelial and endothelial cells, are held together by the tight junctions at contact points,
the desmosomes which serve as anchoring points for intermediate filaments. In the polarized cell,
the apical and the basolateral surfaces have distinct lipid and protein domains (Simons and Fuller,
1985). This distinct composition could not be maintained unless the two were prevented from
exchanging materials and the various components were specifically targeted when newly
Essentially, tight junctions play a dual role as barriers and fences (see Chapter 4) (e.g., see
Gumbiner, 1993; Anderson and Van Itallie, 1995). The barrier function refers to the tight seal that
prevents diffusional exchanges between separate compartments The fence function refers to the
prevention of exchanges between the basolateral and apical domains of the plasma membrane to
maintain specialized functions, such as unidirectional secretion or active transport across the sheets.
In addition to their structural role, cell junctions can also provide signals that initiate cascades
involved in cell growth and differentiation (e.g., see Clark and Brugge, 1995; Takahashi et al.,
(1998); Reichert et al., 2000). For example, note that components of the signaling systems are
present at junctions (see below). In some cases, the role of the cell junction component may more
direct, for example, the adherens junction protein β-catenin (see below) is translocated into the
nucleus and binds to a transcription factor (see Nusse, 1997; Eger et al., 2000). Similarly, the
junctional component CASK (see Hata et al., 1996) is translocated into the nucleus (Hsueh et al.,
2000) and is required for EGF receptor localization and signalling in the nematode Caenorhabditis
elegans. CASK is membrane-associated guanylate kinase that is bound to the adhesion protein,
syndecan, at epithelial cell junctions (Cohen et al., 1998). In the nucleus, a complex of CASK and
Tbr-1 binds to a specific DNA sequence. Tbr-1 is a T-box transcription factor that is involved in
forebrain development. The complex of the CASK and Tbr-1 activates several genes containing the
T-box. T-box gene family have a binding domain, the T-domain which is important for
http://www.albany.edu/~abio304/text/chapter_11.html (24 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
The structure and formation of tight junctions as well as the factor that underlie the formation and
maintenance of distinct membrane domains have been explored.
Maintenance of polarity
Some progess has been made in the study of the maintenance of polarity in Drosophila epithelium.
Proteins which localize to one of three surfaces and suspected to have a role in organizing the
corresponding domain have been called epithelial cell surface organizers (ECSOs) (see Tepass,
1997). These include cadherins (see below) that localize laterally (see Drubin and Nelson, 1996),
Crumbs proteins of Drosophila that localize to the apical surface in (e.g., Wodarz et al., 1995) and
β1, α integrins (see Chapter 6) located in the basal surface of kidney epithelium (Sorokin et al.,
1990) and Madin-Derby canine kidney (MDCK) cells (e.g., Schoenenberger et al., 1994). ECSOs
are thought to be recruited and retained to their domain by external signals such as homophilic
adhesion in the case of cadherin. The interaction of ECSDOs with cytoplasmic factors permit
interactions with the cytoskeletal system.
In Drosophila, the mutation in four genes (stardust, sdt, bazooka, baz, crumbs, crb, and discs lost,
dlt) has been shown to be lethal by eliminating polarity in epithelial cells (see Tepass, 1997). CRB
and DE-cadherin are considered key regulators of polarity. CRB is an integral membrane protein
with a single transmembrane domain. It contains thirty EGF-like (for other examples see Chapter 6,
Fig. 10 and 11) and four laminin AG domain-like repeats in its extracellular segment and a short
cytoplasmic tail of thirty seven amino acids. CRBs is expressed apically in ectodermally derived
epithelia. Interestingly the mRNA for CRBs is found in the apical cytoplasm ( Tepass et al., 1990),
suggesting that it is the mRNA and not the protein which is targeted to this region. Several examples
of proteins targeted via their mRNA are known (see Chapter 10). Overexpression of CRB expands
the apical plasma membrane and reduces the basolateral domain. In contrast, BAZ and DLT are
cytoplasmic proteins with protein-protein interacting domains such as the PDZ domain. At least in
vitro, one of the PDZ domains of DLT binds to the cytoplasmic domains of CRB ( Bhat et al., 1999,
Klebes and Knust, 2000) and the laterally localized Neurexin IV (NRXIV). Interference with DLT
(mutations or introduction of double-stranded RNA) lead to mistargeted CRB and NRX IV and
disruption of epithelial polarity. The apical distribution of DLT depends on the presence of CRB.
These and other observations suggest that CRB and DLT together with other proteins define the
localization of the zonula adherens ( Klebes and Knust, 2000) and independently the loss of cell
polarity (Grawe et al., 1996). An additional protein, Scribble was found to be necessary to maintain
the polarization of cells (Bilder and Perrimon, 2000), as development proceeds in Drosophila
embryogenesis. Scribble is localized to the epithelial septate junction. Without Scribble, adherens
proteins no longer assemble in the apical-basolateral region interface and apical proteins are no
longer localized at the apical region. Scribble, a protein of 195 kDa (calculated from the cDNA),
was found to contain many protein-protein binding domains and is likely to correspond to a scaffold
needed for other proteins to assemble.. The carboxy-end has four PDZ domains (the initials of the
first three proteins that were discovered with the motif). The amino-end contains sixteen leucine-rich repeats. Similar repeats are known to bind RhoA and Rac-GTPases, known to be associated
with polarity and junctions (Jou and Nelson, 1998; see Kaibuchi et al., 1999). Many PDZ proteins,
including Scribble localize at septate junctions, although others localize elsewhere. The actual
molecular role of Scribble is still unknown. It may have a a role in targeting or it might even be part
http://www.albany.edu/~abio304/text/chapter_11.html (25 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
of the fence that prevents exchanges between the two regions.
Components of tight junctions
EM studies of thin sections (Farquhar and Palade, 1963) revealed discrete sites at which tight
junctions produced close contacts between cells (“kissing points”). Freeze-fracture EM showed
anastomosing strands in the cytoplasmic leaflet of the plasma membrane with complementary
grooves in the external face of the bilayer (see Staehelin, 1974).

Fig. 2. Arrangement of epithelial cells forming a sheet.
Occludin, a 60 kDa integral protein was identified in tight junction strands (Furuse et al., 1993;
Ando-Akatsuka et al., 1996). The cloning of the corresponding cDNA (see Chapter 1) and
hydrophobicity plots (see Chapter 4) showed that occludin had four possible transmembrane
domains and three cytoplasmic domains including the amino- and carboxy-terminals (see Ando-Akatsuka et al., 1996). Occludin is involved in forming barriers and fences. The barrier function is
shown, for example, by the increase electrical resistance across the epithelium when chicken
occludin is overexpressed in under conditions in which they formed tight junctions (McCarthy et al.,
1996). Expression of the carboxy-terminally truncated occludin, rather than wild-type occludin
(Balda et al., 1996), was found to render MDCK cells incapable of maintaining a fluorescent lipid in
a specifically labeled cell surface domain, indicating that occludin is also involved in providing an
apical/basolateral intramembrane diffusion barrier.
More recently, claudin-1 and claudin-2, two 23 kDa integral membrane proteins, were found in tight
junctions of chick liver or transfected MDCK cells (Furuse et al., 1998a). Hydrophobicity analysis
(see Chapter 4) indicated four possible transmembrane domains and cDNA analysis showed no
sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy (see Chapter
1) revealed that both claudins were present in the tight junction strands. When introduced in
fibroblasts lacking tight junctions, these proteins induced the formation of a network of strands and
http://www.albany.edu/~abio304/text/chapter_11.html (26 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
grooves at contact sites (Furuse et al., 1998b). The strands more closely resemble those in native
tight junctions, suggesting that the occludins are major components of the junctions. Extensive
studies indicate that the claudin family contains many members, as many as fifteen have been found
in data-base searches of cDNA (Morita et al., 1999a,b,c; Tsukita and Furuse, 1999).
Certain claudins appear to be associated with specific tissues. Claudin-5 and -6 have been found in
tight-junctional strands of endothelial, but not of epithelial cells (Morita et al., 1999b). Clostridium
perfringes enterotoxin, which binds specifically to claudin-3 and -4, were shown to disrupt tight
junction strands in transfected L fibroblast cells and MDCK cells (Sonoda et al., 1999) implicating
their presence in those junctions. The possibility of other molecular components forming tight
junctions cannot be excluded at this time (see Tsukita and Faruse, 1999)
Aside from the proteins forming tight junctions, there must be some structural basis for establishing
polarity. In addition to targeting sequences and corresponding receptors there must be an assembly
that includes cytoskeletal elements and permits the transfer of cargo to specific locations. The
generation of polarity is of fundamental importance in the physiology of a variety of cells. A general
model of the development of cell polarity has been presented (Drubin and Nelson, 1996). In this
model, clues at the surface lead to localized assemblies of submembrane elements, including the
cytoskeleton responsible for the organization of a pathway along an axis of polarity.
In the case of epithelial cells, the position of adhesion receptor proteins is determined by adhesion to
other cells or the extracellular matrix (ECM) (for cell-cell adhesion, E-cadherin; for adhesion to the
ECM, the integrins) (see also Fig. 7C, Chapter 4). This interaction generates localized assembly of
cytoskeletal elements. Integrins are bound by α-actinin and talin which recruit actin and actin-associated proteins. In turn, these serve as a scaffold for the assembly of signaling components, such
as adhesion kinase and components of the Ras pathway (such as SOS and Grb2 and GTP-binding
proteins), and may even lead to regulating gene expression as already discussed. Similarly, cadherin
recruits cytoplasmic proteins such as β-catenin, plakiglobin and P120. The binding of these proteins
to α-catenin (that has some homology with vinculin) may connect the complex to actin. Cadherin-catenin complexes recruit kinases (Src and Yes) and protein-tyrosine phosphatase, components
associated with signaling (see Chapter 7). GTP-binding proteins and Ras may associate with the
complex. In addition, the region that remains free, the apical region, also seems to respond and
assemble a distinct actin network cross-linked with villin, fimbrin and myosin I. Talin, α-actinin and
vinculin are involved in the formation of fibers containing actin at focal contacts (see Chapter 23).
Catenin, vinculin, α-actinin and plakoglobin have a similar mission but are part of an actin
containing belt connecting epithelial cells and are attached to cadherins. Fimbrin and villin are actin
bundling proteins (villin is present only in microvilli) (see Chapter 24). Talin, α-actinin and vinculin
are involved in the formation of fibers containing actin at focal contacts.
The events accompanying cell adhesion also redistribute the microtubules with consequences on the
targeting of secretory products. Depletion of kinesin disrupts the basolateral delivery, and depletion
of kinesin and dynein disrupts apical delivery (Lafont et al., 1994). Kinesin and dynein are motor
molecules that move along microtubules (see Chapter 24). The difference between the microtubules
transport to the two faces suggest that the organization of the microtubules is distinct in the two
http://www.albany.edu/~abio304/text/chapter_11.html (27 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
The determination of specific targeting of secretory vesicles is clearly closely dependent on these
events. In yeast, this is shown by mutations of the genes coding for sec6/sec8 which lead to the
accumulation of secretory vesicles in the bud of a daughter cell (Novick et al., 1990; TerBush,
1996). The proteins are found only at the tip of the bud, hence they determine polarity.
The sec6/sec8 complex, also known as exocyst, has been implicated in exocytosis and is specifically
located at sites of vesicle fusion. In yeast, the complex contains seven subunits of 70 to 155 kDa,
whereas in the rat, the complex has eight components ranging from 71 to 110 kDa (see Hsu et al.,
1996, 1998). In rat brain, sec6 and sec8 are two components of a 17S complex of 743 kDa
homologous to the yeast Sec6/8/15 complex of 834 kDa, which is required for exocytosis. The rat
brain complex associated with the plasma membrane has been implicated in exocytosis by its
immunoprecipitation with syntaxin, a plasma membrane protein critical for neurotransmission (see
above and Chapter 22).
In the rat brain, the complex is present at sites of neurosecretion such as the hippocampal synapses
(Hsu et al., 1996) and is essential for survival. Mice with a mutation in Sec8 die early during
embryogenesis at the primitive streak stage (Friedrich et al., 1997). In the MDCK cell line, the
complex is required for calcium dependent cell adhesion (Grindstaff et al., 1998). When the cells are
rendered permeable by streptolysin, Sec8 antibodies inhibit delivery of LDL receptor to the basal-lateral membrane, but not the delivery of the receptor for the nerve growth factor p75NTR to the
apical membrane. These findings suggest that the complex is needed to recruit vesicles to specific
domains. Similar conclusions were reached in neuronal tissues where the sec6/8 complex seems to
specify sites for targeting vesicles at domains of neurite outgrowth and potential active zones during
synaptogenesis (Hazuka et al., 1999).
B. Targeting of Plasma Membrane Proteins
Viral coat proteins with different plasma membrane targets share the transport pathway through the
Golgi system (Rindler et al., 1984), as shown by immunological EM methods using colloidal gold
markers of different sizes in doubly infected cells. What routes do these glycoproteins follow after
leaving the TGN? Are they targeted directly to their surface of residence or do they make a stopover
at the other surface?
The proteins are sorted out in the TGN (Rodriguez-Boulan and Nelson, 1989). Whether the delivery
is direct or indirect can be determined by growing the cells in sheets so that either the apical or
basolateral surface is separately accessible to antibodies or proteases. A block in the transport would
implicate a stopover in the alternative surface. The answers are not simple. Some of the
glycoproteins are sorted out in the TGN so that they are targeted directly to either the apical or the
basolateral surface. The delivery of the G-protein of VSV to the basolateral surface (Pfeiffer et al.,
1985) and of the hemagglutinin of influenza virus to the apical surface is direct (Matlin et al., 1983;
Matlin and Simons, 1984). However, other patterns are possible. In rat hepatocytes, all membrane
proteins appear to be delivered to the basolateral surface and eventually, the proteins destined to the
http://www.albany.edu/~abio304/text/chapter_11.html (28 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
apical surface are then rerouted to their final destination (Bartles et al., 1987; Schell et al., 1992). In
other cells, such as a polarized intestinal epithelium cell line (Caco-2), apical proteins can either
proceed directly to the apical surface or arrive first to the basolateral surface (e.g., Matter et al.,
1990a, Le Bevic et al., 1990).
How are the membrane proteins targeted? The targeting need not differ in principle from other
targeting processes. The transported protein may have a targeting domain. Targeting could also have
an entirely different mechanism, possibly involving the lipid components of the membrane and acyl
chains attached to the targeted protein. As discussed below, there is evidence in some cases, for a
targeting mechanism involving glycosphingolipids. The sorting machinery (such as the TGN) must
recognize the signal. In addition, the protein must then be targeted to the appropriate membrane site,
possibly by the presence of another targeting domain. A separate but related problem is the
preservation of the makeup of the target membrane itself.
Originally, most investigators assumed that plasma membrane proteins were targeted by a signal to
the apical surface, a basolateral targeting occurring by default. However, sorting signals have been
demonstrated in the cytoplasmic domain of proteins destined to the basolateral surface (Hunzinker et
al., 1991, Casanova et al., 1991, Mostov et al, 1992). One set of signals appears to be contained in
the Tyr-containing signals for endocytosis via clathrin-coated pits (Mostov et al., 1992). Mutations
of the cytoplasmic tail block basolateral targeting and the proteins are delivered to the apical
surface. Internalization signals, required for endocytosis, can substitute for basolateral signals
(Collawn et al., 1991). It has been suggested that the signal consists of the presence of a reverse β-turn in the protein. Some signals are distinct from the endocytotic signal (e.g., Hunziker et al., 1991,
Aroeti et al., 1993) and, in some cases, mutation of the cytoplasmic Tyr that blocks endocytosis has
no effect on basolateral sorting (Hunziker et al., 1991).
A short amino acid sequence (approximately 14 residues) serves as a signal for basolateral
localization of some proteins (Yokode et al., 1992; Mostov et al., 1992). When expressed in livers of
transgenic mice, the LDL receptor containing this sequence is targeted to the basolateral surface.
Mutant receptors lacking the sequence are delivered to the apical side. The probable sequence of a
cytoplasmic domain is Arg Asn X Asp XX Ser/Thr XX Ser, perhaps recognized by an adaptor
molecule of the Golgi.
In polarized MDCK cells the transferrin receptor (TR) is localized in the basolateral surface. After
binding transferrin, ligand and receptor are taken up in coated pits by endocytosis and then returned
to the basolateral surface. TR, whether synthesized de novo or recycled from vesicles, depends on
its cytoplasmic tail for targeting. The targeting signal for both pathways is contained in residues 19
to 41. However, within this region the targeting sequence for the biosynthetic pathway is distinct
from that for the endocytotic pathway (Odorizzi and Trowbridge, 1997)
A variety of observations suggest more complexity than that presented in this discussion. For
example, integrins and laminin have been shown to be transported from the TGN to the basolateral
surface in separate vesicles (Boll et al., 1991), demonstrating the possibility that more than one
pathway may be responsible for the same localization.
http://www.albany.edu/~abio304/text/chapter_11.html (29 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
The glycosyl phosphatidylinositol (GPI) anchor of certain proteins (see Chapter 4) could serve as an
apical signal (see Simons and Ikonen, 1997) by clustering with glycosphingolipids, forming “rafts”
that may correspond, at least in part, to caveolin containing elements (see Chapter 4 and Chapter 9).
After the separation of lipid-linked proteins in the Golgi, vesicle budding could segregate them from
other components. A role of GPI in apical targeting has been demonstrated in Madin-Darby canine
kidney (MDCK) cells and intestinal cells by attaching a GPI anchor to proteins not originally
destined to the apical surface (e.g., see Soole et al., 1985; Brown et al., 1989; Lisanti et al., 1989).
Conversely, replacement of the GPI anchor of placental alkaline phosphatase with the
transmembrane and cytoplasmic domains of VSV G, switched its targeting from the apical to the
basolateral surface (Brown et al., 1989).
The recognition of GPI-anchored proteins could occur in an early step of the cis-Golgi (see Brown
and Rose, 1992) because these proteins were found associated with the glycosphingolipid
microdomains at stages requiring cis-Golgi reactions.
Although many observations are compatible with the raft -GPI model, other significant factors are
likely to come into play. Fisher rat thyroid cells (FRT) (Zurzolo et al., 1993) and MDCK
Concanavalin A-resistant cells (MDCK-ConAr) (Zurzolo et al., 1994) behave differently from other
polarized epithelial cell lines. FRT cells target glycosphingolipids and six out of nine detectable
endogenous GPI-anchored proteins to the basolateral surface. In contrast, two other GPI-anchored
proteins are apical and one is present at either surface. Transfection (see Chapter 1) of several model
GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected
results. The GPI anchored form of Decay accelerating factor (DAF) was targeted to the basolateral
domain. Similarly, the Herpes simplex gD-1 protein attached to GPI in the form of fusion protein,
gD1-DAF, was targeted basolaterally, where gD1-DAF was delivered directly from the Golgi
apparatus to the basolateral surface.
Similar discrepancies are exhibited by MDCK-ConAr cells. In most polarized epithelial cell lines
(e.g., MDCK), both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane. In
contrast, in MDCK-ConAr cells, gD1-DAF was sorted to both surfaces, but GlcCer was still
targeted to the apical surface (Zurzolo et al., 1994). In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In
the FRT cell line gD1-DAF and GlcCer were both targeted basolaterally. Although gD1-DAF and
glucosyl ceramide distributed to the basolateral surface, gDI-DAF did not associate with membrane
clusters. Among several possible alternatives, this surprising finding could be explained by the
presence of a small subset of specialized clusters available for basolateral targeting.
In addition, in some cases, the role of the GPI anchor is in doubt. In MDCK cells, Thy-1 (a
glycoprotein of 25 kDa and unkownn function present in mouse thymocytes, T-cells and neurons)
anchored to GPI was delivered apically. However, a truncated form of Thy-1, lacking 22 out of 31
hydrophobic amino acids at the carboxy-terminal, still resulted in apical secretion of Thy-1 despite
the fact that the GPI anchor was not attached (Powell et al., 1991). It would therefore seem that Thy-1 contains apical targeting information in its protein sector, as well as in the GPI anchor.
GPI anchors are synthesized in the ER and added to primary translation products while they are
http://www.albany.edu/~abio304/text/chapter_11.html (30 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
being translocated across the ER membrane (see Thomas et al, 1990). In the plasma membrane, the
GPI anchors are attached to the external leaflet of the plasma membrane. The motif in the protein
directing attachment to the GPI resides in the amino acid sequence. The signal at the carboxy-end of
the proteins differ with the protein (see Medof et al., 1996). Characteristically, GPI anchored
proteins lack charged amino acids at the carboxy-end of the protein. An additional signal, 15 to 30
amino acids upstream of the terminal hydrophobic stretch of the protein, is needed for GPI
anchoring (see Medof et al., 1996).
In at least some cases, targeting may be a function of the lipid composition of the membrane. The
outer leaflet of the apical membrane of the epithelial Madine-Darby canine kidney (MDCK) cells
contains mainly glycosphingolipids held together by H-bonds. These lipids exclude glycerol-based
phospholipids (Thompson and Tillack, 1985). Similarly, glycosphingolipid clusters are present in
other membranes (see Chapter 4, Section VI).
Signals other than GPIs have also been found (e.g. see Weimbs et al., 1997). Saturated acylated
proteins (e.g., Src family kinases) appear to be targeted to detergent resistant membrane domains
which are thought to correspond to rafts (see Chapter 4). In contrast prenylated proteins (e.g., Ras)
are usually not found in these domains (Melkonian et al., 1999). This is likely to be the consequence
of the ordered environment of lipid rafts or caveolae (see Brown and London, 1998) which is more
likely to favor the incorporation of acyl chains. These chains tend to be present in an extended
configuration, whereas the prenyl moieties are branched and bulky. Studies in intact cells confirm
these observations (Zacharias et al., 2002). This study used FRET (see Chapter 1) a technique which
allows the study the interaction between two chromatophores separated by 10 nm or less and
therefore the proximity of the protein pairs. GFP variant pairs (donor-acceptor) were used. They
were combined to peptides with consensus sequences for either acylation or prenylation or with
caveolin, a protein components of rafts. The fluorescent GFP variants had to be modified to prevent
their dimerization. Again, the acyl proteins but not the prenylated proteins were found in rafts.
Furthermore, in contrast to the acylated proteins, the prenylated proteins were found insensitive to
cholesterol depletion.
The finding of the location of proteins in microdomains or caveolae is significant in relation to
regulatory cascades. The lipid microdomains contain signaling kinases so that when receptors are
bound to their ligand they move to these microdomains initiating a signaling cascade (see Pierce,
N-glycans of secretory (Scheiffele et al., 1995) or in N- and O-glycans of membrane proteins act as
apical signals (Yeaman et al., 1997; Gut et al., 1998). The hypothesis that N-linked carbohydrates
are responsible for apical targeting was tested with three membrane proteins (Gut et al., 1998). Two
are normally not glycosylated and another is a glycoprotein. In all three cases, N-linked
carbohydrates were clearly able to mediate apical targeting. However, the presence of cytoplasmic
basolateral targeting motifs remained basolateral even when attached to N-linked sugars. To
compare the role of GPI and N-glycans, GPI was attached to the rat growth hormone (rGH) which is
normally secreted in non-polarized manner (Benting et al., 1999). This modification did not lead to
an apical delivery. However, the addition of N-glycans to the GPI-anchored rGH did target mostly
to the apical surface. A transmembrane form of rGH accumulated intracellularly unless attached to
http://www.albany.edu/~abio304/text/chapter_11.html (31 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
N-glycans that delivered them to the apical surface. The N-or O-linked oligosaccharide could
possibly attach to lectins present in the rafts (such as VIP36) (see Fiedler and Simons, 1995).
As with other transport systems, it has been possible to partially reconstitute the basolateral targeting
system (Gravotta et al., 1990). The fusion of the vesicles to the surface requires energy and
cytoplasmic extracts. A GTP-binding protein is presumed to be involved because the fusion is
inhibited by GTPγS. As already discussed, GTP-binding proteins of the Rab family are thought to be
involved in fusion of vesicles in the intracellular transport system. There are some indications that
Rab8 is localized in the basolateral transport vesicles in MDCK cells.
Once incorporated into one of the cell membrane faces, the diffusibility of the proteins in the plane
of the membrane appears to be constrained by binding to the cortical cytoskeleton (Vega-Salas,
1987). Na+-K+, ATPase, the Na+-channel, and the anion exchange protein bind to ankyrin/fodrin
complexes (see Nelson and Hammerton, 1989).
The probable interactions involved in establishing and maintaining cell polarity are summarized in
Fig. 3 (Nelson, 1992).
C. Targeting in Secretion and Transcytosis
Secretion may also require targeting to a specific cell surface. This may be a consequence of the
common mechanism accounting for plasma membrane transport and secretion. Although some cells
release secretory products around their perimeter, many polar cells release them only in specific
regions of the membranes. Thus, the same kind of targeting has to be considered in secretion. In
MDCK cells, endogenous constitutively secreted proteins are released in the apical domain (Kondor-Koch et al., 1985; Gottlieb et al., 1986), whereas exogenous proteins (produced after transfection)
release at both the apical and basolateral cell surfaces.
http://www.albany.edu/~abio304/text/chapter_11.html (32 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport

Fig. 3 Mammalian polarized epithelial cell organization of protein trafficking pathways. Protein
sorting and transport between the trans Golgi network (TGN) and different cell surface domains are
regulated. In the TGN, proteins can be sorted by signal-mediated or bulk flow pathways into
different vesicles. Signal-mediated sorting of proteins to the apical plasma membrane (Ap-PM) is
regulated by glycosphingolipid patching, whereas signal-mediated sorting of protein to the
basolateral plasma membrane (BL-PM) is regulated by protein clustering of adaptor proteins; other
signal-mediated pathways may also exist. Docking with targeting patches in each membrane is
regulated by domain-specific GTPase cycles (Ap-GTPase, apical membrane GTPase; BL-GTPase,
basal-lateral membrane GTPase) (from Nelson 1992). Reproduced by permission.
Most cells carry out receptor-mediated endocytosis (Chapter 9), in which the ligand is generally
degraded in the lysosomes and the receptor is either degraded or recycled to the surface. However,
in transcytosis, which is ubiquitous in epithelial cells, the endosomes transfer receptor and ligand to
the surface of opposite polarity, so that the material traverses the cell.
The transcytosis of polymeric immunoglobulin (poly-Ig) is probably the best understood of the
various known cases. Poly-Ig is produced by plasma cells and transported through epithelial cells by
transcytosis. In this process, the epithelial cell adds a polypeptide, the secretory component (SC) or
secretory piece, part of the receptor molecule, to the poly-Ig. The poly-Ig receptor is originally
incorporated in the basolateral surface, where it binds poly-Ig. After transcytosis, the endocytotic
vesicle discharges its contents at the apical surface by exocytosis and the receptor is cleaved. So far,
only one signal has been identified in transcytosis, the phosphorylation of a Ser in the cytoplasmic
domain of the receptor (Casanova et al., 1990).
http://www.albany.edu/~abio304/text/chapter_11.html (33 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
Despite the flow of membranes from one pole of the cell to the other in transcytosis, the two surface
domains remain distinct. This is illustrated by VSV G-protein, which is normally in the basolateral
surface of the infected MDCK cells. When artificially inserted into the apical surface by fusing it to
viral coats at low pH, the VSV-G protein is taken up by endocytosis and delivered to the basolateral
surface (Matlin et al., 1983). Therefore, it would seem possible that portions of the membranes
involved in transcytosis can be recycled to their original location by a process akin to transcytosis in
D. Transport of the Vesicles
What is responsible for the movement of vesicles? The information presently available suggests a
varied pattern (see Bloom and Goldstein, 1998).
The diffusion coefficient of granules in cells is approximately 2.5 x 10-10 cm2/s (Felder and Kam,
1994). It has been calculated that a vesicle 160 nm in diameter can diffuse for a distance of 10 µm in
10 minutes (Bloom and Goldstein, 1998). Therefore, no special mechanism is required in small cells
for a variety of vesicular transport events. For a neuron, the distances from cell body to periphery,
however, is prohibitive (the vesicles may have to travel 1 m or more from the cell body to the
neuron terminal) and microtubular transport is essential.
Normally, the transport from the compartments intermediate between ER and Golgi (ICs) to the
Golgi, occurs on microtubules (Presley et al., 1997; Scales et al., 1997) as shown by direct
observation using conjugates of VSV-G glycoprotein and green fluorescent protein (see Chapter 1,
Chapter 10, Section III). However, in some cases, the absence of MTs does not preclude secretion at
close to normal rates (e.g., van de Moortele et al., 1993). Apparently, in these cases, the Golgi
cisternae segment in ministacks. These ministacks distribute throughout the cytoplasm (Rogalski
and Singer, 1984) adjacent to IC sites and the ER (Cole et al., 1996). Presumably in these cases the
transport can proceed efficiently by diffusion.
These considerations and other presently available information suggest that in anterograde transport
microtubules have no role in transport from ER to IC or in intra-Golgi transport. However, they are
needed for transport from IC to Golgi or from TGN to the cell surface (see Lippincott-Schwartz,
In the retrograde pathway, microtubules are also involved in many steps. Golgi to ER transport is
driven by kinesin (Lippincott-Schwartz et al., 1995), a microtubular motor, and microtubules are
involved in the transport of endosomes and lysosomes toward the centrosome, as shown by direct
observation and the use of microtubular inhibitors (Matteoni and Kreis, 1987).
In polarized epithelial cells, microtubules seem to be involved in the movement of vesicles to the
apical surface (Nelson, 1991, 1992). Depolymerization of microtubules interferes with trafficking
between TGN and the apical membrane (e.g., Rindler et al.,1987; Van Zeijl and Matlin, 1990) and
decreases transcytosis from the basolateral to the apical surface (e.g., Matter et al., 1990b, Breitfeld
et al., 1990). The presence of colchicine, vinblastine or nocodazole (all drugs which interfere with
http://www.albany.edu/~abio304/text/chapter_11.html (34 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
the microtubules) redirects the vesicles from the apical to the basolateral surface. In contrast, the
basolateral traffic is not affected. However, in budding yeast, microtubules do not have a role in
polarized secretion (e.g., Huffaker et al., 1988; Jacobs et al., 1988). In contrast, in these cells, actin
(e.g., see Novick and Botstein, 1985) has been found to be involved in conjunction with Myo2p, a
myosin V (see Chapter 24) (e.g., Santos and Snyder, 1997; Catlett and Weisman, 1998; Schott et al.,
The experiments carried out with mammalian cells indicate a primary role of microtubules in apical
vesicular transport. The presence of myosin I in Golgi derived vesicles (Fath and Burgess, 1993) and
in apical membranes (Mooseker and Coleman, 1989) also suggests a role of the actomyosin system
in at least some of the processes of intracellular transport. Myosin I is found in vesicles in intestinal
brush border cells where they are linked to actin filaments (Drenckhahn and Dermietzel, 1988).
Similarly, myosin I was localized by immunoblotting and immunolabel negative staining of the
isolated vesicles during the assembly of these cells (Fath and Burgess, 1993). This protein was
found at the outer surface of Golgi associated vesicles during the assembly of these cells. The
vesicles contained galactosyl transferase, a trans-Golgi enzyme, as well as alkaline phosphatase, an
apical membrane targeted enzyme. The vesicles were also shown to bundle actin, suggesting that the
actomyosin system functions in the peripheral translocation of vesicles. The results suggest an
involvement of both microtubules and the actomyosin system, the latter perhaps only in the final
step of the delivery in the apical pathway. However, the details are still not clear.
How are the vesicles connected to the transporting systems? Present evidence implicates dynactin.
Dynactin is a 1.2 MDa complex of ten peptides (and includes actin) that is required for cytoplasmic
dynein motility and in vitro vesicle movement (Schroer and Sheetz, 1991).
Electron microscopy of this complex shows an actin-like filament, 37 nm long, with laterally
projecting sidearms (Schafer et al., 1994). A model of dynactin is shown in Fig. 4 (Schroer et al.,
1996). The filamentous part of the molecule is made up of the actin related protein (Arp1) and
probably one molecule of actin. The interaction between dynactin and the MT-dynein system
probably involves the sidearms that contain a microtubule binding site (Waterman-Storer et al.,
1995) and also bind dynein (Collins and Vallee, 1989). The most likely role of the Arp1 portion of
the complex is to provide a connection to the cargo vesicle.
Many myosins of the myosin I family bind directly to phospholipid. Myosin I isoforms associate
with specific membranes (Baines et al., 1995). It is entirely possible then that dynactin binding to
the vesicles is mediated by myosin, although other possibilities are also likely (e.g., ponticulin or an
annexin, see Schroer et al., 1996).
http://www.albany.edu/~abio304/text/chapter_11.html (35 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport

Fig. 4 Current model of dynactin structure. The localization of Arp1, actin-capping protein, p62 and
p150Glued/P135Glued are based on ultrastructural analysis of antibody decorated molecules. The
location of actin, p50, p24 and p27 is uncertain. Because of their similar properties, actin is likely to
be in the Arp1 filament. The overexpression of p50 causes p150Glued to dissociate suggesting the
location in the diagram. From Schroer et al., 1996, reproduced by permission.
E. Recycling of the Plasma Membrane
The process of exocytosis adds a considerable amount of material to the plasma membrane. Much of
this material is recycled through endocytosis. Because of this continuous recycling, the turnover
time of the membranes of granules is relatively long compared to that of ordinary cytoplasmic
proteins (Meldolesi, 1974).
The availability of antibodies to membrane proteins of the luminal side of the secretory vesicle
membrane has permitted following the fate of the secretory vesicles in the regulated secretion of
catecholamine granules (Patzak and Winkler, 1986). Glycoprotein III (gpIII) is exposed to its
fluorescently labelled antibody at exocytosis. The protein appears in coated pits and vesicles in the
first 5 min after exocytosis. Then it passes through the smooth ER and reappears in the trans Golgi
network and in dense-core secretory granules within 30 to 45 min. The protein was never found in
the cisternal lumen, indicating that the membrane itself is being recycled. Similar results were found
for the transferrin receptors (Woods et al. 1986). Their presence was demonstrated in several Golgi
cisternae; therefore, recycling must involve some of the same steps followed by the transport of
newly synthesized protein.
These observations imply that some steps in the recycling process must involve transport in the
opposite direction from that discussed in most of this chapter, that is retrograde transport.
Retrograde transport is beginning to be studied by taking advantage of the effect of the antibiotic
Brefeldin A (Tan et al., 1992). Brefeldin, a macrocyclic lactone synthesized by fungi, prevents the
assembly of nonclathrin coated vesicles and blocks the transport from ER to Golgi (e.g., Klausner et
al., 1992). The retrograde transport, however, is not inhibited, redistributing material such as
enzymes from the Golgi back to the ER. This transport is also blocked by GTPγS, suggesting that
http://www.albany.edu/~abio304/text/chapter_11.html (36 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
GTP-binding proteins are involved in both forward and backward transport.
F. Formation of Lysosomes and Secretory Storage Vesicles
The study of the transport system in the formation of lysosomes received great impetus from the
recognition of at least 30 human lysosomal storage disorders. I-cell disease, a deficiency in
lysosomal enzymes, results from a failure in the recognition marker needed for targeting. Study of I-cell mutants has permitted the identification of the recognition marker, the mannose-6-phosphate
(M6P) residue, and the M6P receptor, MPR (Sahagian et al., 1981). The receptor spans the
membrane and 10 kDa of its carboxy-terminal protrudes into the cytosol (Sahagian and Steer, 1985).
Two distinct but related MPRs are known (see Kornfeld, 1992). One is a type I (amino group
external to the cell) transmembrane glycoprotein of 275 kDa which does not require divalent
cations. The other receptor is also a type I glycoprotein of 46 kDa. The bovine and murine forms of
the latter, but not the human or porcine forms, require divalent cations for optimal binding.
M6PRs bind to lysosomal hydrolases while they are transported from the trans-Golgi to the
lysosomes. In turn, targeting signals in the M6PRs are needed to arrive at their final destination. The
Golgi-localized γ-ear containing, ARF-binding (GGAs) adaptors have been found to bind to the
lysosomal targeting signals of the cation-independent M6PRs, the acidic cluster-dileucine motif of
their cytoplasmic tails. The GGAs, already implicated in protein trafficking between the Golgi and
the endosomes, have all the properties expected for an adaptor mediating the binding of the
receptors to the components needed for transport. They contain a Vps27p/Hrs/STAM (VHS)
domain, binding sites for clathrin, a GTP-ARF binding domain and a domain that binds to proteins
involved in coat assembly such as γ-synergin. The VHS domain of 153 residues is present in various
proteins involved in endocytic trafficking (Lohi and Lehto, 1998). The M6PRs bind to the VHS
domain of the GGAs. The GGAs were found to be present in the TGN, tubules and vesicles which
bud from the TGN and the cell surface as expected from its presumed function (Zhu et al., 2001;
Puertollano et al., 2001).
Lysosomal enzyme precursors are transported through the common pathway, as indicated by
immunocytochemical experiments (Geutze et al., 1984). However, processing continues on arrival
in the cis-Golgi (Fig. 5, Kornfeld, 1987), where lysosomal hydrolases are recognized by GlcNAc-phosphotransferase, which adds GlcNAc-phosphate to α-1,2-mannose residues of the hydrolases.
The phosphotransferase probably recognizes a signal patch, because recognition is very sensitive to
conformation changes. After this modification, M6P residues are exposed by removal of the N-acetylglucosamine and are recognized by MPR. The interaction between receptors and M6P residues
is responsible for the lysosomal enzyme segregation (see von Figura and Hasilik, 1986; Kornfeld,
1987). The two can be detected together in buds and coated vesicles in the TGN, which eventually
form lysosomes (Geutze et al., 1985, Griffiths et al., 1985).
In addition to the processing of the oligosaccharides, lysosomal enzymes are proteolytically cleaved
to their mature form (Gieselman et al., 1983).
http://www.albany.edu/~abio304/text/chapter_11.html (37 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport

Fig. 5 Schematic pathway of lysosomal enzyme targeting to lysosomes. Lysosomal enzymes and
secretory proteins are synthesized in the rough endoplasmic reticulum (RER) and glycosylated by
the transfer of a performed oligosaccharide from dolichol-P-P-oligosaccharide (DOL). In the RER,
the signal peptides (hatching) are excised. The proteins are translocated to the Golgi, where the
oligosaccharides of secretory proteins are processedto complex-type units and the oligosaccharides
of lysosomal enzymes are phosphorylated. Most of the lysosomal enzymes bind to mannose6-phosphate receptors (MPRs) ( ) and are translocated to an acidified prelysosomal
compartment where the ligand dissociates. The receptors recycle back to the Golgi or to the cell
surface, and the enzymes are packaged into lysosomes where cleavage of their propieces is
completed ( ). The Pi may also be cleaved from the mannose residues. A small number of the
lysosomal enzymes fail to bind to the receptors and are secreted along with secretory proteins (
). These enzymes may bind to surface MPRs in coated pits ( ) and be internalized into
the prelysosomal compartment. ( ) N-Acetylglucosamine; ( ) mannose; ( ) glucose; ( )
galactose; ( ) sialic acid. Reprinted with permission from S. Kornfield, Federation of American
Societies for Experimental Biology Journal, Vol.1, No.6: 463, 1987.
The MPRs are recycled. Before the primary lysosomes are formed, the MPRs are sent back to the
trans Golgi. Recovery of receptor probably follows its dissociation from the ligands brought about
by lowering the pH, as in the case of endocytotic vesicles discussed in Chapter 9. Although most
lysosomal enzymes remain segregated in vesicles, a small portion of the lysosomal enzymes are
secreted and are thought to be recovered by endocytotic uptake after binding MPRs present at the
surface (Willingham et al., 1981).
http://www.albany.edu/~abio304/text/chapter_11.html (38 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
Secretory storage granules
Regulated secretion differs from constitutive secretion in the need to store the secreted products in
the secretory vesicles until a physiological signal permits their discharge. Therefore, they must be
separated from the other products destined to the cell surface. This separation occurs in the trans-Golgi, but the selection mechanism is still unknown. The process can be very selective. However, it
also allows for packaging very different proteins in the same vesicle.
The possibility that a receptor is involved in targeting storage secretory products is also supported
by the observation that the precursor of insulin, proinsulin, is bound to Golgi membranes (Munro
and Pelham, 1986), indicating the likelihood that a receptor is present.
G. Synaptic Vesicles
Presynaptic cells discharge neurotransmitters by exocytosis of their synaptic vesicles. Postsynaptic
membrane receptors bind the neurotransmitters. These receptors are channels that open more
frequently after binding the neurotransmitters. The increased conductance of the membrane initiates
the depolarization that can culminate in an action potential (see Chapter 22). The presynaptic
neurotransmitter vesicles are continuously discharged and continuously reformed. Most of the
recycling involves the recovery of synaptic vesicles by endocytosis and their reloading with
neurotransmitter (See Chapter 22). However, there is also a turnover of the synaptic vesicles
themselves; their components are degraded and resynthesized in the cell body. Eventually, the newly
formed vesicles must be targeted to the nerve terminals through axonal transport. The integral
proteins follow the usual path from RER to TGN. These proteins obviously can be found at synaptic
sites, but also (with few exceptions) throughout the Golgi system.
All indications are that the forward or anterograde transport of newly synthesized synaptic vesicle
components is microtubular and is powered by the motor protein of the kinesin family (Chapter 24).
Kinesin is implicated by its association with vesicles (e.g., Morin et al., 1993) and by its
accumulation when the axonal flow is blocked by ligation (Hirokawa et al., 1991). The vesicles and
the kinesin (indentified by immunocytochemistry) accumulate on the cell body side of the ligature.
Evidence from genetic studies also implicates a kinesin-like molecule. The unc-104 gene codes for a
kinesin-like motor, thought to be neuron specific in the nematode Caenorhabditis elegans. This
protein has a kinesin-like motor domain at the amino terminal, but otherwise it has little homology
to other kinesins. Mutant alleles of this gene block the accumulation of synaptic vesicles in axons
(Hall and Hedgecock, 1991). The movement of other vesicles is not affected, suggesting a unique
targeting role for the protein coded by the unc-104 gene.
The study of the biogenesis of synaptic vesicle proteins can be carried out most readily in
neuroendocrine cells and neurons in culture. PC12 cells, derived from pheochromocytoma of the rat
adrenal medulla, have characteristics of both neural and endocrine cells. They synthesize, store and
release the neurotransmitter acetylcholine (Greene and Rein, 1977) and have both regulated and
constitutive secretory pathways. In these cells, regulated secretion involves large dense-core
granules related to chromaffin granules. Small electron-translucent vesicles are also present and are
thought to be related to cholinergic synaptic vesicles. Dense granules were purified and shown to
http://www.albany.edu/~abio304/text/chapter_11.html (39 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
contain the regulated secretory protein secretogranin II. The synaptic protein synaptophysin was
used as a marker for the smaller vesicles (Cutler and Cramer, 1990). Synaptophysin is a major
integral protein of synaptic vesicle membranes. Pulse-chase experiments using immunoprecipitation,
demonstrated that synaptophysin is associated with the smaller vesicles and does not occupy the
dense granules at any time. These findings indicate two separately regulated secretion routes. In
another study, synaptophysin was also traced by pulse-chase (Régnier-Vigouroux et al., 1991). It
was found to follow a route involving the trans Golgi network (TGN). The protein was found to
reach the cell surface from the TGN with a half time of 10 min and was found to cycle between cell
surface and vesicles. The endosomal fraction was identified by exposing the terminals to
peroxidases and tracing this enzyme. Peroxidase is taken up by endocytosis and represents the
material dissolved in the external medium.
The molecular mechanism by which the synaptic vesicle proteins are sorted out is not clear. A
common sequence motif that could be recognized by receptors used in targeting has not been found
in examining the various proteins of the secretory vesicles, and signals involving secondary or
tertiary folding are suspected. A role of specific complexes of synaptic proteins in targeting is also
possible because multimeric complexes can be recovered from synaptic vesicles after detergent
treatment (Bennett et al., 1992).
Bloom, G.S. and Goldstein, L.S.B. (1998) Cruising along microtubule highways: how membranes
move through the secretory pathway, J. Cell Biol. 140:1277-1280. (MedLine)
Kirchhausen, T. (2000) Clathrin, Annu. Rev. Biochem. 69:699-727. (MedLine)
Lippincott-Schwartz, J. (1998) Cytoskeletal proteins and Golgi dynamics, Curr. Opin. Cell Biol.
McNiven, M.A., Cao, I., Pitts, K.R. and Yoon, I. (2000) The dynamin family of mechanoenzymes:
pinching in new places, Trends Biochem Sci 25:115-120.(MedLine)
Mellman, I. and Warren, G. (2000) The road taken: past and future foundations of membrane traffic,
Cell 100:99-112. (MedLine)
Morgan, A. (1995) Exocytosis, Essays in Biochemistry 30:77-95.
Nelson, W. J. (1992) Regulation of cell surface polarity from bacteria to mammals, Science 258:948-955. (MedLine)
Robinson, M.S. (1997) Coats and vesicle budding, Trends in Cell Biol. 7:99-102.
Schekman, R. and Orci, L. (1996) Coat proteins and vesicle budding, Science 271:1526-1533.
http://www.albany.edu/~abio304/text/chapter_11.html (40 of 41) [3/5/2003 7:56:56 PM]
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes, Nature 387:569-572.
Springer, S., Spang, A. and Schekman, R. (1999) A primer on vesicle budding, Cell 97:145-148.
Weimbs, T., Low, S.H., Chapin, S.J. and Mostov, K.E. (1997) Apical targeting in polarized
epithelial cells: there’s more afloat than rafts, Trends in Cell Biol. 7:393-399.
Wieland, F. and Harter, C. (1999) Mechanisms of vesicle formation: insights from the COP system,
Curr. Opin. Cell Biol. 11:440-446. (MedLine)
Kirchhausen, T. and Bruce, A. Clathrin-coat formation in time and space: modelling.
Search the textbook
http://www.albany.edu/~abio304/text/chapter_11.html (41 of 41) [3/5/2003 7:56:56 PM]
Chapter 11: References
Back to Chapter 11
Aalto, M.K., Keränen, S. and Ronne, H. (1992) A family of proteins involved in intracellular transport,
Cell 68:181-182. (Medline)
Aalto, M.K., Ronnel, H. and Carragheenin, S. (1993) Yeast syntaxis Sso1p and Ssop2 belong to a family
of related membrane proteins that function in vesicular transport, EMBO J. 12:4095-4104. (Medline)
Ahle, S. and Ungewickell, E. (1989) Identification of clathrin binding subunit in the HA-2 adaptor protein
complex, J. Biol. Chem. 264:20089-20093. (Medline)
Anderson, J.M. and Van Itallie, C.M. (1995) Tight junctions and the molecular basis for regulation of
paracellular permeability, Am. J. Physiol. 269:G467-475. (Medline)
Ando-Akatsuka, Y., Saitou, M., Hirase, T., Kishi, M., Sakakibara, A., Itoh, M., Yonemura, S., Furuse, M.
and Tsukita, S. (1996) Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse,
dog, and rat-kangaroo homologues, J. Cell Biol. 133:43-47. (Medline)
Aridor, M., Weissman, J., Bannykh, S., Nuoffer, C. and Balch, W.E. (1998) Cargo selection by the COPII
budding machinery during export from the ER, J. Cell Biol. 141:61-70. (MedLine)
Aroeti, B., Kosen, P.A., Kuntz, I.D., Cohen F.E. and Mostov, K.E. (1993) Mutational and secondary
structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor, J. Cell.
Biol. 123:1149-1160. (Medline)
Baines, I.C., Corigliano-Murphy, A. and Korn, E.D. (1995) Quantification and localization of
phosphorylated myosin I isoforms in Acanthamoeba castellanii, J. Cell Biol. 130:591-603. (Medline)
Bhat, M.A., Izaddoost, S., Lu, Y., Cho, K.O., Choi, K.W. and Bellen, H.J. (1999) Discs Lost, a novel
multi-PDZ domain protein, establishes and maintains epithelial polarity, Cell 96:833-845. (MedLine)
Balch, W. E., Dunphy, W. G., Braell, W. A. and Rothman, J. E. (1984a) Reconstitution of the transport of
protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine, Cell 39:405-416. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (1 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Balch, W. E., Glick, B. S. and Rothman, J. E. (1984b) Sequential intermediates in the pathway of
intercompartmental transport in a cell free system, Cell 39:525-536. (Medline)
Balch, W. E., Wagner, R. R. and Keller, D. S. (1987) Reconstitution of transport vesicular stomatitis virus
G protein from the endoplasmic reticulum to the Golgi complex using a cell free system, J. Cell Biol.
104:749-760. (Medline)
Balch, W.E., McCaffery, J.M., Pluttner, H. and Farquhar, M.G. (1994) Vesicular stomatitis virus
glycoprotein is sorted and concentrated during export from the endoplasmic reticulum, Cell 76:841-852.
Balda, M.S., Whitney, J.A., Flores, C., Gonzalez, S., Cereijido, M. and Matter, K. (1996) Functional
dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the
apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane
protein, J. Cell Biol. 134:1031-1049. (Medline)
Bankaitis, V.A., Aitken, J.R., Cleves, A.E. and Dowhan, W. (1990) An essential role for a phospholipid
transfer protein in yeast Golgi function, Nature 347:561-562. (Medline)
Bannykh, S.I. and Balch, E.E. (1997) Membrane dynamics at the endoplasmic reticulum-Golgi interface,
J. Cell Biol.138:1-4. (Medline)
Barlowe, C. (1998) COPII and selective export from the endoplasmic reticulum, Biochim. Biophys. Acta
1404:67-76. (MedLine)
Barlowe, C. and Shekman, R. (1993) SEC12 encodes a guanine-nucleotide-exchange factor essential for
transport vesicle budding from the ER, Nature 365:347-349. (Medline)
Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Ravazzola, M. Amherdt, M. and Shekman, P. (1994)
COPII: a membrane coat formed by SEC proteins that drive vesicle budding from the endoplasmic
reticulum, Cell 77: 895-907. (Medline)
Barnard, R.J., Morgan, A. and Burgoyne, R.D. (1997) Stimulation of NSF ATPase activity by α-SNAP is
required for SNARE complex disassembly and exocytosis, J. Cell Biol. 139:875-883. (Medline)
Barroso, M., Nelson, D.S. and Sztul, E.(1995) Transcytosis-associated protein (TAP)/p115 is a general
fusion factor required for binding of vesicles to acceptor membranes, Proc. Natl. Acad. Sci. USA 92:527-531. (MedLine)
Bartles, J. R., Feracci, H. M., Stieger, B. and Hubbard, A. L. (1987) Biogenesis of the rat hepatocyte
http://www.albany.edu/~abio304/ref/ref11.html (2 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using
subcellular fractionation. J. Cell Biol. 105:1241-1251. (Medline)
Beck, K.A. and Nelson, W.J. (1998) A spectrin membrane skeleton of the Golgi complex, Biochim.
Biophys. Acta. 1404:153-160. (Medline)
Beckers, C.J. and Balch, W.E. (1989) Calcium and GTP: essential components in vesicular trafficking
between the endoplasmic reticulum and Golgi apparatus, J. Cell Biol. 108:1245-1256. (MedLine)
Bednarek, S.Y., Ravazzola, M., Hosobuchi, M., Amherdt, M., Perrelet, A., Shekman, R. and Orci, L.
(1995) COPI and CopII-coated vesicles bud directly from the endoplasmic reticulum in yeast, Cell
83:1183-1196. (Medline)
Bennett, M.K., Calakos, N., Kreiner, T. and Scheller, R.H. (1992) Synaptic vesicle membrane proteins
interact to form a multimeric complex, J. Cell Biol. 116:761-775. (Medline)
Benting, J.H., Rietveld, A.G. and Simons, K. (1999) N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells, J. Cell Biol. 146:313-320.
Berditchevski, F., Tolias, K.F., Wong, K., Carpenter, C.L. and Hemler, M.E. (1997) A novel link between
integrins, transmembrane-4 superfamily proteins (CD63 and CD81), and phosphatidylinositol 4-kinase, J.
Biol. Chem. 272:2595-2598. (Medline)
Bilder, D. and Perriman, N. (2000) Localization of apical epithelial determinants by the basolateral PDZ
protein Scribble, Nature 403:676-680. (Medline)
Block, M.R., Glick, B.S., Wilcox, C.A., Wieland, F.T. and Rothman, J.E. (1988) Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport, Proc. Natl. Acad. Sci. USA 85:7852-7856.
Bloom, G.S. and Goldstein, L.S.B. (1998) Cruising along microtubule highways: how membranes move
through the secretory pathway, J. Cell Biol. 140:1277-1280. (Medline)
Boll, W., Partin, J.S.,Katz, A.I., Caplan, M.J.. and Jamieson, J.D. (1991) Distinct pathways for basolateral
targeting of embrane and secretory proteins in polarized epithelial cells, Proc. Natl. Acad. Sci. USA
88:8592-8596. (Medline)
Braell, W. A., Balch, W. E., Dobbertin, D. C. and Rothman, J. E. (1984a) The glycoprotein that is
transported between successive compartments of the Golgi in a cell free system resides in stacks of
cisternae, Cell 39:511-524. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (3 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Breckenridge, L.J. and Almers, W. (1987) Currents through the fusion pore that forms during exocytosis
of a secretory vesicle, Nature 328:814-817. (MedLine)
Breitfeld, P.P., McKinnon, W.C. and Mostov, K.E. (1990) Effect of nocodazole on vesicular traffic to the
apical and basolateral surfaces of polarized MDCK cells, J. Cell Biol. 111:2365-2373. (Medline)
Bremser, M., Nickel, W., Schweikert, M., Ravazzola, M., Amherdt, M., Hughes, C.A., Sollner, T.H.,
Rothman, J.E. and Wieland, F.T. (1999) Coupling of coat assembly and vesicle budding to packaging of
putative cargo receptors, Cell 96:495-506. (MedLine)
Broadie, K., Prokop, A., Bellen, H.J., O’Kane, C.J., Schulze, K.L. and Sweeney, S.T. (1995) Syntaxin and
synaptobrevin function downstream of vesicle docking in Drosophila, Neuron 1 5:663-673. (MedLine)
Brodsky, F.M. (1988) Living with clathrin: its role in intracellular membrane traffic, Science 242:1396-1402. (Medline)
Brodsky, F.M. (1997) New fashions in vesicle coats, Trends in Cell Biol. 7:175-179.
Brown, D.A. and Rose, J.K. (1992) Sorting of GPI-anchored proteins to the glycolipid-enriched
membrane subdomains during transport to the apical cell surface, Cell 68:533-544. (Medline)
Brown, D.A. and London, E. (1998) Structure and origin of ordered lipid domains in biological
membranes, J. Membr. Biol. 164:103-114. (MedLine)
Brown, D.A., Crise, B., and Rose, J.K. (1989) Mechanism of membrane anchoring affects polarized
expression of two proteins in MDCK cells, Science 245:1499-1501. (Medline)
Brown, H.A., Gutowski, S., Moomaw, C.R., Slaughter, C. and Sternweis, P.C. (1993) ADP-ribosylation
factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity, Cell 75:1137-1144. (Medline)
Cao, X., Ballew, N., and Barlowe, C. (1998a) Initial docking of ER-derived vesicles requires Uso1p and
Ypt1p but is independent of SNARE proteins, EMBO J. 17:2156-2165. (Medline)
Cao, H., Garcia, F. and McNiven, M.A (1998b) Differential distribution of dynamin isoforms in
mammalian cells, Mol. Biol. Cell 9:2595-2609. (MedLine)
Carpenter, C.L. and Cantley, L.C. (1996) Phosphoinositide kinases, Curr. Opin. Cell Biol. 8:153-158.
http://www.albany.edu/~abio304/ref/ref11.html (4 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Casanova, J.E., Breitfeld, P.P., Ross, S.A., and Mostov, K.E. (1990) Phosphorylation of a polymeric
immunoglobulin receptor required for efficient transcytosis, Science 248:742-745. (Medline)
Casanova, J.E., Apodaka, G. and Mostov, K.E. (1991) An autonomous signal for basolateral sorting in the
cytoplasmic domain of the polymeric immunoglobulin receptor, Cell 66:65-75. (Medline)
Catlett, N.L and Weisman, L.S. (1998) The terminal tail region of a yeast myosin-V mediates its
attachment to vacuole membranes and sites of polarized growth, Proc. Natl. Acad. Sci. USA 95:14799-14804.
Chandler, D.E. and Heuser, J.E. (1980) Arrest of membrane fusion events in mast cells by quick-freezing,
J. Cell Biol. 86:666-674. (MedLine)
Chapman, E.R., Au, S., Barton, N. and Jahn, R. (1994) SNAP-25, a t-SNARE which binds to syntaxin
and synaptobrevin via domains that may form coiled coils, J. Biol. Chem. 269:27427-27432. (Medline)
Chaudhary, A., Gu, Q.M., Thum, O., Profit, A.A., Qi, Y., Jeyakumar, L., Fleischer, S. and Prestwich,
G.D. (1998) Specific interaction of Golgi coatomer protein α-COP with phosphatidylinositol 3,4,5-trisphosphate, J. Biol. Chem. 273:8344-8350. (Medline)
Cheever, M.L., Sato, T.K., de Beer, T., Kutateladze, T.G., Emr, S.D. and Overduin, M. (2001) Phox
domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes, Nature Cell Biol.
3:613-618. (MedLine)
Chen, Y.A. and Scheller, R.H. (2001) SNARE-mediated membrane fusion, Nature Rev. Mol. Cell Biol.
2:98-106. (MedLine)
Clark, E.A. and Brugge, J.S. (1995) Integrins and signal transduction pathways: the road taken, Science
268:233-239. (MedLine)
Clary, D.O., Griff, I.C., Rothman, J.E. (1990) SNAPs, a family of NSF attachment proteins involved in
intracellular membrane fusion in animals and yeast, Cell 61:709-721. (Medline)
Cohen, A.R., Woods, D.F., Marfatia, S.M., Walther, Z., Chishti, A.H., Anderson, J.M. and Wood, D.F.
(1998) Human CASK/LIN-2 binds syndecan-2 and protein 4.1 and localizes to the basolateral membrane
of epithelial cells, J. Cell Biol. 142:129-138. (MedLine)
Cole, N.B., Sciaky, N., Marotta, A., Song, J. and Lippincott-Schwartz, J. (1996) Golgi dispersal during
microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites, Mol.
Biol. Cell. 7: 631-650. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (5 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Collawn, J.F., Kuhn, L.A., Liu, L.-F. C., Tainer, J.A. and Trowbridge, I.S. (1991) Transplanted LDL and
mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of transferrin
receptor, EMBO J. 10:3247-3254. (Medline)
Collins, C.A. and Vallee, R.B. (1989)Preparation of microtubules from rat liver and testis: cytoplasmic
dynein is a major microtubule associated protein, Cell Motility and Cytosk. 14:491-500. (Medline)
Colombo, M.I., Beron, W. and Stahl, P.D. (1997) Calmodulin regulates endosome fusion, J. Biol. Chem.
272:7707-7712. (MedLine)
Confalonieri, F. and Duguet, M. (1995) A 200-amino acid ATPase module in search of a basic function,
BioEssays 17:639-650. (Medline)
Cosson, P. and Letourneur, F. (1994) Coatomer interaction with di-lysine endoplasmic reticulum retention
motifs, Science 263:1629-1631. (MedLine)
Cosson, P. and Letourneur, F. (1997) Coatomer (COPI)-coated vesicles: role in intracellular transport and
protein sorting, Curr. Opin. Cell Biol. 9:484-487. (MedLine)
Cosson, P., Demolliere, C., Henneke, S., Duden, R. and Letourneur, F. (1996)δ and ζ-COP, two coatomer
subunits homologous to clathrin-associated proteins are involved in ER retrieval, EMBO J. 15:1792-1798.
Cowles, C.R., Odorizzi, G., Payne, G.S., Emr, S.D. (1997) The AP-3 adaptor complex is essential for
cargo-selective transport to the yeast vacuole, Cell 91:109-118. (Medline)
Cremona, O., Di Paolo, G., Wenk, M.R., Luthi, A., Kim, W.T., Takei, K., Daniell, L., Nemoto, Y.,
Shears, S.B., Flavell, R.A., McCormick, D.A. and De Camilli, P (1999) Essential role of phosphoinositide
metabolism in synaptic vesicle recycling, Cell 99:179-188. (MedLine
Cutler, D.F. and Cramer, L.P. (1990) Sorting during transport to the surface of PC12 cells: divergence of
synaptic vesicle and secretory granule proteins, J. Cell Biol. 110:721-730. (Medline)
De Camilli, P., Emr, S.D., McPherson, P.S. and Novick, P. (1996) Phosphoinositides as regulators in
membrane traffic, Science 271:1533-1539. (Medline)
Dell’Angelica, E.C., Ohno, H., Ooi, C.E., Rabinovich, E., Roche, K.W. and Bonifacino J.S. (1997) AP-3:
an adaptor-like protein complex with ubiquitous expression, EMBO J. 16:917-928. (Medline)
De Matteis, M.A. and Morrow, J.S. (1998) The role of ankyrin and spectrin in membrane transport and
http://www.albany.edu/~abio304/ref/ref11.html (6 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
domain formation, Curr. Opin. Cell Biol. 10:542-549. (Medline)
de Vries, K.J., Heinrichs, A.A., Cunningham, E., Brunink, F., Westerman, J., Somerharju, P.J., Cockcroft,
S., Wirtz, K.W. and Snoek, G.T. (1995) An isoform of the phosphatidylinositol-transfer protein transfers
sphingomyelin and is associated with the Golgi system, Biochem. J. 310:643-649. (Medline)
Diaz, R., Mayorga, L.S., , Weidman, P.J., Rothman, J.E. and Stahl, P.D.(1989) Vesicle fusion following
receptor-mediated endocytosis requires a protein active in Golgi transport, Nature 339:398-400.
Dominguez, M., Dejgaard, K., Fullekrug, J., Dahan, S., Fazel, A., Paccaud, J.P., Thomas, D.Y., Bergeron,
J.J. and Nilsson, T. (1998) gp25L/emp24/p24 protein family members of the cis-Golgi network bind both
COP I and II coatomer, J. Cell Biol. 140:751-765. (MedLine)
Donaldson, J.G., Finazzi, D. and Klausner, R.D. (1992) Brefeldin A inhibits Golgi-membrane catalyzed
exchange of guanine nucleotide into ARF protein, Nature 360:350-352. (Medline)
Drenckhahn, D. and Dermietzel. R. (1988) Organization of the actin filament cytoskeleton in the intestinal
brush border: a quantitative and qualitative immunoelectron microscope study, J. Cell Biol. 107:1037-1048. (Medline)
Drubin, D.G. and Nelson, W.J. (1996) Origins of cell polarity, Cell 84:335-344. (MedLine)
Duden, R., Allan, V. and Kreis, T. (1991a) Involvement of p-COP in membrane traffic through the Golgi
complex, Trends Cell Biol. 1:14-19.
Duden, R., Griffiths, G., Frank, R., Argos, P. and Kreis, T. E. (1991b) p-COP, a 110 kd protein associated
with non-clathrin-coated vesicles and Golgi complex, shows homology to ξβ-adaptin, Cell 64:649-665.
Eger, A., Stockinger, A., Schaffhauser, B., Beug, H. and Foisner, R. (2000) Epithelial mesenchymal
transition by c-Fos estrogen receptor activation involves nuclear translocation of β-catenin and
upregulation of β-catenin/lymphoid enhancer binding factor-1 transcriptional activity, J. Cell Biol.
148:173-188. (MedLine)
Farquhar, M.G. and Palade, G.E. (1963) Junctional complexes in various epithelia, J. Cell Biol. 17: 375-412.
Ferro-Novick, S. and Jahn, R. (1994) Vesicle fusion from yeast to man, Nature 370:191-193. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (7 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Fiedler, K. and Simons, K. (1995) The role of N-glycans in the secretory pathway, Cell 81:309-312.
Frölich K.U. http://yeamob.pci.chemie.uni-tuebingen. de/AAA/Tree.html
Fukuda, R., McNew, J.A., Weber, T., Parlati, F., Engel, T., Nickel, W., Rothman, J.E. and Söllner, T.H.
(2000) Functional architecture of an intracellular membrane t-SNARE, Nature 407:198-202. (MedLine)
Füllekrug, J., Suganuma, T., Tang, B.L., Hong, W., Storrie, B. and Nilsson, T. (1999) Localization and
recycling of gp27 (hp24gamma3): complex formation with other p24 family members, Mol. Biol. Cell
10:1939-1955. (MedLine)
Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S. and Tsukita, S. (1993)
Occludin: a novel integral membrane protein localizing at tight junctions, J. Cell Biol. 123:1777-1788.
Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K. and Tsukita, S.(1998a) Claudin-1 and -2: novel integral
membrane proteins localizing at tight junctions with no sequence similarity to occludin, J. Cell Biol.
141:1539-1550. (Medline)
Furuse, M., Sasaki, H., Fujimoto, K. and Tsukita, S. (1998b) A single gene product, claudin-1 or -2,
reconstitutes tight junction strands and recruits occludin in fibroblasts, J. Cell Biol. 143:391-401.
Faundez, V., Horng, J.T. and Kelly, R.B. (1998) A function for the AP3 coat complex in synaptic vesicle
formation from endosomes, Cell 93:423-432. (Medline)
Fath, K.R. and Burgess, D.R. (1993) Golgi derived vesicles from developing epithelial cells bind actin
filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein, J. Cell Biol.
120:117-27. (Medline)
Felder, S. and Kam, Z. (1994) Human neutrophil motility: time-dependent three-dimensional shape and
granule diffusion, Cell. Motil. Cytoskel. 28: 285-302. (Medline)
Fiedler, K., Veit, M., Stamens, M.A. and Rothman, J.E. (1996) Bimodal interaction of coatamer with p24
family of putative cargo receptors, Science 273:1396-1399. (Medline)
Friedrich, G.A., Hildebrand, J.D. and Soriano, P. (1997) The secretory protein Sec8 is required for
paraxial mesoderm formation in the mouse, Dev. Biol. 192:364-374. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (8 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Gaidarov, I. and Keen, J.H. (1999) Phosphoinositide-AP-2 interactions required for targeting to plasma
membrane clathrin-coated pits, J. Cell Biol. 146:755-764. (Medline)
Gaidarov, I., Chen, Q., Falck, J.R., Reddy, K.K. and Keen, J.H. (1996) A functional phosphatidylinositol
3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 α subunit. Implications
for the endocytic pathway, J. Biol. Chem. 271:20922-20929. (Medline)
Gaidarov, I., Krupnick, J.G., Falck, J.R., Benovic, J.L. and Keen, J.H. (1999) Arrestin function in G
protein-coupled receptor endocytosis requires phosphoinositide binding, EMBO J. 18:871-881. (Medline)
Gallusser, A. and Kirchhausen (1993) The 1 and 2 subunits of the AP complexes are the clathrin coat
assembly components, EMBO J. 12:5237-5244. (Medline)
Gammie, A.E., Kurihara, L.J., Vallee, R.B. and Rose, M.D. (1995) DNM1, a dynamin-related gene,
participates in endosomal trafficking in yeast, J. Cell Biol. 130:553-566. (Medline)
Gaynor, E. C. and Emr, S.D. (1997) COPI-independent anterograde transport: cargo-selective ER to Golgi
protein transport in yeast COPI mutants, J. Cell Biol. 136:789-802. (Medline)
Gaynor, E.C., Graham, T.R., Emr, S.D. (1998) COPI in ER/Golgi and intra-Golgi transport: do yeast
COPI mutants point the way? Biochim. Biophys. Acta 1404:33-51. (MedLine)
Geutze, H. J., Slot, J. W., Strous, J. A. M., Hasilik, A. and von Figura, K. (1984) The ultrastructural
localization of the mannose 6-phosphate receptor in rat liver, J. Cell Biol. 98:2047-2054. (Medline)
Geutze, H. J., Slot, J. W., Strous, J. G., Hasilik, A. and von Figura, K. (1985) Possible pathway for
lysosomal enzyme delivery. J. Cell Biol. 101:2253-2263. (Medline)
Gieselman, V., Pohlmann, R., Hasilik, A. and van Figura, K. (1983) Biosynthesis and transport of
cathepsin D in cultured human fibroblasts, J. Cell Biol. 97:1-5. (Medline)
Girod, A., Storrie, B., Simpson, J.C., Johannes, L., Goud, B., Roberts, L.M., Lord, J.M., Nilsson, T. and
Pepperkok, R. (1999) Evidence for a COP-I-independent transport route from the Golgi complex to the
endoplasmic reticulum, Nature Cell Biol. 1:423-430. (MedLine)
Glick, B.S. and Rothman J.E. (1987) Possible role for fatty acyl-coenzyme A in intracellular protein
transport, Nature 326:309-312. (Medline)
Glickman, J.N., Conibear, E., and Pearse, B.M.F. (1989) Specificity of binding of clathrin adaptors to
signals on the mannose 6-phosphate / insulin-like growth factor II receptor, EMBO J. 4:1041-1047.
http://www.albany.edu/~abio304/ref/ref11.html (9 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Godi, A., Santone, I., Pertile, P., Devarajan, P., Stabach, P.R., Morrow, J.S., Di Tullio, G., Polishchuk, R.,
Petrucci, T.C., Luini, A.and De Matteis, M.A. (1998) ADP ribosylation factor regulates spectrin binding
to the Golgi complex, Proc. Natl. Acad. Sci. USA 95:8607-8612. (Medline)
Godi, A., Pertile, P., Meyers, R., Marra, P., Di Tullio, G., Iurisci,C., Luini, A., Corda, D. and De Matteis,
G. (1999) ARF mediates recrutiment of PtdIns-4-OH kinase-β and stimulates synthesis of PtdIns(4,5)P2
on the Golgi complex Nature Cell Biol. 1:280-287. (Medline)
Gold, E.S., Underhill, D.M., Morrissette, N.S., Guo, J., McNiven, M.A. and Aderem, A. (1999) Dynamin
2 is required for phagocytosis in macrophages, J. Exp. Med. 190:1849-1856. (MedLine)
Goldberg, J. (2000) Decoding of sorting signals by coatamer through GTPase switch in COPI coat
complex, Cell 100:671-679. (Medline)
Goodman, O.B., Jr., Krupnick, J.G., Santini, F., Gurevich, V.V., Penn, R.B., Gagnon, A.W., Keen, J.H.
and Benovic, J.L. (1996) β-arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor,
Nature 383:447-450. (Medline)
Götte, M. and Fischer von Mollard, G. (1998) A new beat for the SNARE drum, Trends Cell Biol. 8:215-218. (MedLine)
Gottlieb, T. A., Beaudry, G., Rizzolo, L., Colman, A., Rindler, M., Adesnik, M. and Sabatini, D. D.
(1986) Secretion of endogenous and exogenous proteins from polarized MDCK cell monolayers, Proc.
Natl. Acad. Sci. USA. 83:2100-2104. (Medline)
Graham, T.R. and Emr, S. (1991) Compartmental organization of Golgi-specific modification and
vacuolar protein sorting events defined in yeast sec18 (NSF) mutant, J. Cell Biol. 114:207-218. (Medline)
Gravotta, D., Adesnik, M. and Sabatini, D.D. (1990) Transport of influenza HA from the trans-Golgi
network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes:
energy dependence and involvement of GTP-binding proteins, J. Cell Biol. 111:2893-2908. (Medline)
Grawe, F., Wodarz, A., Lee, B., Knust, E. and Skaer, H. (1996) The Drosophila genes crumbs and
stardust are involved in the biogenesis of adherens junctions, Development 122:951-959. (MedLine)
Greene, L.A. and Rein, G. (1977) Synthesis, storage and release of acetylcholine by a noradrenergic
phaeochromocytoma cell line, Nature 268:349-351. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (10 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Griffiths, G., Pfeiffer, S., Simon, K. and Matlin, K. (1985) Exit of newly synthesized membrane proteins
from the trans cisternae of the Golgi complex to the plasma membrane, J. Cell Biol. 101:949-964.
Grindstaff, K.K., Yeaman, C., Anandasabapathy, N., Hsu, S.C., Rodriguez-Boulan, E., Scheller, R.H. and
Nelson, W.J. (1998) Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle
delivery to the basal-lateral membrane in epithelial cells, Cell 93:731-740. (Medline)
Gumbiner, B.M. (1993) Breaking through the tight junction barrier, J. Cell Biol. 123:1631-1633.
Guo, W., Roth, D., Walch-Solimena, C. and Novick, P. (1999) The exocyst is an effector for Sec4p,
targeting secretory vesicles to sites of exocytosis, EMBO J.18:1071-1080. (MedLine)
Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S. and Novick, P. (2000) Protein complexes in
transport vesicle targeting, Trends Cell Biol. 10:251-255. (MedLine)
Gut, A., Kappeler, F., Hyka, N., Balda, M.S., Hauri, H.P. and Matter, K. (1998) Carbohydrate-mediated
Golgi to cell surface transport and apical targeting of membrane proteins, EMBO J. 17:1919-1929.
Hall, D.H.and Hedgecock, E.M. (1991) Kinesin related gene unc-104 is required for axonal transport of
synaptic vesicles, Cell 65:837-847. (Medline)
Hao, W., Tan, Z., Prasad, K., Reddy, K.K., Chen, J., Prestwich, G.D., Falck, J.R., Shears, S.B. and Lafer,
E.M. (1997) Regulation of AP-3 function by inositides. Identification of phosphatidylinositol 3,4,5-trisphosphate as a potent ligand, J. Biol. Chem. 272:6393-6398. (Medline)
Harter, C., Pavel, J., Coccia, F., Draken, E., Wegehingel, S., Tschochner, H. and Wieland, F. (1996)
Nonclathrin coat protein γ, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane
proteins of the early secretory pathway, Proc. Natl. Acad. Sci. USA 93:1902-1906. (MedLine)
Hata, Y., Butz, S. and Sudhof, T.C. (1996) CASK: a novel dlg/PSD95 homolog with an N-terminal
calmodulin-dependent protein kinase domain identified by interaction with neurexins, J. Neurosci.
16:2488-2494. (MedLine)
Hay, J.C. and Scheller, R.H. (1997) SNAREs and NSF in targeted membrane fusion, Curr. Opin. Cell
Biol.9:505-512. (Medline)
Hayashi, T., Yamasaki, S., Nauenburg, S., Binz, T. and Niemann, H.(1995) Disassembly of the
reconstituted synaptic vesicle membrane fusion complex in vitro, EMBO J. 14:2317-2325. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (11 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Hazuka, C.D., Foletti, D.L., Hsu, S.C., Kee, Y., Hopf, F.W., and Scheller, R.H. (1999) The sec6/8
complex is located at neurite outgrowth and axonal synapse-assembly domains, J. Neurosci. 19:1324-1334. (Medline)
Heidelberger, R., Heinemann, C., Neher, E. and Matthews, G. (1994) Calcium dependence of the rate of
exocytosis in a synaptic terminal, Nature 371:513-515. (MedLine)
Heilker, R., Manning-Krieg, U., Zuber, J.-F., and Spiess, M. (1996) In vitro binding of clathrin adaptors
to sorting signals correlates with endocytosis and basolateral sorting, EMBO J. 15:2893-2899. (Medline)
Helms, J.B. and Rothman, J.E. (1992) Inhibition by brefeldin A of a Golgi enzyme that catalyzes
exchange of guanine nucleotide bound to ARF, Nature 360:352-354. (Medline)
Henley, J.R. and McNiven, M.A. (1996) Association of a dynamin-like protein with the Golgi apparatus
in mammalian cells, J. Cell Biol 133:761-775. (MedLine)
Henley, J.R., Krueger, E.W., Oswald, B.J. and McNiven, M.A. (1998) Dynamin-mediated internalization
of caveolae, J. Cell Biol. 141:85-99. (MedLine)
Henley, J.R., Cao, H. and McNiven, M.A. (1999) Participation of dynamin in the biogenesis of
cytoplasmic vesicles, FASEB J. 13, Suppl 2:S243-S247. (MedLine)
Herskovits, J.S., Burgess, C.C., Obar, R.A., Vallee, R.B. (1993) Effects of mutant rat dynamin on
endocytosis, J. Cell Biol. 122:565-578. (Medline)
Hinshaw, J. E. and Schmid, S.L. Dynamin self-assembles into rings suggeting a mechanism for coated
vesicle budding, (1995) Nature374:190-192. (Medline)
Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K.K., Bloom, G.S. and Brady, S.T. (1991)
Kinesin associates with anterogradely transported membranous organelles in vivo, J. Cell Biol. 114:295-302. (Medline)
Hohl, T.M., Parlati, F., Wimmer, C., Rothman, J.E., Söllner, T.H. and Engelhardt, H. (1998) Arrangement
of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes, Mol Cell 2:539-548.
Hope, H.R. and Pike, L.J. (1996) Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains, Mol. Biol. Cell 7:843-881. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (12 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Hsu, S.C., Ting, A.E., Hazuka, C.D., Davanger, S., Kenny, J.W., Kee, Y. and Scheller, R.H. (1996) The
mammalian brain rsec6/8 complex, Neuron 17:1209-1219. (Medline)
Hsu, S.C., Hazuka, C.D., Roth, R., Foletti, D.L., Heuser, J. and Scheller, R.H. (1998) Subunit
composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin
filaments, Neuron 20:1111-1122. (Medline)
Hsueh, Y.P., Wang, T.F., Yang, F.C. and Sheng, M. (2000) Nuclear translocation and transcription
regulation by the membrane-associated guanylate kinase CASK/LIN-2, Nature 404:298-302. (MedLine)
Huang, P.-H. and Chiang, H.-L. (1997) Identification of novel vesicles in the cytosol to vacuole protein
degradation pathway, J. Cell Biol. 136:803-810. (Medline)
Huffaker, T.C., Thomas, J.H. and Botstein, D. (1988) Diverse effects of β-tubulin mutations on
microtubule formation and function, J. Cell Biol. 106:1997-2010. (MedLine)
Hunt, J.M., Bommert, K., Charlton, M.P., Kistner, A., Habermann, E., Augustine, G.J. and Betz, H.
(1994) A post-docking role for synaptobrevin in synaptic vesicle fusion, Neuron 12:1269-1279.
Hunziker, W., Harter, C., Matter, K. and Mellman, I. (1991) Basolateral sorting in MDCK cells requires a
distinct cytoplasmic domain determinant, Cell 66:907-920. (Medline)
Jacobs, C.W., Adams, A.E., Szaniszlo, P.J. and Pringle, J.R. (1988) Functions of microtubules in the
Saccharomyces cerevisiae cell cycle, J. Cell Biol. 1071409-1426. (MedLine)
Jahn, R. and Südhof, T.C. (1999) Membrane fusion and exocytosis, Annu. Rev. Biochem. 68:863-911.
Jones, S.M., Howell, K.E., Henley, J.R., Cao, H. and McNiven, M.A. (1998) Role of dynamin in the
formation of transport vesicles from the trans-Golgi network, Science 279:573-577. (MedLine)
Jou, T.S. and Nelson, W.J. (1998) Effects of regulated expression of mutant RhoA and Rac1 small
GTPases on the development of epithelial (MDCK) cell polarity, J. Cell Biol. 142:85-100. (Medline)
Kaibuchi, K., Kuroda, S., Fukata, M. and Nakagawa, M. (1999) Regulation of cadherin-mediated cell-cell
adhesion by the Rho family GTPases, Curr. Opin. Cell Biol. 11:591-596. (Medline)
Kaiser, C.A. and Shekman, R.(1990) Distinct sets of SEC genes govern transport vesicle formation and
junction early in the secretory pathway, Cell 61:723-733. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (13 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Kanai,F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E.,Cantley, L.C. and Yaffe, M.B. (2001)
The PX domains of p47phox and p40phox bind to lipid products of PI(3)K, Nature Cell Biol. 3:675-678 .
Klausner, R.D., Donaldson, J.G. and Lippincott-Schwartz, J. (1992) Brefeldin A: insights into the control
of membrane traffic and organelle structure, J. Cell Biol. 116:1071-1080. (Medline)
Klebes, A. and Knust, E. (2000) A conserved motif in Crumbs is required for E-cadherin localisation and
zonula adherens formation in Drosophila, Curr. Biol. 10:76-85. (MedLine)
Klenchin, V.A. and Martin, T.F. (2000) Priming in exocytosis: attaining fusion-competence after vesicle
docking, Biochimie 82:399-407. (MedLine)
Kondor-Koch, C., Bravo, R., Fuller, S. D., Cutler, D. and Garoff, H. (1985) Exocytotic pathways exist to
both apical and basolateral cell surface of the polarized epithelial cell MDCK, Cell 43:297-306. (Medline)
Kornfeld, S. (1987) Trafficking of lysosomal enzymes, FASEB J. 1:462-468. (Medline)
Kornfeld, S. (1992) Structure and function of the mannose 6-phosphate/insulinlike growth factor II
receptors, Annu. Rev. Biochem. 61:307-330. (MedLine)
Kreis, T.E., Lowe, M. and Pepperkok, R. (1995) COPs regulating membrane traffic, Ann. Rev. Cell Biol.
11:677-706. (Medline)
Kreitzer, G., Marmorstein, A., Okamoto, P., Vallee, R. and Rodriguez-Boulan, E. Kinesin and dynamin
are required for post-Golgi transport of a plasma-membrane protein, Nature Cell Biol. 2:125-127.
Ladinsky, M.S, Kremer, J.R, Furcinitti, P. S., McIntosh, J.R. and Howell, K.E. (1994), HVEM
tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat, J.
Cell Biol. 127:29-38. (Medline)
Lafont, F., Burkhardt, J.K. and Simons, K. (1994) Involvement of microtubule motors in basolateral and
apical transport in kidney cells, Nature 372:801-803. (Medline)
Lanoix, J., Ouwendijk, J., Lin, C.C., Stark, A., Love, H.D., Ostermann, J. and Nilsson T. (1999) GTP
hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I
vesicles, EMBO J. 18:4935-4948. (MedLine)
Lavoie, C., Paiement, J., Dominguez, M., Roy, L., Dahan, S., Gushue, J.N. and Bergeron, J.J. (1999)
http://www.albany.edu/~abio304/ref/ref11.html (14 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Roles for alpha(2)p24 and COPI in endoplasmic reticulum cargo exit site formation, J. Cell Biol.146:285-299. (MedLine)
Le Bevic, A., Quaroni, A., Nichols, B. and Rodriguez-Boulan, E. (1990) Biogenetic pathways of plasma
membrane proteins in Caco-2, a human intestinal epithelial cell line, J. Cell Biol. 111:1351-1361.
Le Borgne, R., Griffiths, G. and Hoflack, B. (1996) Mannose 6-phosphate receptors and ADP-ribosylation
factors cooperate for high affinity interaction of the AP-1 Golgi assembly with membranes, J. Biol. Chem.
271:2162-2170. (Medline)
Le Borgne, R. and Hoflack, B. (1997) Mannose 6-phosphate receptors regulated the formation of clathrin-coated vesicles in the TGN, J. Cell Biol. 137:335-345. (Medline)
Le Borgne, R. and Hoflack, B. (1998) Mechanisms of protein sorting and coat assembly: insights from
clathrin coated vesicle pathway, Curr. Opin. Cell Biol. 10:499-503. (Medline)
Le Borgne, R., Alconada, A., Bauer, U. and Hoflack, B. (1998) The mammalian AP-3 adaptor-like
complex mediates the intracellular transport of lysosomal membrane glycoproteins, J. Biol. Chem.
273:29451-29461. (Medline)
Lee, J. and Lentz, B.R. (1997) Evolution of lipidic structures during model membrane fusion and the
relation of this process to cell membrane fusion, Biochemistry 36:6251-6259. (MedLine)
Letourneur, F., Gaynor, E.C., Hennecke, S., Démollière, C., Duden, R., Emr, S.D., Riezman, H. and
Cosson, P. (1994) Coatamer is essential for retrieval of dilysine-tagged proteins to the endoplasmic
reticulum, Cell 79:1199-1207. (Medline)
Lewis, M.J. and Pelham, H.R. (1992) Ligand-induced redistribution of a human KDEL receptor from the
Golgi complex to the endoplasmic reticulum, Cell 68:353-364. (MedLine)
Lin, C.-C., Love, H.D., Gushue, J.N., Bergeron, J.J. and Ostermann, J. (1999) ER/Golgi intermediates
acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro, J. Cell Biol. 147:1457-1472.
Lippincott-Schwartz, J. (1998) Cytoskeletal proteins and Golgi dynamics, Curr. Opin. Cell Biol.10:52-59.
Lippincott-Schwartz, J., Cole, N.B., Marotta, A., Conrad, P.A and Bloom, G.S. (1995) Kinesin is the
motor for microtubule-mediated Golgi-to-ER membrane traffic, J. Cell. Biol. 128: 293-306. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (15 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Lisanti, M.P., Caras, I.W., Davitz, M.A. and Rodriguez-Boulan, E. (1989) A glycophospholipid
membrane anchor acts as an apical targeting signal in polarized epithelial cells, J. Cell Biol. 109:2145-2156. (Medline)
Llorente, A., Rapak, A., Schmid, S.L., van Deurs, B. and Sandvig, K. (1998) Expression of mutant
dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus, J. Cell Biol. 140:553-563. (MedLine)
Lohi, O., and Lehto, V.-P. (1998) VHS domain marks a group of proteins involved in endocytosis and
vesicular trafficking, FEBS Lett. 440: 255-257. (MedLine)
Love, H.D., Lin, C.C., Short, C.S. and Ostermann, J. (1998) Isolation of functional Golgi-derived vesicles
with a possible role in retrograde transport, J. Cell Biol. 140:541-551. (Medline)
Maier, O., Knoblich, M. and Westermann, P. (1996) Dynamin II binds to the trans-Golgi network,
Biochem. Biophys. Res. Commun. 223:229-233. (MedLine)
Malhotra V., Orci, L., Glick, B.S.. Block, M.R. and Rothman, J.E. (1988) Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack,
Cell 54:221-227. (Medline)
Malhotra, V., Serafini, T., Orci, L., Shepherd, J.G. and Rothman, J.E. (1989) Purification of a novel class
of coated vesicles mediating the biosynthetic protein transport through the Golgi stacks, Cell 58:329-336.
Martin, T.F. (1997) Phosphoinositides as spatial regulators of membrane traffic, Curr. Opin. Neurobiol.
7:331-338. (Medline)
Martínez-Menárguez, J.A., Geuze, H.J., Slot, J.W. and Klumperman, J. (1999) Vesicular tubular clusters
between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles, Cell 98:81-89. (MedLine)
Marzioch, M., Henthorn, D.C., Herrmann, J.M., Wilson, R., Thomas, D.Y., Bergeron, J.J. and Solari, R.C.
and Rowley, A. (1999) Erp1p and Erp2p, partners for Emp24p and Erv25p in a yeast p24 complex, Mol.
Biol. Cell 10:1923-1938. (MedLine)
Matlin, K. and Simons, K. (1984) Sorting of a plasma membrane glycoprotein occurs before it reaches the
cell surface in cultured epithelial cells, J. Cell Biol. 99:2131-2139. (Medline)
Matlin, K., Bainton, D. F., Pesonen, M., Louvard, D., Genty, N. and Simons, K. (1983) Transepithelial
transport of viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby
http://www.albany.edu/~abio304/ref/ref11.html (16 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
canine kidney cells. I. Morphological evidence, J. Cell Biol. 97:627-637. (Medline)
Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S.Y., Hamamoto, S., Schekman, R. and Yeung, T. (1998)
COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined
liposomes, Cell 93:263-275. (MedLine)
Matteoni, R. and Kreis, T.E. (1987) Translocation and clustering of endosomes and lysosomes depends on
microtubules, J. Cell. Biol. 105:1253-1265 (Medline)
Matter, K., Brauchbar, M. and Hauri, H.-P. (1990a) Sorting of endogenous plasma membrane proteins
occurs from two sites in cultured human intestinal epithelial cells (Caco-2), Cell 60:429-437. (Medline)
Matter, K., Bucher, K. and Hauri, H.-P.(1990b) Microtubule perturbation retards both the direct and the
indirect pathway but does not affect sorting of plasma membrane proteins in intestinal cells (Caco-2)
EMBO J. 9:3163-3170. (Medline)
May, A.P., Misura, K.M.S., Whiteheart, S.W. and Weiss, W.I. (1999) Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein, Nature Cell Biol. 1:175-182. (Medline)
Mayer, A., Wickner, W. and Haas, A. (1996) Sec18p (NSF)-driven release of Sec17p (α-SNAP) can
precede docking and fusion of yeast vacuoles, Cell 85:83-94. (Medline)
McCarthy, K.M., Skare, I.B., Stankewich, M.C., Furuse, M., Tsukita, S., Rogers, R.A., Lynch, R.D. and
Schneeberger, E.E. (1996) Occludin is a functional component of the tight junction, J. Cell Sci. 109:2287-2298. (Medline)
McNew, J.A., Parlati, F., Fukuda, R., Johnston, R.J., Paz, K., Paumet, F., Söllner, T.H. and Rothman, J.E.
(2000) Compartmental specificity of cellular membrane fusion encoded in SNARE proteins, Nature
407:153-159. (MedLine)
McNiven, M.A., Cao, I., Pitts, K.R. and Yoon, I. (2000) The dynamin family of mechanoenzymes:
pinching in new places, Trends Biochem Sci 25:115-120. MedLine
McPherson, P.S., Garcia, E.P., Slepnev, V.I., David, C., Zhang, X., Grabs, D., Sossin, W.S., Bauerfeind,
R., Nemoto, Y. and De Camilli, P. (1996) A presynaptic inositol-5-phosphatase, Nature 379:353-357.
Medof, M.E., Nagarajan, S. and Tykocinski, M.L. (1996) Cell-surface engineering with GPI-anchored
proteins, FASEB J. 10:574-586. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (17 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Melançon, P., Glick, B.S., Malhotra, V., Weidman, P.J., Serafini, T., Gleason, M.L., Orci, L. and
Rothman, J.E. (1987) Involvement of GTP-binding “G” proteins in transport through the Golgi stack, Cell
51:1053-1062. (Medline)
Meldolesi, J. (1974) Dynamics of cytoplasmic membranes in guinea pig pancreatic acinar cells. I.
Synthesis and turnover of membrane proteins, J. Cell Biol. 61:1-13.
Melkonian, K.A., Ostermeyer, A.G., Chen, J.Z., Roth, M.G. and Brown, D.A. (1999) Role of lipid
modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated,
while few are prenylated, J. Biol. Chem. 274:3910-3917. (MedLine)
Mellman, I. and Warren, G. (2000) The road taken: past and future foundations of membrane traffic, Cell
100:99-112. (MedLine)
Monck, J.R. and Fernandez, J.M. (1994) The exocytotic fusion pore and neurotransmitter release, Neuron
12:707-716. (MedLine)
Monck, J.R. and Fernandez, J.M. (1996) The fusion pore and mechanisms of biological membrane fusion,
Curr. Opin. Cell Biol. 8:524-533. (MedLine)
Mooseker, M.S. and Coleman, T.R. (1989) The 110-kD protein-calmodulin complex of the intestinal
microvillus (brush border myosin I) is a mechanoenzyme, J. Cell Biol. 108:2395-2400. (Medline)
Morgan, A., Dimaline, R. and Burgoyne, R.D. (1994) The ATPase activity of N-ethylmaleimide-sensitive
fusion protein (NSF) is regulated by soluble NSF attachment proteins, J. Biol. Chem. 269:29347-29350.
Morin, P.J., Johnson, R.J. and Fine, R.E. (1993) Kinesin is rapidly transported in the optic nerve as a
membrane associated protein, Biochim. Biophys. Acta 1146:275-281. (Medline)
Morita, K., Furuse, M., Fujimoto, K. and Tsukita, S. (1999a) Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands, Proc. Natl. Acad. Sci. USA 96:511-516. (Medline)
Morita, K., Sasaki, H., Furuse, M. and Tsukita, S. (1999b) Endothelial claudin. claudin-5/TMVCF
constitutes tight junction strands in endothelial cells, J. Cell Biol. 147:185-194. (Medline)
Morita, K., Sasaki, H., Fujimoto, K., Furuse, M. and Tsukita, S. (1999c) Claudin-11/OSP-based tight
junctions of myelin sheaths in brain and Sertoli cells in testis, J. Cell Biol. 145:579-588. (Medline)
Mostov, K., Apodaca, G., Aroeti, B. and Okamoto, C. (1992) Plasma membrane protein sorting in
http://www.albany.edu/~abio304/ref/ref11.html (18 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
polarized epithelial cells, J. Cell Biol. 116:577-583. (Medline)
Muñiz, M., Nuoffer. C., Hauri, H.P. and Riezman, H. (2000) The Emp24 complex recruits a specific
cargo molecule into endoplasmic reticulum-derived vesicles, J. Cell Biol. 148:925-930. (MedLine)
Munro, S. and Pelham, H. R. B. (1986) An H5P70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein, Cell 46:291-300. (Medline)
Nakagawa, T., Goto, K. and Kondo, H. (1996) Cloning, expression, and localization of 230-kDa
phosphatidylinositol 4-kinase, J. Biol. Chem. 271:12088-12094. (Medline)
Narula, N. and Snow, J.C.(1995) Distinct coat vesicles labeled for p200 bud from trans-Golgi neet work
membranes, Proc. Natl. Acad. Sci. USA 92:2874-2878. (Medline)
Nelson, W.J. (1991) Cytoskeleton functions in membrane traffic in polarized cells, Seminars in Cell Biol.
2:375-385. (Medline)
Nelson, W. J. (1992) Regulation of cell surface polarity from bacteria to mammals, Science 258:948-955.
Nelson, W. J. and Hammerton, R. W. (1989) A membrane-cytoskeleton complex containing Na+, K+
ATPase, ankyrin and fodrin in Madin-Darby canine kidney (MDCK) cells: implications from the
biogenesis of epithelial cell polarity. J. Cell Biol. 108:893-902. (Medline)
Nelson, D.S., Alvarez, C., Gao, Y.S., Garcia-Mata, R., Fialkowski, E. and Sztul, E. (1998) The membrane
transport factor TAP/p115 cycles between the Golgi and earlier secretory compartments and contains
distinct domains required for its localization and function, J. Cell Biol. 143:319-331. (MedLine)
Neuwald, A.F. (1999) The hexamerization domain of N-ethylmaleimide-sensitive factor: structural clues
to chaperone function, Structure Fold. Des. 7:R19-23. (Medline)
Neuwald, A.F., Aravind, L., Spouge, J.L. and Koonin, E.V. (1999) AAA+: A class of chaperone-like
ATPases associated with the assembly, operation, and disassembly of protein complexes, Genome Res.
9:27-43. (Medline)
Newman, L.S., McKeever, M.O., Okano, H.J. and Darnell, R.B. (1995) β-NAP, a cerebellar degeneration
antigen, is a neuron-specific vescile coat protein, Cell 82:773-783. (Medline)
Nichols, B.J., Ungermann, C., Pehham, H.R.B., Wickner, W.T. and Haas, A. (1997) Homotypic vacuolar
fusion mediated by t- and v-SNAREs, Nature 386:199-902. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (19 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Nickel, W., Sohn, K., Bunning, C. and Wieland, F.T. (1997) p23, a major COPI-vesicle membrane
protein, constitutively cycles through the early secretory pathway, Proc. Natl. Acad. Sci. USA 94:11393-11398. (MedLine)
Nicoziani, P., Vilhardt, F., Llorente, A., Hilout, L., Courtoy, P.J., Sandvig, K. and van Deurs, B. (2000)
Role for dynamin in late endosome dynamics and trafficking of the cation-independent Mol. Biol. Cell
11:481-495. (MedLine)
Novick, P. and Zerial, M. (1997) The diversity of Rab proteins in vesicle transport, Curr. Opin. Cell Biol.
9:496-504. (MedLine)
Novick, P., Field, C. and Schekman, R. (1980) Identification of 23 complementation groups required for
post-translational events in the yeast secretory pathway, Cell 21:205-215. (Medline)
Novick, P. and Botstein, D. (1985) Phenotypic analysis of temperature-sensitive yeast actin mutants, Cell
40:405-416. (MedLine)
Nusse, R. (1997) A versatile transcriptional effector of Wingless signaling, Cell 89:321-323. (MedLine)
Odorizzi, G. and Trowbridge, I.S. (1997) Structural requirements for basolateral sorting of human
transferrin receptor in biosynthetic and endocytotic pathways in Madin-Darby canine kidney cells, J. Cell
Biol. 137:1255-1264. (Medline)
Oh, P., McIntosh, D.P. and Schnitzer, J.E. (1998) Dynamin at the neck of caveolae mediates their budding
to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium, J. Cell Biol.
141:101-114. (MedLine)
Ohashi, M., Jan de Vries, K., Frank, R., Snoek, G., Bankaitis, V., Wirtz, K. and Huttner, W.B. (1995) A
role for phosphatidylinositol transfer protein in secretory vesicle formation, Nature 377:544-547.
Ohno, H., Stewart, J.,m Fournier, M.C., Bosshart, H., Rhee, I., Miyatake, S., Saito, T., Gallusser, A.,
Kichhausen, T. Banifacion, J.S. (1995) Interactions of tyrosine-based sorting signals with clathrin-associated proteins, Science 269:1872-1875. (Medline)
Orci, L., Glick, B. S. and Rothman, J. E. (1986) A new type of coated vesicular carrier that appears not to
contain clathrin: its possible role in protein transport within the Golgi stacks, Cell 46:171-184. (Medline)
Orci, L., Malhotra, V., Amherdt, M., Serafini, T. and Rothman, J.E. (1989) Dissection of a single round of
vesicular transport: sequential intermediates for cisternal movement in Golgi stack, Cell 56:357-368.
http://www.albany.edu/~abio304/ref/ref11.html (20 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Orci, L., Stamnes, M., Ravazzola, M., Amherdt, M., Perrelet, A., Söllner, T.H. and Rothman, J.E. (1997)
Bidirectional transport by distinct populations of COPI-vesicles, Cell 90:335-349. (Medline)
Ostermann, J., Orci, L., Tani, K. Amherdt. M., Ravazzola, M., Elazar, Z. and Rothman, J.E. (1993)
Stepwise assembly of functionally active transport vesicles, Cell 75: 1015-1025. (MedLine)
Panaretou, C., Domin, J., Cockcroft, S. and Waterfield, M.D. (1997) Characterization of p150, an adaptor
protein for the human phosphatidylinositol (PtdIns) 3-kinase. Substrate presentation by
phosphatidylinositol transfer protein to the p150.Ptdins 3-kinase complex, J. Biol. Chem. 272:2477-2485.
Parlati, F., McNew, J.A., Fukuda, R., Miller, R., Sollner, T.H. and Rothman, J.E. (2000) Topological
restriction of SNARE-dependent membrane fusion, Nature 407:194-198. (MedLine)
Patel, S. and Latterich, M. (1998) The AAA team: related ATPases with diverse functions, Trends Cell
Biol. 8:65-71. (Medline)
Patzak, A. and Winkler, H. (1986) Exocytotic exposure and recycling of membrane antigens of
chromaffin granules: ultrastructural evaluation after immunolabeling, J. Cell Biol. 102:510-515.
Pearse, B.M. (1988) Receptors compete for adaptors found in plasma membrane coated pits, EMBO J.
11:3331-3336. (Medline)
Pelham, H.R.B. (1999) SNAREs and the secretory pathway-lessons from yeast, Exp. Cell Res. 247:1-8.
Pelham, H.R.B. (2001) SNAREs and the specificity of membrane fusion, Trends Cell Biol. 99-101.
Peter, F., Plutner, H., Zhu, H., Kreis, T.E. and Balch, W.E. (1993) β-COP is essential for transport of
protein from the endoplasmic reticulum to the Golgi in vitro, J. Cell Biol. 122:1155-1167. (Medline)
Peters, C. and Mayer, A (1998) Ca2+/calmodulin signals the completion of docking and triggers a late
step of vacuole fusion, Nature 396:575-580. (MedLine)
Pevner, J., Hsu, S.-C. and Scheller, R.H. (1994) n-Sec1: a neural-specific syntaxin-binding protein, Proc.
Natl. Acad. Sci. USA 91:1445-1449. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (21 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Pfeffer, S.R. (1999) Transport-vesicle targeting: tethers before SNAREs, Nature Cell Biol. 1:E17-22.
Pfeiffer, S., Fuller, S. D. and Simons, K. (1985) Intracellular sorting and basolateral appearance of the G
protein of vesicular stomatitis virus in Madin-Darby canine kidney cells. J. Cell Biol. 101:470-476.
Pierce, S.K. (2002) Lipid rafts and B-cell activation, Nature Rev. Immunol. 2:96-105. (MedLine)
Pike, L.J. and Casey, L. (1996) Localization and turnover of phosphatidylinositol 4,5-bisphosphate in
caveolin-enriched membrane domains, J. Biol. Chem. 271:26453-26456. (Medline)
Powell, S.K., Lisanti, M.P. and Rodriguez-Boulan, E.J. (1991) Thy-1 expresses two signals for apical
localization in epithelial cells, Am. J. Physiol. Cell Physiol. 29:C715-720. (Medline)
Presley, J.F, Cole, N.B., Schroer, T.A., Hirschberg K., Zaal K.J., Lippincott-Schwartz J. (1997) ER-to-Golgi transport visualized in living cells, Nature 389:81-85. (Medline)
Propopov, V., Govindan, B., Novick, P., and Gerst, J.E. (1993) Homologs of the syaptobrevin/VAMP
family of synaptic vesicle proteins function on the late secretory pathway of S. cerevisiae, Cell 74:855-861. (Medline)
Puertollano, R., Aguilar, R.C., Gorshkova, I., Crouch, R.J and Bonifacino, J.S. Sorting of mannose 6-phosphate receptors mediated by the GGAs, Science 292:1712-1716. (MedLine)
Qualmann, B., Roos, J., DiGregorio, P.J. and Kelly, R.B. (1999) Syndapin I, a synaptic dynamin-binding
protein that associates with the neural Wiskott-Aldrich syndrome protein, Mol. Biol. Cell 10:501-513.
Régnier-Vigouroux,A., Tooze, S.A., and Huttner, W.B.(1991) Newly synthesized synaptophysin is
transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane,
EMBO J. 10:3589-35601. (Medline)
Reichert M, Muller T, Hunziker W (2000) The PDZ domains of zonula occludens-1 induce an epithelial
to mesenchymal transition of madin-darby canine kidney I cells. Evidence for a role of β-catenin/tcf/lef
signaling, J. Biol. Chem. 275:9492-9500. (MedLine)
Reinhard, C., Harter, C., Bremser, M., Br gger, B., Sohn, K., Helms, J.B. and Wieland, F. (1999)
Receptor-induced polymerization of coatomer, Proc. Natl. Acad. Sci. USA 96:1224-1228. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (22 of 32) [3/5/2003 7:57:08 PM]
Chapter 11: References
Rice, L.M. and Brunger, A.T. (1999) Crystal structure of the vesicular transport protein Sec17:
implications for SNAP function in SNARE complex disassembly, Mol. Cell. 4:85-95. (Medline)
Rindler, M. J.. Ivanov, I. E., Plesken, H., Rodriguez-Boulan, E. and Sabatini, D. D. (1984) Viral
glycoproteins destined for apical or basolateral plasma membrane domains traverse the Golgi apparatus
during the intracellular transport in doubly infected Madine-Darby canine kidney cells (MDCK), J. Cell
Biol. 98:1304-1319. (Medline)
Rindler, M.J., Ivanov, I.E. and Sabatini, D.D. (1987) Microtubule -acting drugs lead to the non-polarized
delivery of influenza hemagglutinin to the cell surface of polarized Madin-Darby canine idney cells, J.
Cell Biol. 104:231-241. (Medline)
Robinson, M.S. (1997) Coats and vesicle budding, Trends in Cell Biol. 7:99-102
Rodriguez-Boulan, E. and Nelson, W.J. (1989) Morphogenesis of the polarized epithelial cell phenotype,
Science 245:718-724. (Medline)
Rogalski, A.A. and Singer, S.J. (1984) Associations of elements of the Golgi apparatus with microtubules,
J. Cell. Biol. 99: 1092-1100 (Medline)
Rossi, G., Jiang, Y., Newman, A. and Ferro-Novick (1991) Dependence of Ypt1 and Sec 4 membrane
attachement on Bet2, Nature 351:158-161. (Medline)
Rothman, J.E. (1994) Mechanisms of intracellular protein transport (1994) Nature 372:55-62. (Medline)
Rothman, J. E. and Orci, L. (1992) Molecular dissection of the secretory pathway Nature 355:409-415.
Rothman, J.E. and Wieland, F.T. (1996) Protein sorting by transport vesicles, Science 272:227-234.
Rowe, T., Aridor, M., McCaffery, J.M., Plutner, H. and Nuoffer, C. and Balch, W.E. (1996) COPII
vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI, J. Cell Biol. 135:895-911. (Medline)
Sacher, M., Jiang, Y., Barrowman, J., Scarpa, A., Burston ,J., Zhang, L., Schieltz, D., Yates, J.R. 3rd,
Abeliovich, H. and Ferro-Novick, S. (1998) TRAPP, a highly conserved novel complex on the cis-Golgi
that mediates vesicle docking and fusion, EMBO J. 17:2494-2503. (Medline)
Sahagian, G. G. and Steer, C. J. (1985) Transmembrane orientation of a mannose-6-phosphate receptor in
http://www.albany.edu/~abio304/ref/ref11.html (23 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
isolated clathrin coated vesicles, J. Biol. Chem. 260:9838-9842. (Medline)
Sahagian, G. G., Distler, J. and Jourdian, G. W. (1981) Characterization of a membrane-associated
receptor from bovine liver that binds phosphomannosyl residues of bovine testicular -galactosidase, Proc.
Natl. Acad. Sci. USA. 78:4289-4293. (Medline)
Salama, N.R., Yeung, T. and Shekman, R.W. (1993) The sec 13p complex and reconstitution of vesicle
budding from ER with purified cytosolic proteins, EMBO J. 12:4073-4082. (Medline)
Sandoval , I.V. and Bakke, O. (1994) Targeting of membrane proteins to endosomes and lysosomes,
Trends in Cell Biol. 4:292-297.
Sandvig, K. and van Deurs, B. (1996) Endocytosis, intracellular transport, and cytotoxic action of Shiga
toxin and ricin, Physiol. Rev. 76:949-966. (MedLine)
Santos, B. and Snyder, M. (1997) Targeting of chitin synthase 3 to polarized growth sites in yeast requires
Chs5p and Myo2p, J. Cell Biol. 136:95-110. (MedLine)
Scales, S.J., Pepperkok, R. and Kreis, T.E. (1997) Visualization of ER-to-Golgi transport in living cells
reveals a sequential mode of action for COPII and COPI, Cell 90:1137-1148. (Medline)
Schimmoller, F., Singer-Kruger, B., Schroder, S., Kruger, U., Barlowe, C. and Riezman, H. (1995) The
absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of
selected proteins to the Golgi, EMBO J. 14:1329-1339. (MedLine)
Schoenenberger, C.A., Zuk, A., Zinkl, G.M., Kendall, D. and Matlin, K.S. (1994) Integrin expression and
localization in normal MDCK cells and transformed MDCK cells lacking apical polarity, J. Cell Sci.
107:527-541. (MedLine)
Schafer, D.A., Gill, S.R., Cooper, J.A., Heuser, J.E. and Schroer, T.A. (1994) Ultrastructural analysis of
the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin, J. Cell
Biol.126:403-412. (Medline)
Scheiffele, P., Peranen, J. and Simons, K. (1995) N-glycans as apical sorting signals in epithelial cells,
Nature 378:96-98. (Medline)
Schell, M.J., Maurice, M., Stieger, B. and Hubbard. A.L. (1992) 5′ nucleotidase is sorted to the apical
domain of hepatocytes via an indirect route, J. Cell Biol., 119:1173-1182. (Medline)
Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A.V., Witke, W., Huttner, W.B.
and Soling, H.D. (1999) Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to
http://www.albany.edu/~abio304/ref/ref11.html (24 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
lysophosphatidic acid, Nature 401:133-141. (MedLine)
Schott, D., Ho, J., Pruyne, D. and, Bretscher, A. (1999) The COOH-terminal domain of Myo2p, a yeast
myosin V, has a direct role in secretory vesicle targeting, J. Cell Biol. 147:791-808. (MedLine)
Schröder, S. and Ungewickell, E. (1991) Subunit interaction and function of clathrin-coated vesicle
adaptors for the Golgi and the plasma membrane, J. Biol. Chem. 266:7910-7918. (Medline)
Schroer, T.A. and Sheetz, M.P. (1991) Two activators of microtubule-based vesicle transport, J. Cell Biol.
115:1309-1318. (Medline)
Schroer, T.A., Bingham, J.B. and Gill S.R. (1996) Actin-related protein 1 and cytoplasmic dynein based
motility-what’s the connection, Trends Cell Biol. 6:212-215.
Schulze-Lohoff. E., Hasilik, A. and von Figura, K. (1985) Cathepsin D precursors in clathrin coated
organelles from human fibroblasts, J. Cell Biol. 101:824-829. (Medline)
Schwaninger, R., Plutner, H., Bokoch, G.M. and Balch, W.E. (1992) Multiple GTP-binding proteins
regulate vesicular transport from the ER to Golgi membranes, J. Cell Biol. 119:1077-1096. (Medline)
Seaman, M.N., McCaffery, J.M., Emr, S.D. (1998) A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast, J. Cell. Biol. 142:665-681. (Medline)
Serafini, T. and Rothman, J.E. (1992) Purification of Golgi cisternae-derived non-clathrin-coated vesicles,
Methods Enzymol. 219:286-299. (MedLine)
Sever, S., Muhlberg, A.B. and Schmid, S.L.(1999) Impairment of dynamin’s GAP domain stimulates
receptor-mediated endocytosis, Nature 398:481-486. (MedLine)
Shaywitz, D.A., Espenshade, P.J., Gimeno, R.E. anf Kaiser, C.A. (1997) COPII subunit interactions in the
assembly of the vesicle coat, J. Biol. Chem. 272:25413-25416. (Medline)
Shih, W., Galluser, A. and Kirchhausen, T. (1995) A clathrin binding site in the hinge of the β2 chain of
mammalian AP-2 complexes, J. Biol. Chem. 270:31083-31090. (Medline)
Simons, K. and Fuller, S. D. (1985) Cell surface polarity in epithelium, Annu. Rev. Cell Biol. 1:243-288.
Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes, Nature 387:569-572. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (25 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
Simons, K. and Zerial, M. (1993) Rab proteins and the road map for intracellular transport, Neuron
11:789-799. (Medline)
Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J.M., Brech, A., Callaghan, J., Toh, B.H., Murphy,
C., Zerial, M. and Stenmark, H. (1998) EEA1 links PI(3)K function to Rab5 regulation of endosome
fusion, Nature 394:494-498. (MedLine)
Simpson, F., Bright, N.A., West, M.A., Newman, L.S., Darnell, R.B. and Robinson, M.S. (1996) A novel
adaptor-related protein complex, J. Cell Biol. 133:749-760. (Medline)
Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., Fiedler, K., Helms, J.B. and
Wieland, F.T. (1996) A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in
coatomer binding, J. Cell Biol. 135:1239-1348. (MedLine)
Sönnichsen, B., Watson, R., Clausen, H., Misteli, T. and Warren, G. (1996) Sorting by COP I-coated
vesicles under interphase and mitotic conditions, J. Cell Biol. 134:1411-1425. (MedLine)
Soole, K.L., Jepson, M.A., Hazlewood, G.P., Gilbert, H.J., and Hirst, B.H. (1985) Epithelial sorting of a
glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and
intestinal Caco-2 cells, J. Cell Sci. 108:369-377. (Medline)
Sorokin, L., Sonnenberg, A., Aumailley, M., Timpl, R. and Ekblom, P. (1990) Recognition of the laminin
E8 cell-binding site by an integrin possessing the α6 subunit is essential for epithelial polarization in
developing kidney tubules, J. Cell Biol. 111:1265-1273. (MedLine)
Søgaard, M., Tani, K., Ye, R.R., Geromanos, S., Tempst, P., Kirchhausen, T. Rothman, J.E. and Söllner,
T. (1994) A rab protein is required for the assembly of SNARE complexes in the docking of transport
vesicles, Cell 78:937-948. (Medline)
Söllner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H., Geromanos, S. Tempst, P. and
Rothman, J.E. (1993a) SNAP receptors implicated in vesicle targeting and fusion, Nature 362:318-324.
Söllner,T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H. and Rothman, J.E. (1993b) A protein
assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle
docking, activation and fusion. Cell 75:409-418. (Medline)
Sonoda, N., Furuse, M., Sasaki, H., Yonemura, S., Katahira, J., Horiguchi, Y. and Tsukita, S. (1999)
Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands.
Evidence for direct involvement of claudins in tight junction barrier, J. Cell Biol. 147:195-204. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (26 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
Springer, S. and Schekman, R. (1998) Nucleation of COPII vesicular coat complex by endoplasmic
reticulum to Golgi vesicle SNAREs, Science 281:698-700. (MedLine)
Springer, S., Spang, A. and Schekman, R. (1999) A primer on vesicle budding, Cell 97:145-148.
Stack, J.H. and Emr, S.D. (1994) Vps34p required for yeast vacuolar protein sorting is a multiple
specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities,
J. Biol. Chem. 269:31552-31562. (Medline)
Staehelin, L.A. (1974) Structure and function of intercellular junctions, Int. Rev. Cytol. 39:191-283.
Stepp, J.D., Pellicena-Palle, A., Hamilton, S., Kirchhausen, T. and Lemmon, S. K. (1995) A late Golgi
sorting function for Saccharomyces cerevisiae Apm1p, but not Apm2p, a second yeast clathrin AP
medium chain-related protein, Mol. Biol. Cell 6:41-58. (Medline)
Stepp, J.D., Huang, K. and Lemmon, S.K. (1997)The yeast adaptor protein complex, AP-3, is essential for
the efficient delivery of alkaline phosphatase by the alternate pathway to the vacuole, J. Cell Biol.
139:1761-1774. (Medline)
Stone, S., Sacher, M., Mao, Y., Carr, C., Lyons, P., Quinn, A.M. and Ferro-Novick, S. (1997) Bet1p
activates the v-SNARE Bos1p, Mol. Biol. Cell 8:1175-1181 (Medline)
Stoorvogel, W., Oorschot, V. and Geutze, H.J. (1996) A novel class of clathrin-coated vesicles budding
from endosomes, J. Cell Biol. 132:21-33. (Medline)
Storrie, B., Pepperkok, R. and Nilsson, T. (2000) Breaking the COPI monopoly on Golgi recycling,
Trends Cell Biol. 10:385-390. (MedLine)
Stow, J.L. de Almeida, J.B., Narula, N., Holzman, E.J., Ercolani, L. and Ausiello, D.A. (1991) A
heterotrimeric G protein G1-3, on Golgi membranes regulates the secretion of a heparan sulfate
proteoglycan in LLC-PK1 epithelial cells, J. Cell Biol. 114:1113-11124. (Medline)
Takahashi, K., Matsuo, T., Katsube, T., Ueda, R. and Yamamoto, D. (1998) Direct binding between two
PDZ domain proteins Canoe and ZO-1 and their roles in regulation of the jun N-terminal kinase pathway
in Drosophila morphogenesis, Mech. Dev. 78:97-111. (MedLine)
Takei, K., McPherson, P.S., Schmid, S.L. and De Camilli, P. (1995) Tubular membrane invaginations
coated by dynamin rings are induced by GTP-S in nerve terminals, Nature 374:186. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (27 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
Tan, A., Bolscher, J., Feltkamp, C. and Ploegh, H. (1992), Retrograde transport from the Golgi region to
the endoplasmic reticulum is sensitive to GTPγS, J. Cell Biol. 116, 1357-1367. (Medline)
Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B. and Rothman, J.E. (1993) Hydrolysis of
bound GTP by ARF protein triggers uncoating of Golgi-derived COP-coated vesicles, J. Cell Biol.
123:1365-1371. (Medline)
Tepass, U. (1997) Epithelial differentiation in Drosophila, BioEssays 19:673-682. (MedLine)
Tepass, U., Theres, C. and Knust, E. (1990) crumbs encodes an EGF-like protein expressed on apical
membranes of Drosophila epithelial cells and required for organization of epithelia, Cell 61:787-799.
TerBush, D.R., Maurice, T., Roth, D. and Novick, P. (1996) The Exocyst is a multiprotein complex
required for exocytosis in Saccharomyces cerevisiae, EMBO J. 15:6483-6494. (Medline)
Thomas, J.R., Dwek, R.A. and Rademacher, T.W. (1990) Structure, biosynthesis and function of
glycosylphosphatidylinositols, Biochemistry 29:5413-5422. (Medline)
Thompson, T.E. and Tillack, T.W. (1985) Organization of glycosphingolipids in bilayers and plasma
membranes of mammalian cells, Ann. Rev. Biophys. Biophys. Chem. 14:361-386. (Medline)
Traub, L.M. (1997) Clathrin-associated adaptor proteins-putting it all together, Trends in Cell Biol. 7:43-46.
Traub, L.M., Kornfeld, S. and Ungewickell, E. (1995) Different domains of the AP-1 adaptor complex are
required for Golgi membrane binding and clathrin recruitment, J.Biol. Chem. 270:4933-4942. (Medline)
Tsukita, S. and Furuse, M. (1999) Occludin and claudins in tight-junction strands: leading or supporting
players? Trends Cell Biol. 9:268-273. (Medline)
Umeda, A., Meyerholz, A. and Ungewickell, E. (2000) Identification of the universal cofactor (auxilin 2)
in clathrin coat dissociation, Eur. J. Cell Biol. 79:336-342. (MedLine)
Ungewickell, E., Ungewickell, H., Holstein, S.E., Lindner, R., Prasad, K., Barouch, W., Martin, B.,
Greene, L.E. and Eisenberg E. (1995) Role of auxilin in uncoating clathrin-coated vesicles, Nature
378:632-635. (MedLine)
van de Moortele, S., Picart, R., Tixier-Vital, A., and Tougard, C. (1993) Nocodazole and taxol affect
subcellular compartments but not secretory activity of GH3B6 prolactin cells, Eur. J. Cell Biol. 60:217-http://www.albany.edu/~abio304/ref/ref11.html (28 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
227. (Medline)
van der Bliek, A.M., Redelmeier, T.E., Damke, H., Tisdale, E.J., Meyerowitz, E.M. and Schmid, S.L.
(1993) Mutations in human dynamin block an intermediate stage in coated vesicle formation, J. Cell Biol.
122:553-563. (MedLine)
VanRheenen, S.M., Cao, X., Sapperstein, S.K., Chiang, E.C., Lupashin, V.V., Barlowe, C. and Waters,
M.G. (1999) Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex
with Sec35p, J. Cell Biol. 147:729-742. (MedLine)
Van Zeijl, M.J.A.H. and Matlin, K.S (1990) Microtubule perturbation inhibits intracellular transport of an
apical membrane glycoprotein in a substrate dependent manner in polarized Madin-Darby canine kidney
cells, Cell Reg. 1:921-936. (Medline)
Vega-Salas, D.E., Salas, P.J.I., Gundersen, D. and Rodriguez-Boulan, E. (1987) Formation of the apical
pole of the epithelial (Madin-Darby canine kidney) cells:polarity of an apical protein is independent of
tight junctions while segregation of a basolateral marker requires cell-cell interactions, J. Cell Biol.
von Figura. K. and Hasilik, A. (1986) Lysosomal enzymes and their receptors. Annu. Rev. Biochem.
55:167-193. (Medline)
von Mollard, F.G.and Stevens, T.H. (1999) The Saccharomyces cerevisiae v-SNARE Vti1p is required
for multiple membrane transport pathways to the vacuole, Mol. Biol. Cell. 10:1719-1732. (MedLine)
von Mollard, G.F., Nothwehr, S.F. and Stevens, T.H. (1997) The yeast v-SNARE Vti1p mediates two
vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p, J. Cell Biol.
137:1511-1524. (MedLine)
Vowels, J.J. and Payne, G.S. (1998) A dileucine-like sorting signal directs transport into an AP-3-dependent, clathrin-independent pathway to the yeast vacuole, EMBO J. 17:2482-2493. (Medline)
Waterman-Storer, C.M., Karki, S. and Holzbauer, E.L.F. (1995) The p150Glued component of the dynactin
complex binds to both microtubules and the actin related protein centractin (Arp1) Proc. Natl. Acad. Sci.
USA 92:1634-1638. (Medline)
Waters, M.G. and Pfeffer, S.R. (1999) Membrane tethering in intracellular transport, Curr. Opin. Cell
Biol. 11:453-459. (MedLine)
Waters, M. G., Serafini, T. and Rothman. J. E. (1991) ‘Coatamer’: a cytosolic protein complex containing
subunits of non-clathrin-coated Golgi transport vesicles, Nature 349:248-251. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (29 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
Waters, M.G., Clary, D.O. and Rothman, J.E. (1992) A novel 115-kD peripheral membrane protein is
required for intercisternal transport in the Golgi stack, J. Cell Biol. 118:1015-1026. (MedLine).
Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl, M., Parlati, F., Söllner, T.H. and
Rothman, J.E. (1998) SNAREpins: minimal machinery for membrane fusion, Cell 92:759-972. (MedLine)
Weidman, P.J., Melançon, P., Block, M.R. and Rothman, J.E. (1989) Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both soluble protein(s) and an integral membrane
receptor, J. Cell Biol. 108:1589-1596. (Medline)
Weigert, R., Silletta, M.G., Spano, S., Turacchio, G., Cericola, C., Colanzi, A., Senatore, S., Mancini, R.,
Polishchuk, E.V., Salmona, M., Facchiano, F., Burger, K.N., Mironov, A., Luini, A. and Corda, D. (1999)
CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid, Nature 402:429-433. (MedLine)
Weimbs, T., Low, S.H., Chapin, S.J. and Mostov, K.E. (1997a) Apical targeting in polarized epithelial
cells: there’s more afloat than rafts, Trends in Cell Biol. 7:393-399.
Weimbs, T., Low, S.H., Chapin, S.J., Mostov, K.E., Bucher, P. and Hofmann K.(1997b) A conserved
domain is present in different families of vesicular fusion proteins: a new superfamily, Proc. Natl. Acad.
Sci. USA 94:3046-3051. (MedLine)
Whiteheart, S.W., Brunner, M., Wilson, D.W., Wiedmann, M. and Rothman, J.E. (1992) Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) bind to a multi-SNAP receptor complex in
Golgi membranes, J. Biol. Chem. 267:1239-12243.
Whitney, J.A., Gomez, M., Sheff, D., Kresi, T.E. and Mellman, I. (1995) Cytoplasmic coat proteins
involved in endosome function, Cell 83:703-713. (Medline)
Wickner, W. and Haas, A. (2000) Yeast homotypic vacuole fusion: a window on organelle trafficking
mechanisms, Annu. Rev. Biochem. 69:247-275. (MedLine)
Wienke, D.C., Knetsch, M.L., Neuhaus, E.M., Reedy, M.C. and Manstein, D.J. (1999) Disruption of a
dynamin homologue affects endocytosis, organelle morphology, and cytokinesis in Dictyostelium
discoideum Mol. Biol. Cell 10:225-243. (MedLine)
Willingham, M. C., Pastan, I. H., Sahagian, G. G., Jourdian, G. W. and Neufeld, E. F. (1981)
Morphologic study of the internalization of a lysosomal enzyme by mannose-6-phosphate receptor in
cultured Chinese hamster ovary cells, Proc. Natl. Acad. Sci. USA 78:6967-6971. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (30 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
Wilson, D.W., Wilcox, C.A., Flynn, G.C., Chen, E., Kuang, W.-J., Henzel, W.J., Block, M.R., Ulrich, A.
and Rothman, J.E. (1989) A fusion protein required for vesciel-mediated transport in both mammalian
cells and yeast, Nature 339:355-359. (Medline)
Wilson, D.W., Whiteheart, S.W., Wiedmann, M., Brunner, M. and Rothman, J.E. (1992) A multisubunit
particle implicated in membrane fusion, J. Cell Biol. 117:531-538. (Medline)
Wirtz, K.W. (1991) Phospholipid transfer proteins, Annu. Rev. Biochem. 60:73-99. (Medline)
Witke, W., Podtelejnikov, A.V., Di Nardo, A., Sutherland, J.D., Gurniak, C.B., Dotti, C. and Mann, M.
(1998) In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and
actin assembly, EMBO J. 17:967-976. (MedLine)
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995) Expression of crumbs confers apical character
on plasma membrane domains of ectodermal epithelia of Drosophila, Cell 82:67-76. (MedLine)
Woods, J.W., Doriaux, M. and Falquhar, M.G. (1986) Transferrin receptors recycle to the cis and middle
as well as trans Golgi cisternae in Ig-secreting myeloma cells, J. Cell Biol. 103, 277-286. (Medline)
Woodward, M.P. and Roth, T.F. (1978) Coated vesicles: characterization, selective dissociation, and
reassembly, Proc. Natl. Acad. Sci. USA 75:4394-4398. (Medline)
Yang, B., Gonzalez, L. Jr., Prekeris, R., Steegmaier, M., Advani, R.J. and Scheller, R.H. (1999) SNARE
interactions are not selective. Implications for membrane fusion specificity, J. Biol. Chem. 274:5649-5653. (Medline)
Yeaman, C., Le Gall, A.H., Baldwin, A.N., Monlauzeur, L., Le Bivic, A. and Rodriguez-Boulan, E.
(1997) The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in
polarized MDCK cells, J. Cell Biol. 139:929-940. (Medline)
Yokode, M., Pathak, R.K., Hammer, R.E., Brown, M.S.. Goldstein, J.L. and Anderson, R.G.W. (1992)
Cytoplasmic sequence required for basolateral targeting of LDL receptor in liver of transgenic mice, J.
Cell Biol. 117:39-46. (Medline)
Yu, R.C., Hanson, P.I., Jahn, R. and Brunger, A.T. (1998) Structure of the ATP-dependent
oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP, Nature Struct. Biol.
5:803-8011. (Medline)
Yu, R.C., Jahn, R. and Brunger, A.T. (1999) NSF N-terminal domain crystal structure: models of NSF
function, Mol. Cell 4:97-107. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (31 of 32) [3/5/2003 7:57:09 PM]
Chapter 11: References
Zacharias, D.A., Violin, J.D., Newton, A.C. and Tsien, R.Y. (2002) Partitioning of lipid-modified
monomeric GFPs into membrane microdomains of live cells, Science 296:913-916. 9MedLine)
Zerial, M. and McBride, H. (2001) Rab proteins as membrane organizers, Nature Rev. Mol. Cell Biol.
2:107-117. (MedLine)
Zhao, L., Helms, J.B., Brugger, B., Harter, C., Martoglio, B., Graf, R., Brunner, J. and Wieland, F.T.
(1997) Direct and GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit β, Proc.
Natl. Acad. Sci. USA 94:418-423. (MedLine)
Zhao, L., Helms, J.B., Brunner, J. and Wieland, F.T. (1999) GTP-dependent binding of ADP-ribosylation
factor to coatomer in close proximity to the binding site for dilysine retrieval motifs and p23, J. Biol.
Chem. 274:14198-14203. (MedLine)
Zhu, Y., Traub, L.M. and Kornfeld, S. (1998) ADP-ribosylation factor 1 transiently activates high-affinity
adaptor protein complex AP-1 binding sites on Golgi membranes, Mol. Biol. Cell 9:1323-1337.
Zhu, Y., Doray, B., Poussu, A., Lehto, V.P. and Kornfeld, S. (2001) Binding of GGA2 to the lysosomal
enzyme sorting motif of the mannose 6-phosphate receptor, Science 292:1716-1718. (MedLine)
Zimmerberg, J. (2000) Are the curves in the right places? Traffic 1:366-368.
Zurzolo, C., Lisantu, M.P., Caras, I.W., Nitsch, L. and Rodriguez-Boulan, E. (1993)
Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in
Fischer rat thyroid epithelial cells, J. Cell. Biol. 121:1031-1039. (Medline)
Zurzolo, C., van’t Hof, W., van Meer, G. and Rodriguez-Boulan, E. (1994) VIP21/caveolin,
glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial
cells, EMBO J. 13:42-53. (Medline)
http://www.albany.edu/~abio304/ref/ref11.html (32 of 32) [3/5/2003 7:57:09 PM]


Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: