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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
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11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
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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.
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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
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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
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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.
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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
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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
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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).
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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
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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
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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.
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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;
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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).
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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
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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
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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
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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.
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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.
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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
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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).
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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.
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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.
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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).
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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
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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).
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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
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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).
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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).
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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
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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
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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,
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