Vesicle tethering complexes in membrane traffic

Abstract

Despite the recent progress in the field of membrane traffic, the question of how the specificity of membrane fusion is achieved has yet to be resolved. It has become apparent that the SNARE proteins, although central to the process of fusion, are often not the first point of contact between a vesicle and its target. Instead, a poorly understood tethering process physically links the two before fusion occurs. Many factors that have an apparent role in tethering have been identified. Among these are several large protein complexes. Until recently, these seemed unrelated, which was a surprise since proteins involved in membrane traffic often form families, members of which function in each transport step. Recent work has shown that three of the complexes are in fact related. We refer to these as the 'quatrefoil' tethering complexes, since they appear to share a fourfold nature. Here we describe the quatrefoil complexes and other, unrelated, tethering complexes, and discuss ideas about their function. We propose that vesicle tethering may have separate kinetic and thermodynamic elements and that it may be usefully divided into events upstream and downstream of the function of Rab GTPases. Moreover, the diversity of tethering complexes in the cell suggests that not all tethering events occur through the same mechanisms.

Full text

Introduction
Fusion of intracellular membranes is mediated in many, if not
all, cases by SNARE proteins (Chen and Scheller, 2001). The
final stage of fusion involves the formation of a bundle of four
parallel core SNARE domains, one contributed by the vesicle
and three contributed by the target membrane (Fig. 1). Such a
trans SNARE complex bridges the two membranes, and its
formation is thought to overcome the energy barrier preventing
two membranes from fusing. Only particular combinations of
four core SNAREs are able to promote fusion in vitro (McNew
et al., 2000; Parlati et al., 2000). A simple model is therefore
that the SNARE complement of a vesicle and a potential target
membrane is necessary and sufficient to determine their
compatibility for fusion.
Several aspects of this model have recently been questioned.
In particular, it is unclear whether further factors provide
specificity, help in SNARE assembly or even assist in the
fusion event itself. The rate of trans complex formation is too
slow in vitro to account for the rate of membrane fusion
observed in vivo, which suggests that accelerating factors are
involved (Fasshauer et al., 2002). There is also evidence that
in some cases fusion events are regulated downstream of
SNARE complex assembly, although the generality of this
is unclear (Muller et al., 2002). Most debate, however, has
focused on whether interactions between v- and t-SNAREs can
account for the specificity of membrane transport events
(Pelham, 2001). Biochemical and genetic studies have
identified several proteins that appear to play a role in
membrane transport steps after vesicle formation. These
factors could contribute to the fidelity of vesicle fusion and
function in a process that has become known as tethering
(Fig. 1). This is the formation of physical links, often over
considerable distances, between two membranes that are due
to fuse, before trans SNARE complex formation (for reviews,
see Guo et al., 2000; Lowe, 2000; Waters and Hughson, 2000).
Tethering might represent the earliest stage at which specificity
is conferred on a fusion reaction. Both yeast and mammalian
systems have been used in the discovery of tethering factors,
and, although our understanding of the process is still limited,
the emerging picture is of a series of perhaps inter-related steps
that determine the specificity of membrane fusion.
What is the evidence that SNAREs do not provide all the
specificity in vivo? The ubiquitous distribution of SNAREs on
some membranes is not sufficient to account for the fusion of
vesicles to localised regions of those membranes. For example,
the yeast plasma membrane SNAREs Sso1p and Sso2p are
distributed over the entire plasma membrane, and yet vesicles
fuse with only certain parts of the membrane during periods of
polarised growth (Brennwald et al., 1994). Cleavage of squid
synaptic SNAREs with toxins prevents SNARE complex
formation but results in the association of more, not fewer,
vesicles with the membrane (Hunt et al., 1994). Similarly, the
percentage of tethered neuronal vesicles is significantly higher
in flies lacking syntaxin or synaptobrevin than in wild-type
flies (Broadie et al., 1995). These results are consistent with
SNAREs being involved in membrane fusion but dispensable
for a prior tethering event that initially attaches the vesicle to
its target without causing it to fuse. Vesicle tethering has
been observed in an in vitro reconstituted system, which
demonstrated that ER-derived vesicles attach to the Golgi
apparatus, losing their ability to diffuse freely, in a reaction that
is independent of functional SNARE proteins (Cao et al.,
1998). A growing number of factors proposed to be involved
in tethering have been identified. In many cases, the mode of
action, interactions and indeed identities of these factors
remain obscure. Although it is still far from clear how tethering
2627
Despite the recent progress in the field of membrane traffic,
the question of how the specificity of membrane fusion is
achieved has yet to be resolved. It has become apparent that
the SNARE proteins, although central to the process of
fusion, are often not the first point of contact between a
vesicle and its target. Instead, a poorly understood
tethering process physically links the two before fusion
occurs. Many factors that have an apparent role in
tethering have been identified. Among these are several
large protein complexes. Until recently, these seemed
unrelated, which was a surprise since proteins involved in
membrane traffic often form families, members of which
function in each transport step. Recent work has shown
that three of the complexes are in fact related. We refer to
these as the ‘quatrefoil’ tethering complexes, since they
appear to share a fourfold nature. Here we describe the
quatrefoil complexes and other, unrelated, tethering
complexes, and discuss ideas about their function. We
propose that vesicle tethering may have separate kinetic
and thermodynamic elements and that it may be usefully
divided into events upstream and downstream of the
function of Rab GTPases. Moreover, the diversity of
tethering complexes in the cell suggests that not all
tethering events occur through the same mechanisms.
Key words: Vesicle tethering, Membrane traffic, Exocyst, Sec34/35
complex, COG complex, GARP complex, TRAPP
Summary
Vesicle tethering complexes in membrane traffic
James R. C. Whyte and Sean Munro*
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
*Author for correspondence (e-mail sean@mrc-lmb.cam.ac.uk)
Journal of Cell Science 115, 2627-2637 (2002) © The Company of Biologists Ltd
Commentary

2628
occurs at a molecular level, connections between the variety of
seemingly disparate tethering factors are beginning to become
apparent and may prove useful in elucidating their roles.
Protein complexes and coiled-coil proteins as
potential tethering factors
Two broad classes of molecules are proposed to have a role in
tethering: a group of long, coiled-coil proteins and several
large, multisubunit complexes. The former have the potential
to form homodimeric coiled coils with lengths up to several
times the diameter of a vesicle. In yeast, one example is Uso1p,
whose importance in tethering is clear since it is an essential
protein and the only cytosolic factor required for the tethering
of ER-derived vesicles to washed acceptor membranes in the
assay mentioned above (Barlowe, 1997). The formation of long
coiled coils by Uso1p (Yamakawa et al., 1996) and its
mammalian homologue p115 has been observed by electron
microscopy (Sapperstein et al., 1995). Likewise, X-ray
crystallography has shown that a part of the endosomal
tethering protein EEA1 forms long coiled coils (Dumas et al.,
2001), and this is generally supposed to hold for the other
mammalian examples, the coiled-coil proteins of the Golgi
(often called golgins), and their yeast counterparts. These
structures have led to the idea that the large coiled-coil proteins
are anchored at one end to a membrane, which allows them to
‘search’ the surrounding space for a passing vesicle, which is
then bound by the other end. Although this is an attractive
model, there is currently a lack of direct evidence for it.
Although the coiled-coil proteins are known in many cases
to bind to a target membrane and/or vesicle, their receptors on
the membranes are generally not known. One exception is
p115, which tethers COPI vesicles to the Golgi. Its receptors
are two other coiled-coil proteins: giantin on the vesicles and
GM130 on the Golgi (Sonnichsen et al., 1998). However, the
simultaneous binding of p115 to both these molecules has been
called into question by the finding that they share, and compete
for, a common binding site on p115 (Linstedt et al., 2000).
Nevertheless, the in vivo significance of the interaction
between p115 and GM130 is demonstrated by the
accumulation of transport vesicles and reduction of secretory
transport when this interaction is inhibited (Seemann et al.,
2000). Giantin is membrane anchored, and GM130 interacts
with Golgi membranes through another protein, GRASP65
(Barr et al., 1998). Even in the case of p115, stripped of its
complications, the idea that a long, coiled-coil tether links the
vesicle and the target has yet to be confirmed. p115 and Uso1p
may not even be typical of other large, coiled-coil proteins,
since they are unique in having a large globular domain at one
end.
Apart from their involvement in the Golgi and in endosomal
fusion (Nielsen et al., 2000), long, coiled-coil proteins are not
associated with other transport steps. We therefore focus here
on the second class of tethering factor, multisubunit complexes,
since there has been recent and rapid progress in their
characterisation. The overall molecular function of the
complexes is still unknown, but biochemical studies have
revealed their subunit compositions and interactions with other
membrane-trafficking components. The complexes can thus
now be grouped and distinguished on the basis of sequence
similarity. The resulting classification suggests that the
functions of some complexes differ and that tethering is a
complex process that encompasses several steps both upstream
and downstream of a stable, physical attachment of a vesicle
to a target membrane.
Multisubunit tethering complexes
Seven large, conserved complexes have been proposed to have
roles in vesicle tethering at distinct trafficking steps (Fig. 2).
In most cases these complexes were initially identified and
characterised in yeast. Frequently, the same complex has been
identified by more than one laboratory and characterised in
more than one organism, which has led to a confusing variety
of names. It is to be hoped that a standard nomenclature for
each complex will soon be adopted, as has recently been agreed
for the COG complex (Table 1); we shall use a single name for
each complex here. The seven complexes can be divided into
two groups: those that have a common domain at the N-
terminus of at least some, if not all, of their subunits (Whyte
and Munro, 2001), and those that do not (Fig. 3). The family
of complexes related to each other by virtue of their shared
domain, which we shall call the ‘quatrefoil’ complexes,
consists of the exocyst (termed the Sec6/8 complex in
mammalian studies), the conserved oligomeric Golgi (COG)
complex [previously known as the Sec34/35 complex, and in
mammals also as the Golgi transport complex (GTC) or ldlCp
complex] and the Golgi-associated retrograde protein (GARP)
complex (which is also known in yeast as the Vps52/53/54
[VFT]). The other group consists of complexes that seem to
Journal of Cell Science 115 (13)
3. SNARE assembly 4. Fusion1. Approach
v-SNARE
t-SNARE
2. Vesicle tethering
Fig. 1. Steps in the delivery of vesicles to the correct organelle. (1) An intracellular transport vesicle approaches its destination organelle either
by diffusion or motor-mediated directed transport. (2) The vesicle is then proposed to be tethered to the organelle by protein complexes and
long coiled-coil proteins. (3) A v-SNARE protein on the vesicle then engages a t-SNARE on the target, forming a four-helical bundle whose
assembly drives the two bilayers into close proximity, (4) thereby causing membrane fusion. Both vesicle tethering and SNARE assembly have
been referred to by others as ‘docking’, so to avoid confusion we use only the former terms here.

2629Vesicle tethering complexes in membrane traffic
bear no relation to each other or to the quatrefoil complexes
and comprises TRAPP, the Class C Vps complex (also known
as the Pep3p/Pep5p complex or the homotypic fusion and
vacuole protein sorting [HOPS] complex) and the Dsl1p
complex.
A family of quatrefoil tethering complexes
The exocyst, COG complex and GARP complex not only share
an N-terminal domain in their subunits but all contain a
multiple of four subunits. Moreover, the eight components of
the COG complex can be divided into two distinct sets of four.
Although this use of multiples of four subunits might be
coincidental, we tentatively propose that these complexes be
collectively termed “quatrefoil” tethering complexes. More
speculatively, their quatrefoil nature might reflect interaction
with a set of fourfold-symmetric components, such as the core
SNARE domains that form the trans SNARE complex.
The exocyst
Probably the best-characterised tethering complex is the
exocyst. Most of its eight subunits were originally identified as
products of genes whose mutation causes yeast to accumulate
vesicles destined for plasma membrane (Guo et al., 1999a;
TerBush et al., 1996). The complex is localised to sites of
polarised exocytosis in yeast, to which one of the subunits,
Sec3p, is localised independently of the cytoskeleton and the
secretory pathway (Finger et al., 1998). This contrasts with the
localisation of the other components to such sites, which
requires both actin and a functional secretory pathway. Sec3p
might thus act as a landmark for polarised secretion
independently of the rest of the exocyst. One of the other
components, Sec15p, binds to Sec4p, the Rab GTPase present
on secretory vesicles (Guo et al., 1999b). Moreover, it appears
to bind preferentially to the activated, GTP-bound form of
Sec4p. Sec10p and Sec15p co-immunoprecipitate from the
soluble fraction of cytosol, which indicates that they form a
subcomplex. These findings lead to a model in which activated
Sec4p on a Golgi-derived vesicle binds to the Sec10p-Sec15p
subcomplex, which results in its assembly with Sec3p and the
remainder of the exocyst on the plasma membrane and thereby
tethers the vesicle (Guo et al., 1999b).
The localisation of Sec3p appears to be mediated by its
interaction with the GTP-bound form of two Rho family
GTPases, namely Rho1p (Guo et al., 2001) and Cdc42p (Zhang
et al., 2001), which also have a role in the polarisation of actin.
Vesicles travel along actin filaments en route to the sites of
polarised secretion (Karpova et al., 2000; Pruyne and
Bretscher, 2000); the Rho GTPases might therefore coordinate
the cytoskeletal tracks along which vesicles travel with the
machinery that tethers them to their target. The synthetic
lethality of the combination of sec3 and mutations in profilin,
which regulates actin polymerisation and depolymerisation,
illustrates the importance of the actin cytoskeleton in this
process (Finger and Novick, 1997; Haarer et al., 1996).
nucleus
ER
Golgi
vacuole
Class C
Class C
exocyst
TRAPP II
GARP
COG
Dsl1p
COG
endo.
TRAPP I
Fig. 2. Putative tethering complexes in the yeast secretory pathway.
Protein complexes that have been found to have a role in particular
vesicular transport steps are indicated next to those steps. The role of
early and late endosomes in yeast is contentious, and so for
simplicity this compartment has been shown as a single organelle.
Table 1. Revised nomenclature for the COG complex
Yeast Mammals
New name**** Previous names New name**** Previous name
Cog1p* Cod3p†† Sec36p§§ Tfi1p¶¶ Cog1* ldlBp***
Cog2p* Sec35p§Cog2* ldlCp†††
Cog3p Sec34p§,¶ Gdr20p** Cog3 hSec34‡‡‡
Cog4p Cod1p†† Sgf1p‡‡ Sec38p§§ Tfi3p¶¶ Cog4 hCod1††,§§§
Cog5p Cod4p†† Cog5 GTC-90¶¶¶
Cog6p Cod2p†† Sec37p§§ Tfi2p¶¶ Cog6 hCod2††, §§§
Cog7p* Cod5p†† Cog7* –†
Cog8p Dor1p†† Cog8 hDor1††,§§§
*Assignment of the same name to yeast and mammalian versions of Cog1, Cog2 and Cog7 is on functional grounds, since sequence similarity is low. †(Ungar
et al., 2002); ‡(VanRheenen et al., 1998); §(VanRheenen et al., 1999); ¶(Kim et al., 1999); **(Spelbrink and Nothwehr, 1999); ††(Whyte and Munro, 2001);
‡‡(Kim et al., 2001b); §§(Ram et al., 2002); ¶¶(Suvorova et al., 2002); ***(Chatterton et al., 1999); †††(Podos et al., 1994); ‡‡‡(Suvorova et al., 2001); §§§(Loh and
Hong, 2002); ¶¶¶(Walter et al., 1998); ****(Ungar et al., 2002).

2630
The mammalian counterpart of the exocyst consists of
homologues of the eight yeast proteins and is also localised to
sites of polarised growth (Brymora et al., 2001; Hsu et al.,
1996; Kee et al., 1997). In some non-polarised cell types, the
exocyst is associated both with the trans Golgi network (TGN)
and with the plasma membrane, and antibody inhibition of
each pool affects cargo exit from the TGN and delivery to the
plasma membrane, respectively (Yeaman et al., 2001). In
polarised epithelial cells, antibodies inhibit basolateral but not
apical traffic, and overexpression of human Sec10 results in
increased synthesis and delivery of secretory and basolateral,
but not apical, plasma membrane proteins (Grindstaff et al.,
1998; Lipschutz et al., 2000). The mammalian exocyst has
been reported to interact with microtubules (Vega and Hsu,
2001) and septins (Hsu et al., 1998), although the localisation
of Sec3p in yeast is not dependent on, or coincident with, the
septins (Finger et al., 1998). The mammalian exocyst also
interacts with Ca2+ signalling proteins (Shin et al., 2000) and,
as in yeast, it is probably regulated by small GTPases. Sec5 is
an effector of the GTPase RalA, and inhibition of Ral function
leads to a decrease in the formation or stability of a mammalian
exocyst subcomplex containing Sec6 and Sec10 (Brymora et
al., 2001; Moskalenko et al., 2002; Sugihara et al., 2002).
The COG complex
The COG complex has been proposed to act as a tether at the
Golgi apparatus, although it is unclear which vesicles are its
substrates. Wuestehube et al. identified sec34 and sec35
mutants in a screen designed to identify yeast genes involved
specifically in early stages of the secretory pathway
(Wuestehube et al., 1996). Two groups subsequently showed
that Sec34p (now Cog3p; Table 1) and Sec35p (Cog2p)
associate as part of a large complex (Kim et al., 1999;
VanRheenen et al., 1999). Identification of the other six
components of the complex (Whyte and Munro, 2001) showed
that the eight components fall into two phenotypic groups. The
data are best explained by a model in which two distinct classes
of vesicle are tethered by the complex to the early Golgi:
vesicles recycling within the Golgi, and vesicles recycling
to the Golgi from later, endosomal compartments (Fig. 2).
Defects in the former process could lead indirectly to a failure
in ER-to-Golgi transport, as observed for sec34 and sec35
mutants in vivo (Wuestehube et al., 1996) and in vitro
(VanRheenen et al., 1998).
A function for the COG complex in recycling of Golgi
components is supported by the identification of COG3 (as
GRD20) as a gene required for the proper localisation of a
TGN protein (Spelbrink and Nothwehr, 1999). Two reports
reiterating the identification of a subset of the subunits did not
resolve the issue of anterograde versus retrograde transport
(Kim et al., 2001b; Ram et al., 2002), but a third shows
interactions of the COG complex that support a recycling role
(Suvorova et al., 2002). These interactions are with SNAREs
involved in intra-Golgi recycling and with the COPI vesicle
coat, which is involved in retrograde transport, but not with a
Journal of Cell Science 115 (13)
Exocyst
(Sec6/8) COG complex
(Sec34/35)GARP complex
(VPS52/53/54, VFT)
Exo84
Sec3
Sec8
Sec10
Sec15
Sec6
Exo70
Sec5
Cog7
Cog4
Cog6
Cog1
Cog8
Cog5
Cog2
Cog3Sac2 (Vps52)
Luv1 (Vps54)
Vps53
Vps51
Trs23
Trs120
Trs130
Trs31
Kre11 (Trs65)
Trs33
Bet3
Gsg1 (Trs85)
TRAPP I and TRAPP II
Trs20
Bet5
Sec20
Dsl1
Tip20
Class C Vps complex
(HOPS, Pep3/5)
Vam2 (Vps41)
Pep5 (Vps11)
Vps16
Pep3 (Vps18)
Vps33
Vam6 (Vps39)
Quatrefoil tethering complexes
Other complexes
400 aa
I
II
Dsl1p complex
TMD
clathrin rpt.
RING-H2
Sec1-like
required for
growth
*
*******
*
*
*
*
**
*
*
*
*
**
*
**
*
Fig. 3. Composition of proposed tethering
complexes. For each complex the known
components in the yeast S. cerevisiae are
shown, arranged by size, and identifiable
domains indicated. In each case the
standard gene name in the Saccharomyces
Genome Database is given first, followed
by alternative names that have also been
used in recent publications. Vps51p is
encoded by the open reading frame
YKR020w (Elizabeth Conibear, personal
communication). The two sets of related
subunits of the TRAPP complexes are
indicated by different colours.
Homologues of most of these proteins
exist in higher eukaryotes, but in some
cases have extra domains. Thus in
mammals Sec5 has an N-terminal TIG
domain, Exo84 a PH domain, Vam6 a
CNH domain (Caplan et al., 2001) and
Vam2 a C-terminal RING-H2 domain
(Radisky et al., 1997). Vps54 has an N-
terminal zinc-finger-like domain in
Drosophila and C. elegans, but not in
mammals. Vam6 in both yeast and higher
eukaryotes has a conserved half RING
domain (C2HC) at its C-terminus. The ‘p’
has been removed from the yeast protein
names for clarity.

2631Vesicle tethering complexes in membrane traffic
component of the ER-to-Golgi COPII coat. Mutants show
Golgi-associated glycosylation and sorting defects at
temperatures permissive for the in vitro ER-to-Golgi tethering
assay (Suvorova et al., 2002), and indeed a mutant of one of
the subunits, Cog1p, shows no defect when tested in the in vitro
assay for ER-to-Golgi transport at restrictive temperature (Ram
et al., 2002). The ER-to-Golgi tethering defect may therefore
be an indirect effect, but a recent report shows a defect in a
different in vitro assay that measures tethering of ER-derived
vesicles, whether homotypic or to another membrane
(Morsomme and Riezman, 2002). The same report suggests
that, in addition to a tethering function, the complex could have
a separate and unexpected involvement in a sorting event that
occurs in vitro to create two classes of ER-derived vesicle
(Morsomme and Riezman, 2002). The exact role of the
complex remains contentious, but, by analogy with other
tethering complexes, the COG complex is expected to interact
with a Rab. The most likely interaction would be as an effector
of an early Golgi Rab such as Ypt1p (Short and Barr, 2002),
and this is supported by an in vitro interaction of the complex
with Ypt1p that occurs preferentially in the presence of GTP
(Suvorova et al., 2002).
Identification of the eight yeast components of the
complex revealed homology to several, characterised and
uncharacterised, mammalian proteins (Whyte and Munro,
2001). In particular, it led to the prediction that the mammalian
Sec34p-containing complex (Suvorova et al., 2001) is the same
as the GTC (Walter et al., 1998), which was purified from
bovine brain cytosol on the basis of its stimulatory activity in
an intra-Golgi-transport assay. The GTC in turn was already
suspected to be the same as the ldlCp complex, a Golgi-
associated complex thought to contain ldlBp and ldlCp, two
proteins that complement mutant cultured cell lines that have
a range of Golgi defects (Chatterton et al., 1999; Podos et
al., 1994). Identification of the eight components of the
mammalian COG complex has shown these predictions to be
correct (Loh and Hong, 2002; Ungar et al., 2002). Interestingly,
some of the components appear to be in a subcomplex, which
may represent one lobe of the two-lobed structure seen by
electron microscopy of the whole complex (Ungar et al., 2002).
This division is consistent with the phenotypic division of
the yeast proteins into two halves, but the existence of a
subcomplex in yeast has not been investigated.
Perhaps the most interesting finding to come from analysis
of the components of the COG complex is that some show
extensive, albeit distant, sequence similarity to components of
the exocyst and the GARP complex, and many of the subunits
of all three complexes appear to have at their N-termini a
common domain (Whyte and Munro, 2001). An alignment of
these domains is shown in Fig. 4A. The domain is predicted to
form two short stretches of potential coiled coil or amphipathic
helix (not to be confused with the sequences in the long, coiled-
coiled tethering proteins such as Uso1p). For at least some of
these components, the sequence similarity is likely to reflect
more than simply a shared structure, because similarity
searches find other components before any other coiled-coil
proteins (Whyte and Munro, 2001). The presence of the
common domain is not discernible in all of the components by
sequence similarity searches. It remains to be seen whether this
is because of its absence in some cases or because of an
inability to detect a structural similarity at the sequence level
owing to sequence divergence. The latter is at least suggested
by the presence of regions of predicted short coiled-coil near
the N-termini of all components of the human COG complex
and exocyst (Fig. 4B). Similar predictions are seen for the yeast
COG complex, exocyst and GARP complex (TerBush et al.,
1996) (data not shown). The significance of this putative
domain is not yet known; it may be involved in assembly of
the complex or have some other function. Nevertheless, its
existence reveals a similarity between some of the tethering
complexes and suggests that they have similar modes of action.
The GARP complex
Retrograde traffic from endosomes to the Golgi has not been
as extensively characterised as other transport steps, but the
GARP complex localises to the TGN and is required for this
process (Conboy and Cyert, 2000; Conibear and Stevens,
2000). The GARP complex consists of four proteins (Vps51p,
Vps52p, Vps53p and Vps54p) (Conibear and Stevens, 2001;
Gavin et al., 2002) (E. Conibear, personal communication),
some of which show extensive sequence similarity to other
quatrefoil complex components (Whyte and Munro, 2001).
Whether it functions as a tethering complex has yet to be
established, but strong evidence for this comes from the finding
that it is an effector of the Rab Ypt6p and interacts with the
SNARE Tlg1p (Siniossoglou and Pelham, 2001). That its
components share the domain found in the exocyst and COG
complex additionally suggests a tethering function, but
confirmation of such a role awaits development of an in vitro
assay for this transport step. Genes encoding mammalian
homologues of Vps52p, Vps53p and Vps54p are discernible in
the databases but have not been characterised.
Non-quatrefoil tethering complexes
Although no enzymatic activity has yet been ascribed to the
three similar complexes already described, two of the
remaining complexes, although dissimilar in subunit
composition, possess guanine-nucleotide-exchange factor
(GEF) activity towards Rabs. Yeast contains 11 Rab proteins,
whereas humans have at least 60 (Bock et al., 2001). Apart
from vesicle tethering, their functions involve interactions of
vesicles with the cytoskeleton and possibly vesicle budding
(reviewed in Zerial and McBride, 2001). As in the cases of
other regulatory GTPases, their function depends on a
GDP/GTP cycle. The transition to their active, GTP-bound
form is promoted by the corresponding GEF, and they then
exert their effects by binding to target molecules (effectors).
The TRAPP complexes
The TRAPP (transport protein particle) complex was originally
described as a large protein complex functioning in the later
stages of ER-to-Golgi traffic in yeast (Sacher et al., 2000;
Sacher et al., 1998). After its initial identification, it transpired
that TRAPP represents two distinct complexes. TRAPP I is
~300 kDa in size and contains seven subunits, whereas TRAPP
II is ~1000 kDa and contains an additional three subunits (Fig.
3) (Sacher et al., 2001). Subunits of these complexes share
some sequence similarity, the six smallest subunits falling into
two families of three (Bet3p, Trs33p and Trs31p; and Bet5p,

2632
Trs20p and Trs23p) (Sacher et al., 2000). TRAPP I and TRAPP
II both cofractionate with an early Golgi marker but not late-
Golgi markers and are also present in a cytosolic pool. The
Golgi association is stable, in that Bet3p does not relocate to
the ER when anterograde ER-to-Golgi traffic is blocked, unlike
components that cycle between the ER and Golgi, and
the complex remains assembled under these conditions
(Barrowman et al., 2000). The Golgi receptor for TRAPP is
not known.
TRAPP I, but not TRAPP II, is required for an in vitro ER-
to-Golgi transport assay (Sacher et al., 2001). TRAPP I binds
to COPII vesicles formed in vitro from permeabilised yeast
cells. This binding appears to be independent of other factors,
since it occurs even when the COPII vesicles are formed from
purified coat components in the absence of cytosol and Golgi.
Moreover, if Bet3p is depleted from both vesicles and Golgi,
no tethering occurs, which indicates that other factors are not
sufficient to tether vesicles in the absence of TRAPP I
(Barrowman et al., 2000). TRAPP II, in contrast, might be
required for a later transport step. A temperature-sensitive
mutant of a TRAPP II-specific subunit accumulates Golgi
forms of invertase and CPY, as well as aberrant Golgi
structures. Its subunits also show synthetic interactions with
mutants of ARF1 and components of COPI but not COPII
(Sacher et al., 2001).
Immobilised TRAPP I and TRAPP II both act as exchange
factors for the early Golgi Rab Ypt1p (Sacher et al., 2001), and
there is conflicting evidence about whether either acts as an
exchange factor for the late Golgi Rabs Ypt31p and Ypt32p
(Jones et al., 2000; Wang et al., 2000). It is also not clear
whether or how the Ypt1p exchange activity is regulated, and
why Uso1p (a long coiled-coil protein and Ypt1p effector) is
required in vitro to tether COPII vesicles to the Golgi (Cao et
al., 1998) when TRAPP I alone can bind to COPII vesicles.
Journal of Cell Science 115 (13)
Cog1
Cog2
Cog3
Cog4
Cog5
Cog6
Cog7
Cog8
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0100 300200 400 500 600 700 800 900 1000
..DGDRDLQEHRQRIQALAEETAQNLKRNVYQNYRQFIETAREISY-LESEMYQLSHLLTEQKS S LESIPLTLLPAA A A A G
..TSFEQLKMAVTNLKRQANK K SEGSLAYVKG G LSTFFEAQDALSAIHQKLEADGTEKVEG- - SMTQKLENVLNRASNTAD
..D D VEDRENEKGRLE E AYEKCDRDLDELIVQHYTELT T AIRTYQS-ITERITNSRNKIKQVKENLLSCKMLLHCKRDELR
..HGAEEIRGLERQVRAEIEHK K E E LRQMVGERYRDLIEA A DTIGQ-MR R CAVGLVDAVKATDQYCARLRQAGSAAPRP P R
..RKRVQLEELRD D LELY Y KLLKTAMVELINKDYADFVNLSTNLVG-MDKALNQLSVPLGQLRE E VLSLRS S VSEGIRAVD
..IHQAVIAEQLAKLAQGISQLDRELHLQVVARHEDL L AQATGIES-LEGVLQM M QTRIGALQGAVDRIKAKIVEPYNKIV
..SGLERLR R EPERLAE E RAQLLQ Q TRDLAFANYKTFIRGAECTER-IHRLFGDVEASLGRLLDRLPSFQQSCRNFVKEAE
..QSLANIDEV V NKIRLKIR R LDDNIRTVVRGQTNVGQDGRQALEE-AQKAIQQLFGKIKDIKDKAEKSEQMVKEITRDIK
..RDA A S S KLLQEKLSHYLDIVEVNIAHQISLRSEAFFHAMTSQHE-LQDYLRKTSQAVKMLRDKIAQIDKVMCEGSLHIL
Exo84 36
Sec5178
Sec835
Cog125
Cog237
Cog583
Cog856
Vps5357
Vps54198
..
..
..
..
..
..
..
..
..
Exo70
Exo84
Sec3
Sec5
Sec6
Sec8
Sec10
Sec15
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0.5
1.0
0.0
0100 300200 400 500 600 700 800 900 1000
A
B
Fig. 4. The presence of a shared domain in components of the COG complex and the exocyst. (A) Sequence alignment of the N-terminal
amphipathic helical regions of the indicated components of the human COG complex and exocyst. Residues are shaded if identical (black) or
conserved (grey) in at least three proteins. Grey bars show the regions predicted to form coiled-coil (the hydrophobic heptad repeat indicated by
black circles). (B) Prediction of the propensity of the subunits of the human COG complex and human exocyst to form coiled coils. The length
of the x-axis corresponds to the length of the proteins, with residue numbers indicated at the bottom of the figure. On the y-axis is plotted the
probability of a coiled-coil being at each residue of the protein, as determined by the algorithm of Lupas (Lupas, 1996) using the MacStripe
program (v2.0b1) with a window length of 28 residues, the MTIDK matrix and weighting of hydrophobic residues. Red bars indicate regions
that are aligned in (A). Blue bars indicate longer regions of sequence similarity between Cog3, Cog6 and Exo70 [see alignment in Whyte and
Munro (Whyte and Munro, 2001)].

2633Vesicle tethering complexes in membrane traffic
Perhaps the combined interactions are required for proper
tethering; binding of TRAPP I to vesicles could stimulate its
GEF activity, resulting in recruitment of Uso1p by Ypt1p and
additional binding of the vesicle by Uso1p.
At least seven of the TRAPP subunits are well conserved in
mammals and are present in a large complex (Gavin et al.,
2002; Sacher et al., 2000). The mammalian complex(es) has
not been extensively characterised but has been shown to
localise to the Golgi (Gecz et al., 2000; Sacher et al., 2000).
Mutations in the homologue of yeast Trs20p are responsible
for the human disease spondyloepiphyseal dysplasia tarda
(SEDL) (Gecz et al., 2000). The non-lethality of this X-linked
skeletal disorder might be caused by the presence of an
additional, autosomal, version of the SEDL gene that appears
to be a processed pseudogene but is nonetheless expressed.
The Class C Vps complex
The Class C Vps complex was identified through
characterisation of the many yeast mutants that show defects
in the sorting of proteins to the vacuole. Such vacuolar protein
sorting (vps) mutants have been classified on the basis of their
phenotypes, and Class C mutants are those that lack coherent
vacuoles altogether (Raymond et al., 1992). All four of the
mutants in this class (Pep3p, Pep5p, Vps16p and Vps33p) are
part of a very large (38S) complex that appears to function at
two distinct transport steps (Rieder and Emr, 1997). It was first
identified as being involved in fusion to the vacuole of both
transport vesicles and other vacuoles. At the vacuolar surface,
the Class C Vps complex has another two components: Vam2p
(Vps41p) and Vam6p (Vps39p). The latter is a GEF for the Rab
Ypt7p (Wurmser et al., 2000). The complex is also an effector
of Ypt7p (Seals et al., 2000) and also binds to the unpaired
vacuolar SNARE Vam3p. This binding is probably through
Vps33p, which is a member of the Sec1 (or Munc18) family
of SNARE-binding proteins (Jahn, 2000; Sato et al., 2000).
This has led to a model in which the Class C complex recruits
Vps39p to both the vacuole and incoming vesicles. Vps39p
activates Ypt7p, which in turn acts on the complex to promote
a tethering interaction. Inhibition of Vam3p is then relieved,
which allows trans SNARE complex formation and fusion to
proceed.
The components of the complex do not show any clear
homology to other known Rab GEFs or to other tethering
complexes. However, several of the components have a domain
related to the repeating structure of clathrin (Conibear and
Stevens, 1998) and also contain RING-H2 domains, which are
zinc-binding motifs that mediate a number of protein-protein
interactions. In yeast and Drosophila, mutations in the RING-
H2 domain abrogate function (Rieder and Emr, 1997;
Sevrioukov et al., 1999). In many other proteins, RING
domains serve to recruit ubiquitin-ligases (Borden and
Freemont, 1996; Joazeiro and Weissman, 2000), and given the
recent revelation of ubiquitin as a key sorting determinant in
the endocytic/vacuolar pathway, they might have a similar
function here (Hicke, 2001). Human homologues of Class C
Vps subunits have recently been characterised and found to be
in a complex localized on late endosomes/lysosomes (Caplan
et al., 2001; Kim et al., 2001a; McVey Ward et al., 2001).
The same complex also appears to function in Golgi-to-
endosome transport. This is indicated by genetic and physical
interactions between Class C mutants and genes encoding
proteins known to promote tethering and fusion at the
endosome (Peterson et al., 1999; Tall et al., 1999).
Furthermore, Class C mutants show allele-specific defects in
either Golgi-to-endosome or endosome-to-vacuole transport
(Peterson and Emr, 2001). Parallels with the vacuolar function
of the Class C complex have yet to be addressed. However, the
Class C Vps complex is not the only factor that has been
proposed to contribute to tethering in the endosomal system.
In mammalian cells the long coiled-coil protein EEA1 appears
to act as a tether during homotypic fusion of early endosomes
(Christoforidis et al., 1999). There is no clear homologue of
EEA1 in yeast, although it was initially proposed that Vac1p
(Pep7p) might be related, and both are effectors for Rab5
GTPase (Vps21p in yeast) (Peterson et al., 1999; Tall et al.,
1999). However a second Rab5-effector, Rabenosyn-5, appears
to be more related to Vac1p, although the latter has a RING
domain that is absent from the mammalian protein (Nielsen
et al., 2000). The function of Vac1p and Rabenosyn-5 is
unknown, but yeast lacking Vac1p show defects in delivery of
proteins from the Golgi to the endosome (Weisman and
Wickner, 1992). Vac1p interacts genetically and physically
with components of the Class C Vps complex, and so it is
possible that Vac1p and Rabenosyn-5 are involved in recruiting
the Class C Vps complex to endosomal membranes (Peterson
and Emr, 2001; Srivastava et al., 2000; Webb et al., 1997).
However both Vac1p and Rabenosyn-5 also bind to the Sec1-
related protein Vps45p (Burd et al., 1997; Nielsen et al., 2000).
Thus it is also possible that there are further proteins stably
associated with Vac1p or Rabenosyn-5, which form another
entirely distinct tethering complex that acts on endosomal
membranes.
Dsl1p complex
So far, there is no clearly described mechanism to tether Golgi-
derived vesicles to the ER in this vital recycling pathway. If
there is such a mechanism, it may well involve Dsl1p, a
peripheral membrane protein of the ER that is required for
retrograde traffic and binds to another peripheral membrane
protein, Tip20p (Andag et al., 2001; Reilly et al., 2001). Tip20p
is part of a complex that contains the SNARE-like membrane
protein Sec20p, and together they bind to the ER SNARE
Ufe1p (Lewis et al., 1997; Sweet and Pelham, 1993). It
therefore seems likely that a complex consisting of at least
Dsl1p-Tip20p-Sec20p exists and that it interacts with the
SNARE Ufe1p. Mutants of TIP20 are synthetically lethal in
combination with those of several subunits of the Golgi-to-ER
(COPI) vesicle coat (Frigerio, 1998), and Dsl1p interacts with
a COPI subunit in two-hybrid assays (Reilly et al., 2001).
Clarification of the function of the Dsl1p complex awaits
further investigation of its biochemical role and protein-protein
interactions, but interestingly Dsl1p has a short stretch of
predicted coiled coil near its N-terminus, in common with
components of quatrefoil tethering complexes.
Mechanistic ideas
An important but unresolved question is how tethering factors
promote fusion of the vesicle to the correct organelle. The
mechanism may be purely kinetic, with the vesicle being

2634
tethered within the vicinity of its destination and having an
increased probability of undergoing SNARE-mediated fusion.
Alternatively, the mechanism may be thermodynamic, the
tethering factors actively promoting SNARE-mediated fusion
in response to vesicle binding. This might be either by release
of inhibition of SNAREs or by activation of the SNAREs on
the vesicle and target (see below). Of course, both kinetic and
thermodynamic mechanisms could operate simultaneously,
mediated by the same or different sets of tethering factors. One
possibility is that the long, coiled-coil proteins are kinetic
tethers that passively hold the vesicle and do not need to
transduce signals about vesicle binding along their length,
whereas the multi-protein complexes are thermodynamic
tethers that actively promote interaction between vesicle and
target. Whether there is any cross-talk between the two classes
of tethering factor has yet to be explored.
Interactions with SNAREs
Some t-SNAREs possess, in addition to the core SNARE
domain, an N-terminal domain that can bind to the core domain
and thereby prevent its participation in a trans SNARE
complex. An attractive idea is that tethering complexes might
relieve this autoinhibition by promoting an open conformation
of the N-terminal domain relative to the core domain; recent
evidence supports the idea that tethering complexes bind to
such N-terminal domains although not necessarily that the
complexes thereby relieve autoinhibition.
Deletion of the N-terminal domain of Vam3p results in a
significant reduction in homotypic vacuolar fusion in yeast
and a significant reduction in the formation of trans SNARE
complexes (Laage and Ungermann, 2001). The Class C Vps
complex also binds much less efficiently to the truncated
Vam3p, which suggests that the reduction in fusion is caused
by an inability of the Class C Vps complex to promote trans
SNARE complex formation. The GARP complex provides
another example of the binding of a putative tethering complex
to an N-terminal SNARE domain, that of Tlg1p (Siniossoglou
and Pelham, 2001).
The N-terminal domain of the yeast plasma membrane t-
SNARE Sso1p is essential. However, a constitutively open
mutant of Sso1p is viable (Munson and Hughson, 2002). Thus
the requirement for growth is not that the N-terminal domain
be able to bind to the core domain, although the constitutively
open mutant does indeed form complexes with other SNAREs
more readily. This implies that the N-terminal domain has an
activating function as well as an inhibitory role. The activating
function could be provided by the binding of the exocyst or
another tethering factor, although there is currently no direct
evidence for this.
A variation on this idea is that tethering factors relieve the
inhibition of SNAREs by Sec1 family proteins (for details, see
Waters and Hughson, 2000), which in some cases hold the
SNARE N-terminal domain in a closed conformation. In
neuronal exocytosis, for instance, nSec1 binds to the
monomeric form of the SNARE syntaxin1A, preventing it from
interacting with its t-SNARE partners (Misura et al., 2000;
Yang et al., 2000). This sort of mechanism may be employed
by the Class C Vps complex, one subunit of which is a Sec1
homologue. As ever, the real situation may be more
complicated, since some Sec1 proteins might themselves have
an activating role in SNARE assembly (Carr and Novick, 2000;
Jahn, 2000).
Conclusion
The concept of tethering has grown around the realisation that
the specificity of membrane fusion is conferred by more than
just the SNAREs. The definition and ordering of events in
tethering will be a crucial advance. In this respect, it is useful
to consider where a tethering complex lies in relation to Rab
activation. The TRAPP complexes and the Class C Vps
complex lie upstream of Rab activation, whereas the quatrefoil
complexes lie downstream. This reaffirms the idea that the
complexes function differently. There could thus be two sorts
of tethering event. In the first, a large complex would respond
to a vesicle by activating a Rab, causing Rab effectors such as
long, coiled-coil proteins to tether the vesicle. TRAPP would
operate in this way, and its interactions with vesicles would
reflect the start of a signalling event rather than a stable tether.
The second sort of event in tethering would rely on large
complexes that are Rab effectors and fulfil some function that
promotes SNARE-mediated fusion. This function could
operate in parallel with long, coiled-coil effectors. The COG
complex, the GARP complex and the exocyst would operate in
this manner. Of course, the same tethering event could involve
a different large complex both before and after Rab activation
– for instance, TRAPP II and the COG complex in Golgi
recycling. The same complex could even act both upstream and
downstream of Rab activation, as described above for the Class
C Vps complex. The notion that tethering could involve
different mechanisms in different parts of the pathway would
account for the fact that, unlike Rabs and SNAREs, tethering
complexes and coiled-coil proteins are not ubiquitous in the
pathway and distinct protein complexes act in different places.
Whatever their exact functions turn out to be, rapid and
exciting progress is expected in this area.
We thank Elizabeth Conibear, Chris Kaiser, Vladimir Lupashin and
Gerry Waters for communication of results prior to publication, and
Alison Gillingham, Bernhard Sommer and the referees for helpful
comments on the manuscript.
References
Andag, U., Neumann, T. and Schmitt, H. D. (2001). The coatomer-
interacting protein Dsl1p is required for Golgi-to-endoplasmic reticulum
retrieval in yeast. J. Biol. Chem. 276, 39150-39160.
Barlowe, C. (1997). Coupled ER to Golgi transport reconstituted with purified
cytosolic proteins. J. Cell Biol. 139, 1097-1108.
Barr, F. A., Nakamura, N. and Warren, G. (1998). Mapping the interaction
between GRASP65 and GM130, components of a protein complex involved
in the stacking of Golgi cisternae. EMBO J. 17, 3258-3268.
Barrowman, J., Sacher, M. and Ferro-Novick, S. (2000). TRAPP stably
associates with the Golgi and is required for vesicle docking. EMBO J. 19,
862-869.
Bock, J. B., Matern, H. T., Peden, A. A. and Scheller, R. H. (2001). A
genomic perspective on membrane compartment organization. Nature 409,
839-841.
Borden, K. L. and Freemont, P. S. (1996). The RING finger domain: a recent
example of a sequence-structure family. Curr. Opin. Struct. Biol. 6, 395-401.
Brennwald, P., Kearns, B., Champion, K., Keranen, S., Bankaitis, V. and
Novick, P. (1994). Sec9 Is a SNAP-25-like component of a yeast SNARE
complex that may be the effector of Sec4 function In exocytosis. Cell 79,
245-258.
Broadie, K., Prokop, A., Bellen, H. J., O’Kane, C. J., Schulze, K. L. and
Journal of Cell Science 115 (13)

2635Vesicle tethering complexes in membrane traffic
Sweeney, S. T. (1995). Syntaxin and synaptobrevin function downstream of
vesicle docking in Drosophila. Neuron 15, 663-673.
Brymora, A., Valova, V. A., Larsen, M. R., Roufogalis, B. D. and Robinson,
P. J. (2001). The brain exocyst complex interacts with RalA in a GTP-
dependent manner: Identification of a novel mammalian Sec3 gene and a
second Sec15 gene. J. Biol. Chem. 13, 13.
Burd, C. G., Peterson, M., Cowles, C. R. and Emr, S. D. (1997). A novel
Sec18p/NSF-dependent complex required for Golgi-to- endosome transport
in yeast. Mol. Biol. Cell 8, 1089-1104.
Cao, X. C., Ballew, N. and Barlowe, C. (1998). Initial docking of ER-derived
vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.
EMBO J. 17, 2156-2165.
Caplan, S., Hartnell, L. M., Aguilar, R. C., Naslavsky, N. and Bonifacino,
J. S. (2001). Human Vam6p promotes lysosome clustering and fusion in
vivo. J. Cell Biol. 154, 109-122.
Carr, C. M. and Novick, P. J. (2000). Membrane fusion. Changing partners.
Nature 404, 347-349.
Chatterton, J. E., Hirsch, D., Schwartz, J. J., Bickel, P. E., Rosenberg, R.
D., Lodish, H. F. and Krieger, M. (1999). Expression cloning of LDLB, a
gene essential for normal Golgi function and assembly of the ldlCp complex.
Proc. Natl. Acad. Sci. USA 96, 915-920.
Chen, Y. A. and Scheller, R. H. (2001). SNARE-mediated membrane fusion.
Nat. Rev. Mol. Cell. Biol. 2, 98-106.
Christoforidis, S., McBride, H. M., Burgoyne, R. D. and Zerial, M. (1999).
The Rab5 effector EEA1 is a core component of endosome docking. Nature
397, 621-625.
Conboy, M. J. and Cyert, M. S. (2000). Luv1p/Rki1p/Tcs3p/Vps54p, a yeast
protein that localizes to the late Golgi and early endosome, is required for
normal vacuolar morphology. Mol. Biol. Cell 11, 2429-2443.
Conibear, E. and Stevens, T. H. (1998). Multiple sorting pathways between
the late Golgi and the vacuole in yeast. Biochim. Biophys. Acta 1404, 211-
230.
Conibear, E. and Stevens, T. H. (2000). Vps52p, Vps53p, and Vps54p form
a novel multisubunit complex required for protein sorting at the yeast late
Golgi. Mol. Biol. Cell 11, 305-323.
Conibear, E. and Stevens, T. H. (2001). The GARP complex is required for
retrograde transport from both early and late endosomes to the Golgi. Yeast
18, S135-S135.
Dumas, J. J., Merithew, E., Sudharshan, E., Rajamani, D., Hayes, S.,
Lawe, D., Corvera, S. and Lambright, D. G. (2001). Multivalent
endosome targeting by homodimeric EEA1. Mol. Cell 8, 947-958.
Fasshauer, D., Antonin, W., Subramaniam, V. and Jahn, R. (2002). SNARE
assembly and disassembly exhibit a pronounced hysteresis. Nat. Struct. Biol.
9, 144-151.
Finger, F. P. and Novick, P. (1997). Sec3p is involved in secretion and
morphogenesis in Saccharomyces cerevisiae. Mol. Biol. Cell 8, 647-662.
Finger, F. P., Hughes, T. E. and Novick, P. (1998). Sec3p is a spatial landmark
for polarized secretion in budding yeast. Cell 92, 559-571.
Frigerio, G. (1998). The Saccharomyces cerevisiae early secretion mutant
tip20 is synthetic lethal with mutants in yeast coatomer and the SNARE
proteins Sec22p and Ufelp. Yeast 14, 633-646.
Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A.,
Schultz, J., Rick, J. M., Michon, A. M., Cruciat, C. M. et al. (2002).
Functional organization of the yeast proteome by systematic analysis of
protein complexes. Nature 415, 141-147.
Gecz, J., Hillman, M. A., Gedeon, A. K., Cox, T. C., Baker, E. and Mulley,
J. C. (2000). Gene structure and expression study of the SEDL gene for
spondyloepiphyseal dysplasia tarda. Genomics 69, 242-251.
Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C.,
Rodriguez-Boulan, E., Scheller, R. H. and Nelson, W. J. (1998). Sec6/8
complex is recruited to cell-cell contacts and specifies transport vesicle
delivery to the basal-lateral membrane in epithelial cells. Cell 93, 731-740.
Guo, W., Grant, A. and Novick, P. (1999a). Exo84p is an exocyst protein
essential for secretion. J. Biol. Chem. 274, 23558-23564.
Guo, W., Roth, D., Walch-Solimena, C. and Novick, P. (1999b). The exocyst
is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis.
EMBO J. 18, 1071-1080.
Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S. and Novick, P.
(2000). Protein complexes in transport vesicle targeting. Trends Cell Biol.
10, 251-255.
Guo, W., Tamanoi, F. and Novick, P. (2001). Spatial regulation of the exocyst
complex by Rho1 GTPase. Nat. Cell Biol. 3, 353-360.
Haarer, B. K., Corbett, A., Kweon, Y., Petzold, A. S., Silver, P. and Brown,
S. S. (1996). SEC3 mutations are synthetically lethal with profilin mutations
and cause defects in diploid-specific bud-site selection. Genetics 144, 495-
510.
Hicke, L. (2001). A new ticket for entry into budding vesicles – ubiquitin. Cell
106, 527-530.
Hsu, S. C., Hazuka, C. D., Roth, R., Foletti, D. L., Heuser, J. and Scheller,
R. H. (1998). Subunit composition, protein interactions, and structures of
the mammalian brain sec6/8 complex and septin filaments. Neuron 20, 1111-
1122.
Hsu, S. C., Ting, A. E., Hazuka, C. D., Davanger, S., Kenny, J. W., Kee, Y.
and Scheller, R. H. (1996). The mammalian brain rsec6/8 complex. Neuron
17, 1209-1219.
Hunt, J. M., Bommert, K., Charlton, M. P., Kistner, A., Habermann, E.,
Augustine, G. J. and Betz, H. (1994). A post-docking role for
synaptobrevin in synaptic vesicle fusion. Neuron 12, 1269-1279.
Jahn, R. (2000). Sec1/Munc18 proteins: mediators of membrane fusion
moving to center stage. Neuron 27, 201-204.
Joazeiro, C. A. and Weissman, A. M. (2000). RING finger proteins:
mediators of ubiquitin ligase activity. Cell 102, 549-552.
Jones, S., Newman, C., Liu, F. L. and Segev, N. (2000). The TRAPP complex
is a nucleotide exchanger for Ypt1 and Ypt31/32. Mol. Biol. Cell 11, 4403-
4411.
Karpova, T. S., Reck-Peterson, S. L., Elkind, N. B., Mooseker, M. S.,
Novick, P. J. and Cooper, J. A. (2000). Role of actin and Myo2p in
polarized secretion and growth of Saccharomyces cerevisiae. Mol. Biol. Cell
11, 1727-1737.
Kee, Y., Yoo, J. S., Hazuka, C. D., Peterson, K. E., Hsu, S. C. and Scheller,
R. H. (1997). Subunit structure of the mammalian exocyst complex. Proc.
Natl. Acad. Sci. USA 94, 14438-14443.
Kim, D. W., Sacher, M., Scarpa, A., Quinn, A. M. and Ferro-Novick, S.
(1999). High-copy suppressor analysis reveals a physical interaction
between Sec34p and Sec35p, a protein implicated in vesicle docking. Mol.
Biol. Cell 10, 3317-3329.
Kim, B. Y., Kramer, H., Yamamoto, A., Kominami, E., Kohsaka, S. and
Akazawa, C. (2001a). Molecular characterization of mammalian
homologues of class C Vps proteins that interact with syntaxin-7. J. Biol.
Chem. 276, 29393-29402.
Kim, D. W., Massey, T., Sacher, M., Pypaert, M. and Ferro-Novick, S.
(2001b). Sgf1p, a new component of the Sec34p/Sec35p complex. Traffic 2,
820-830.
Laage, R. and Ungermann, C. (2001). The N-terminal domain of the t-
SNARE Vam3p coordinates priming and docking in yeast vacuole fusion.
Mol. Biol. Cell 12, 3375-3385.
Lewis, M. J., Rayner, J. C. and Pelham, H. R. B. (1997). A novel SNARE
complex implicated in vesicle fusion with the endoplasmic reticulum.
EMBO J. 16, 3017-3024.
Linstedt, A. D., Jesch, S. A., Mehta, A., Lee, T. H., Garcia-Mata, R.,
Nelson, D. S. and Sztul, E. (2000). Binding relationships of membrane
tethering components – The giantin N terminus and the GM130 N terminus
compete for binding to the p115 C terminus. J. Biol. Chem. 275, 10196-
10201.
Lipschutz, J. H., Guo, W., O’Brien, L. E., Nguyen, Y. H., Novick, P. and
Mostov, K. E. (2000). Exocyst is involved in cystogenesis and tubulogenesis
and acts by modulating synthesis and delivery of basolateral plasma
membrane and secretory proteins. Mol. Biol. Cell 11, 4259-4275.
Loh, E. and Hong, W. (2002). Sec34 is implicated in traffic from the
endoplasmic reticulum to the Golgi and exists in a complex with GTC-90
and ldlBp. J. Biol. Chem. (in press).
Lowe, M. (2000). Membrane transport: Tethers and TRAPPs. Curr. Biol. 10,
R407-R409.
Lupas, A. (1996). Prediction and analysis of coiled-coil structures. Methods
Enzymol 266, 513-525.
McNew, J. A., Parlati, F., Fukuda, R., Johnston, R. J., Paz, K., Paumet,
F., Sollner, T. H. and Rothman, J. E. (2000). Compartmental specificity
of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153-
159.
McVey Ward, D., Radisky, D., Scullion, M. A., Tuttle, M. S., Vaughn, M.
and Kaplan, J. (2001). hVPS41 is expressed in multiple isoforms and can
associate with vesicles through a RING-H2 finger motif. Exp. Cell Res. 267,
126-134.
Misura, K. M., Scheller, R. H. and Weis, W. I. (2000). Three-dimensional
structure of the neuronal-Sec1-syntaxin 1a complex. Nature 404, 355-362.
Morsomme, P. and Riezman, H. (2002). The Rab GTPase Ypt1p and
tethering factors couple protein sorting at the ER to vesicle targeting to the
Golgi apparatus. Dev. Cell 2, 307-317.

2636
Moskalenko, S., Henry, D. O., Rosse, C., Mirey, G., Camonis, J. H. and
White, M. A. (2002). The exocyst is a Ral effector complex. Nat. Cell Biol.
4, 66-72.
Muller, O., Bayer, M. J., Peters, C., Andersen, J. S., Mann, M. and Mayer,
A. (2002). The Vtc proteins in vacuole fusion: coupling NSF activity to V(0)
trans-complex formation. EMBO J. 21, 259-269.
Munson, M. and Hughson, F. M. (2002). Conformational regulation
of SNARE assembly and disassembly in vivo. J. Biol. Chem. 277, 9375-
9381.
Nielsen, E., Christoforidis, S., Uttenweiler-Joseph, S., Miaczynska, M.,
Dewitte, F., Wilm, M., Hoflack, B. and Zerial, M. (2000). Rabenosyn-5,
a novel Rab5 effector, is complexed with hVPS45 and recruited to
endosomes through a FYVE finger domain. J. Cell Biol. 151, 601-612.
Parlati, F., McNew, J. A., Fukuda, R., Miller, R., Sollner, T. H. and
Rothman, J. E. (2000). Topological restriction of SNARE-dependent
membrane fusion. Nature 407, 194-198.
Pelham, H. R. B. (2001). SNAREs and the specificity of membrane fusion.
Trends Cell Biol. 11, 99-101.
Peterson, M. R., Burd, C. G. and Emr, S. D. (1999). Vac1p coordinates Rab
and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle
docking/fusion at the endosome. Curr. Biol. 9, 159-162.
Peterson, M. R. and Emr, S. D. (2001). The Class C Vps complex functions
at multiple stages of the vacuolar transport pathway. Traffic 2, 476-486.
Podos, S. D., Reddy, P., Ashkenas, J. and Krieger, M. (1994). LDLC encodes
a brefeldin A-sensitive, peripheral Golgi protein required for normal Golgi
function. J. Cell Biol. 127, 679-691.
Pruyne, D. and Bretscher, A. (2000). Polarization of cell growth in yeast. J.
Cell Sci. 113, 571-585.
Radisky, D. C., Snyder, W. B., Emr, S. D. and Kaplan, J. (1997).
Characterization of VPS41, a gene required for vacuolar trafficking and
high-affinity iron transport in yeast. Proc. Natl. Acad. Sci. USA 94, 5662-
5666.
Ram, R. J., Li, B. and Kaiser, C. (2002). Identification of Sec36p, Sec37p
and Sec38p: components of the yeast complex that contains Sec34p and
Sec35p. Mol. Biol. Cell. 13, 1484-1500.
Raymond, C. K., Howaldstevenson, I., Vater, C. A. and Stevens, T. H.
(1992). Morphological classification of the yeast vacuolar protein sorting
mutants – evidence for a prevacuolar compartment in class-E vps mutants.
Mol. Biol. Cell 3, 1389-1402.
Reilly, B. A., Kraynack, B. A., VanRheenen, S. M. and Waters, M. G.
(2001). Golgi-to-endoplasmic reticulum (ER) retrograde traffic in yeast
requires Dsl1p, a component of the ER target site that interacts with a COPI
coat subunit. Mol. Biol. Cell 12, 3783-3796.
Rieder, S. E. and Emr, S. D. (1997). A novel RING finger protein complex
essential for a late step in protein transport to the yeast vacuole. Mol. Biol.
Cell 8, 2307-2327.
Sacher, M., Jiang, Y., Barrowman, J., Scarpa, A., Burston, J., Zhang, L.,
Schieltz, D., Yates, J. R., Abeliovich, H. and Ferro-Novick, S. (1998).
TRAPP, a highly conserved novel complex on the cis-Golgi that mediates
vesicle docking and fusion. EMBO J. 17, 2494-2503.
Sacher, M., Barrowman, J., Schieltz, D., Yates, J. R. and Ferro-Novick, S.
(2000). Identification and characterization of five new subunits of TRAPP.
Eur. J. Cell Biol. 79, 71-80.
Sacher, M., Barrowman, J., Wang, W., Horecka, J., Zhang, Y., Pypaert,
M. and Ferro-Novick, S. (2001). TRAPP I implicated in the specificity of
tethering in ER-to-Golgi transport. Mol. Cell 7, 433-442.
Sapperstein, S. K., Walter, D. M., Grosvenor, A. R., Heuser, J. E. and
Waters, M. G. (1995). p115 is a general vesicular transport factor related
to the yeast endoplasmic reticulum to Golgi transport factor Uso1p. Proc.
Natl. Acad. Sci. USA 92, 522-526.
Sato, T. K., Rehling, P., Peterson, M. R. and Emr, S. D. (2000). Class C Vps
protein complex regulates vacuolar SNARE pairing and is required for
vesicle docking/fusion. Mol. Cell 6, 661-671.
Seals, D. F., Eitzen, G., Margolis, N., Wickner, W. T. and Price, A. (2000).
A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is
required for homotypic vacuole fusion. Proc. Natl. Acad. Sci. USA 97, 9402-
9407.
Seemann, J., Jokitalo, E. J. and Warren, G. (2000). The role of the tethering
proteins p115 and GM130 in transport through the Golgi apparatus in vivo.
Mol. Biol. Cell 11, 635-645.
Sevrioukov, E. A., He, J. P., Moghrabi, N., Sunio, A. and Kramer, H.
(1999). A role for the deep orange and carnation eye color genes in
lysosomal delivery in Drosophila. Mol. Cell 4, 479-486.
Shin, D. M., Zhao, X. S., Zeng, W., Mozhayeva, M. and Muallem, S. (2000).
The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes
and regulates their activity. J. Cell Biol. 150, 1101-1112.
Short, B. and Barr, F. A. (2002). Membrane traffic: exocyst III – makes a
family. Curr. Biol. 12, R18-R20.
Siniossoglou, S. and Pelham, H. R. (2001). An effector of Ypt6p binds the
SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi
membranes. EMBO J. 20, 5991-5998.
Sonnichsen, B., Lowe, M., Levine, T., Jamsa, E., Dirac-Svejstrup, B. and
Warren, G. (1998). Role for giantin in docking COPI vesicles to Golgi
membranes. J. Cell Biol. 140, 1013-1021.
Spelbrink, R. G. and Nothwehr, S. F. (1999). The yeast GRD20 gene is
required for protein sorting in the trans-Golgi network/endosomal system
and for polarization of the actin cytoskeleton. Mol. Biol. Cell 10, 4263-4281.
Srivastava, A., Woolford, C. A. and Jones, E. W. (2000). Pep3p/Pep5p
complex: a putative docking factor at multiple steps of vesicular transport
to the vacuole of Saccharomyces cerevisiae. Genetics 156, 105-122.
Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K. and Ohta,
Y. (2002). The exocyst complex binds the small GTPase RalA to mediate
filopodia formation. Nat. Cell Biol. 4, 73-78.
Suvorova, E. S., Duden, R. and Lupashin, V. V. (2002). The Sec34/Sec35p
complex, a Ypt1p effector required for retrograde intra-Golgi trafficking,
interacts with Golgi SNAREs and COPI vesicle coat proteins. J. Cell Biol.
157, 631-643.
Suvorova, E. S., Kurten, R. C. and Lupashin, V. V. (2001). Identification of
a human orthologue of Sec34p as a component of the cis-Golgi vesicle
tethering machinery. J. Biol. Chem. 276, 22810-22818.
Sweet, D. J. and Pelham, H. R. (1993). The TIP1 gene of Saccharomyces
cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the
cytoplasmic domain of Sec20p. EMBO J. 12, 2831-2840.
Tall, G. G., Hama, H., DeWald, D. B. and Horazdovsky, B. F. (1999). The
phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab
GTPase and a Sec1p homologue to facilitate vesicle-mediated vacuolar
protein sorting. Mol. Biol. Cell 10, 1873-1889.
TerBush, D. R., Maurice, T., Roth, D. and Novick, P. (1996). The Exocyst
is a multiprotein complex required for exocytosis in Saccharomyces
cerevisiae. EMBO J. 15, 6483-6494.
Ungar, D., Oka, T., Brittle, E. E., Vasile, E., Lupashin, V. V., Chatterton,
J. E., Heuser, J. E., Krieger, M. and Waters, M. G. (2002).
Characterization of a mammalian Golgi-localized protein complex, COG,
that is required for normal Golgi morphology and function. J. Cell Biol. 157,
405-415.
VanRheenen, S. M., Cao, X., Lupashin, V. V., Barlowe, C. and Waters, M.
G. (1998). Sec35p, a novel peripheral membrane protein, is required for ER
to Golgi vesicle docking. J. Cell Biol. 141, 1107-1119.
VanRheenen, S. M., Cao, X., Sapperstein, S. K., Chiang, E. C., Lupashin,
V. V., Barlowe, C. and Waters, M. G. (1999). Sec34p, a protein required
for vesicle tethering to the yeast Golgi apparatus, is in a complex with
Sec35p. J. Cell Biol. 147, 729-742.
Vega, I. E. and Hsu, S. C. (2001). The exocyst complex associates with
microtubules to mediate vesicle targeting and neurite outgrowth. J. Neurosci.
21, 3839-3848.
Walter, D. M., Paul, K. S. and Waters, M. G. (1998). Purification and
characterization of a novel 13 S hetero-oligomeric protein complex that
stimulates in vitro Golgi transport. J. Biol. Chem. 273, 29565-29576.
Wang, W., Sacher, M. and Ferro-Novick, S. (2000). TRAPP stimulates
guanine nucleotide exchange on Ypt1p. J. Cell Biol. 151, 289-295.
Waters, M. G. and Hughson, F. M. (2000). Membrane tethering and fusion
in the secretory and endocytic pathways. Traffic 1, 588-597.
Webb, G. C., Zhang, J., Garlow, S. J., Wesp, A., Riezman, H. and Jones,
E. W. (1997). Pep7p provides a novel protein that functions in vesicle-
mediated transport between the yeast Golgi and endosome. Mol. Biol. Cell
8, 871-895.
Weisman, L. S. and Wickner, W. (1992). Molecular characterization of
VAC1, a gene required for vacuole inheritance and vacuole protein sorting.
J. Biol. Chem. 267, 618-623.
Whyte, J. R. and Munro, S. (2001). The Sec34/35 Golgi transport complex
is related to the exocyst, defining a family of complexes involved in multiple
steps of membrane traffic. Dev. Cell 1, 527-537.
Wuestehube, L. J., Duden, R., Eun, A., Hamamoto, S., Korn, P., Ram, R.
and Schekman, R. (1996). New mutants of Saccharomyces cerevisiae
affected in the transport of proteins from the endoplasmic reticulum to the
Golgi complex. Genetics 142, 393-406.
Wurmser, A. E., Sato, T. K. and Emr, S. D. (2000). New component of the
vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7
Journal of Cell Science 115 (13)

2637Vesicle tethering complexes in membrane traffic
GTPase to SNARE-dependent docking and fusion. J. Cell Biol. 151, 551-
562.
Yamakawa, H., Seog, D. H., Yoda, K., Yamasaki, M. and Wakabayashi, T.
(1996). Uso1 protein is a dimer with two globular heads and a long coiled-
coil tail. J. Struct. Biol. 116, 356-365.
Yang, B., Steegmaier, M., Gonzalez, L. C., Jr and Scheller, R. H. (2000).
nSec1 binds a closed conformation of syntaxin1A. J. Cell Biol. 148, 247-
252.
Yeaman, C., Grindstaff, K. K., Wright, J. R. and Nelson, W. J. (2001).
Sec6/8 complexes on trans-Golgi network and plasma membrane regulate
late stages of exocytosis in mammalian cells. J. Cell Biol. 155, 593-604.
Zerial, M. and McBride, H. (2001). Rab proteins as membrane organizers.
Nat. Rev. Mol. Cell. Biol. 2, 107-117.
Zhang, X., Bi, E., Novick, P., Du, L., Kozminski, K. G., Lipschutz, J. H.
and Guo, W. (2001). Cdc42 interacts with the exocyst and regulates
polarized secretion. J. Biol. Chem. 276, 46745-46750.