MembraneTrafficking:Tethering the assembly of SNARE complexes. By Wan Jin Hong and Sima Lev. The fusion of transport vesicles with the irtargetmem-branes is fundamental for intracellula..

1. Special Issue: Membrane Trafficking
Tethering the assembly of SNARE
complexes
WanJin Hong1,2
and Sima Lev3
1
School of Pharmaceutical Sciences, Xiamen University, Xiamen, People’s Republic of China
2
Institute of Molecular and Cell Biology, Singapore
3
Molecular Cell Biology Department, Weizmann Institute of Science, Rehovot 76100, Israel
The fusion of transport vesicles with their target mem-
branes is fundamental for intracellular membrane traf-
ficking and diverse physiological processes and is driven
by the assembly of functional soluble N-ethylmaleimide-
sensitive factor attachment protein receptor (SNARE)
complexes. Prior to fusion, transport vesicles are physi-
cally linked to their target membranes by various teth-
ering factors. Recent studies suggest that tethering
factors also positively regulate the assembly of function-
al SNARE complexes, thereby coupling tethering with
fusion events. This coupling is mediated, at least in part,
by direct physical interactions between tethering fac-
tors, SNAREs, and Sec1/Munc18 (SM) proteins. In this
review we summarize recent progress in understanding
the roles of tethering factors in the assembly of specific
and functional SNARE complexes driving membrane-
fusion events.
Membrane fusion
Membrane fusion along the secretory and endocytic path-
ways is mediated by a large group of membrane-bound
proteins known as SNAREs. All SNAREs contain a char-
acteristic coiled-coil SNARE motif of approximately 70
amino acids comprising heptad repeats and can function
on either target membranes (t-SNAREs) or transport vesi-
cles (v-SNAREs). The interaction of cognate v- and t-
SNAREs leads to the formation of a trans-SNARE complex
or SNAREpin, in which four SNARE motifs assemble as a
twisted parallel four-helix bundle that brings the opposing
membranes together and eventually catalyzes membrane
fusion [1,2]. Following fusion, the remaining SNARE com-
plex on the fused membranes is referred to as a cis-SNARE
complex, which undergoes disassembly catalyzed by
ATPase N-ethylmaleimide-sensitive fusion protein (NSF)
and its cofactor soluble NSF attachment protein (SNAP) [3]
to recycle the SNAREs for a new fusion event [4].
Pairing of distinct SNAREs and subsequent assembly of
functional SNAREpins are employed by different mem-
brane-trafficking events [5]. In many cases, a single
SNARE can be assembled into more than one SNAREpin
and thus can regulate several fusion events [6,7]. Hence,
the assembly of a functional SNAREpin is remarkably
important for both membrane fusion specificity and coor-
dinating different membrane-trafficking pathways. Recon-
stitution studies suggest that numerous SNARE
complexes can be formed in vitro, but only a few can
functionally drive membrane fusion [8,9]. Furthermore,
the assembly of a functional SNAREpin occurs in a step-
wise manner and is attributed to the formation of SNARE
complex intermediates, such as t-SNARE subcomplexes on
the target membranes that provide templates for v-
SNARE binding [7]. These findings raise several critical
questions. What are the mechanisms that regulate the
assembly of specific SNAREpins? How is the promiscuous
assembly of nonfunctional SNARE complexes prevented in
intact cells? How are the intermediate steps of the assem-
bly process regulated?
Numerous studies have shown that membrane-fusion
specificity is largely dependent on SNARE proteins, be-
cause different SNAREpins can catalyze distinct fusion
events [1,6]. Recent studies, however, suggest that both
SM proteins and tethering factors play an active role in
SNAREpin formation and thus contribute to the specificity
of the fusion as well as to its speed and high fidelity [10–
15]. Tethering factors appear to influence SNAREpin as-
sembly in several different ways including stabilization of
SNARE proteins or the entire SNARE complex, gathering
of t-SNAREs on the target membranes, or activating the
assembly process by interacting with SM proteins. The
direct physical interactions of tethering factors with
SNAREs and SM proteins mediate many of these func-
tions, as will be discussed in this review. Tethering factors
also interact with Rab GTPases and vesicle coats and are
believed to bridge the transport vesicles with their target
membranes [16,17], thereby functionally coupling tether-
ing with fusion events. This review summarizes recent
advances in understanding the role of tethering factors
in the assembly of functional SNARE complexes.
SNARE: general features, classification, and complexes
The SNARE hypothesis was first introduced in 1993 [3]
and suggested that a v-SNARE on the vesicle pairs with
cognate t-SNAREs on the target membrane to form a
complex that not only determines the specificity of the
Review
0962-8924/$ – see front matter
ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.09.006
Corresponding author: Lev, S. (Sima.Lev@weizmann.ac.il).
Keywords: SNAREs; SM proteins; tethering factors; SNAREpin; membrane fusion.
Trends in Cell Biology, January 2014, Vol. 24, No. 1 35

2. fusion but also catalyzes the fusion process. Since then
many SNAREs have been identified, of which 38 are known
to exist in humans [6,18] (Figure 1). Most SNAREs contain
one SNARE motif, whereas four SNAREs (SNAP-23, SNAP-
25, SNAP-29/GS32, and SNAP-47) contain two SNARE
motifs [6,18–20] (Figure 1). Initially, SNAREs were classi-
fied as v- or t-SNAREs according to their subcellular locali-
zation. However, many SNAREs can be found on both
vesicles and target membranes; therefore, an alternative
classification based on the crystal structure of the synaptic
SNARE complex (Syntaxin [Stx]1A, synaptobrevin2, and
Synaptosome-associated protein [SNAP]-25B) has been for-
mulated [21]. The crystal structure revealed that an ionic
layer comprising an arginine (R) (contributed by synapto-
brevin) and three glutamine (Q) residues (contributed by
Stx1A and SNAP-25) is present in the central position of the
four-helix bundle. These findings led to the structural clas-
sification of R- and Q-SNAREs [22] and suggested that a
functional SNARE complex generally comprises three Q-
SNARE motifs and one R-SNARE motif. Most R-SNAREs
act as v-SNAREs and most Q-SNAREs act as t-SNAREs.
However, there are exceptions: the R-SNAREs Ykt6 and
Sec22B act as part of the t-SNARE subcomplexes, whereas
GS15, Bet1, and Slt1 are Q-SNAREs that function as v-
SNAREs. In addition to the SNARE motif, most SNAREs
contain a C-terminal hydrophobic transmembrane domain
(TM) (Figure 1). Seven of the 38 SNAREs do not have a TM
domain and instead associate with membranes by various
lipid modifications. SNAP-23, SNAP-25, SNAP-29,
SNAP47, Stx9/19, and Stx11 are palmitoylated at multiple
cysteineresidues, whereasYkt6 containsa C-terminalmotif
that mediates its prenylation (farnesylation) and subse-
quent palmitoylation [23]. Moreover, many SNAREs con-
tain N-terminal regulatory regions preceding the SNARE
motif (Figure 1). These N-terminal regions regulate impor-
tant properties of SNAREs. For example, the short N-ter-
minal peptide of Stx1, Stx5, and Stx16 interacts with SM
proteins [6]. The Habc region ofStx16folds intoa three-helix
bundle and intramolecularily interacts with the SNARE
motif, thereby preventing its assembly into a SNARE com-
plex [24]. Similarly, the longin domain of Vamp7, an
R-SNARE involved in both endocytic and secretory path-
ways, interacts with its SNARE motif via hydrophobic inter-
actions [25]. The N-terminal extension of VAMP4 contains a
dileucine motif and acidic clusters that mediate its recycling
fromtheendosometothetrans-Golginetwork(TGN)[26,27].
Because the formation of a functional SNAREpin is the
key driver for membrane fusion, identification of functional
SNARE complexes in vivo has been an important area of
SNARE research. The major SNARE complexes function-
ing in the secretory and endocytic pathways are shown in
Figure 2 [6]. As can be seen, most SNAREpins contain at
least one t-SNARE belonging to the Stx family, and a given
SNARE can participate in several distinct SNARE com-
plexes. For example, Stx5 is present in at least three
different SNAREpins regulating Golgi trafficking and thus
can be envisioned as the master SNARE of the Golgi
apparatus [28].
Regulation of SNAREpin assembly and function
Given that SNAREpins mediate fusion events, it is no
surprise that several mechanisms regulate their formation
and/or functionality. Below we discuss the role of SM
proteins and tethering factors in SNAREpin assembly.
SM proteins
SM proteins are evolutionarily conserved soluble periph-
eral membrane proteins of 60–90 kDa. Four major classes
of SM proteins have been identified in mammals: Sly1,
VPS45, VPS33 (VPS33A, VPS33B), and Munc18 (Munc18-
1, Munc18-2, and Munc18-3) [6,10]. SM proteins are con-
sidered key universal components of the fusion machinery.
They function in distinct intracellular transport steps and
physically interact with different SNARE proteins
(Figure 2) [7,10,29]. Sly1, for example, functions at the
endoplasmic reticulum (ER)–Golgi, Vps45 at the endo-
some–TGN, Vps33 at the endocytic/lysosomal system,
and Muc18 at the plasma membrane (Figure 2). In vitro
reconstitution studies convincingly showed that SM pro-
teins strongly accelerate the rate of SNARE-mediated
fusion and contribute to the specificity of various fusion
events [11,30,31]. Indeed, SM proteins are required for all
Stx1, 2, 3, 4, 5, 7, 12/13, 16, 17, 18Ha SNARE moƟfHb Hc TMN-Pep
VƟ1a, VƟ1b, GS27, GS28, Stx6, 8, 10Ha SNARE moƟfHb Hc TM
VAMP7, Sec22b, Sec22a, Sec22cSNARE moƟfLongin TM
Slt1/Use1, Sec20
SNARE moƟf TM VAMP1, 2, 3, 5, 8, GS15, Bet1
Ha SNARE moƟfHb HcN-Pep
SNARE moƟfLongin
VAMP4SNARE moƟfTGN S TM
SNARE moƟf TMUnknown
SNAP-23, 25, 29, 47 (mulƟple Cys for palmitoylaƟon)
Ykt6 (C-terminal prenylaƟon and palmitoylaƟon)
Stx9/19, 11 (mulƟple Cys for palmitoylaƟon)
SNARE moƟfSNARE MoƟf
TRENDS in Cell Biology
Figure 1. Domain organization of human soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The indicated SNAREs along with their
corresponding domains are shown. Abbreviation: TM, transmembrane domain.
Review Trends in Cell Biology January 2014, Vol. 24, No. 1
36

3. SNARE-mediated fusion events and recent studies on
Vps33 suggest that SM proteins promote the opening of
a fusion pore by enhancing the activity of a SNARE com-
plex [32].
SM proteins can bind individual SNAREs or SNARE
complexes and regulate SNAREpin assembly [10]. Sly1
binds to a short, evolutionarily conserved N-terminal pep-
tide of the mammalian t-SNAREs Stx5 and Stx18 and their
yeast homologs Sed5p and Ufe1p, respectively [33]. Vps45
similarly interacts with the N-terminal peptide of Stx16
(Tlg2p in yeast) and regulates endosome-to-TGN retro-
grade trafficking [34]. Interestingly, Vps45p also interacts
with the v-SNARE Snc2p, which participates in SNARE
complexes containing Tlg2p in yeast. Hence, Vps45p can
bind Tlg2p-containing SNARE complexes independent of
its Tlg2p binding [35], suggesting that SM proteins can
interact with their cognate SNAREs through distinct
mechanisms and at different stages in the SNARE assem-
bly/disassembly cycle. Consistent with the multiple modes
of binding, yeast Sec1p interacts with a fully assembled
exocytic SNARE complex [36]. Mammalian Munc18-1 pro-
tein, however, binds the t-SNARE Stx1 in a closed confor-
mation [37,38], in which an intramolecular interaction
between the N-terminal Habc domain of Stx1 and its
SNARE domain preclude its assembly into a SNARE
complex. Nevertheless, Munc18-1 stimulates SNARE-me-
diated fusion in vitro. It was proposed that Munc18-1
facilitates functional SNAREpin assembly by interacting
with the preassembled Stx1–SNAP25 t-SNARE subcom-
plex, which serves as a platform for VAMP2 binding [31].
The different modes of interaction between SM proteins
and SNAREs demonstrate the important regulatory roles
of SM proteins in SNAREpin assembly. Indeed, in vitro
binding assays suggested that the interaction of SM pro-
teins with SNAREs prevents the formation of non-physio-
logical SNARE complexes and stimulates specific SNARE
pairing [39]. For additional information on SM proteins
and their mechanisms of action, see these recent reviews
[40,41].
Tethering factors
Tethering factors are a large group of proteins or multiple-
protein complexes that link transport vesicles to their
cognate target membranes. As such, they regulate funda-
mental cellular processes and are implicated in various
diseases (Box 1). Tethering factors can be divided into two
major groups: homodimeric long coiled-coil proteins and
multisubunit tethering complexes (MTCs) [16,42–44].
Coiled-coil tethers are large, hydrophilic, dimeric proteins
comprising two globular heads connected by long coiled-
coil domains that can interact with vesicles over distances
of more than 200 nm [42]. MTCs, which contain three to ten
Bet1
Stx5
GS28
Ykt6
GS15
Stx5
GS28
Ykt6
Slt1
Stx18
Sec20
Sec22b
Vamp7
Stx7
VƟ1b
Stx8
Bet1
Stx5
GS27
Sec22b
ER
ERGIC
PM
cis-Golgi
Golgi stack
Vamp2
Stx1
SNAP-25
Vamp8
Stx4
SNAP-23
Recycling
endosome
TGN
MVB
Lysosome
Vamp2/3
Stx13
SNAP-25/29
Vamp8
Stx7
VƟ1b
Stx8
Early
endosome
Vamp4
Stx6
Stx16
VƟ1a
Munc18
Sly1
Vps45
Vps33
Vps33
Sly1
Sly1
B
TRENDS in Cell Biology
SynapƟc
vesicles
Secretory
granule
Figure 2. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes along the secretory and endocytic pathways in mammals. Schematic
summary of known mammalian SNARE complexes and their sites of action along the exocytic and endocytic pathways. Potential v-SNAREs are indicated in red; the Sec1/
Munc18 SM proteins for some SNARE complexes are marked in purple. Abbreviations: ER, endoplasmic reticulum; ERGIC, ER–Golgi intermediate compartment; MVBs,
multivesicular bodies; PM, plasma membrane; TGN, trans-Golgi network.
Review Trends in Cell Biology January 2014, Vol. 24, No. 1
37

4. subunits with an overall molecular weight of approximate-
ly 250–800 kDa, can interact with vesicles over much
shorter distances (up to 30 nm) [45].
Many long coiled-coil tethers including GM130, Golgin-
45, Golgin-97, Golgin-245, and p115 localize to the Golgi
apparatus and belong to the golgin family. The golgins
(approximately 20 members) are regulated by small
GTPases of the Rab and Arl families and function in
membrane–membrane and membrane–cytoskeleton teth-
ering at the Golgi apparatus [46]. Other long coiled-coil
tethers such as EEA1, Rabaptin-5, Rabenosyn-5, and
Rabip4 associate with endosomal compartments [44].
MTCs can be divided into two major classes: complexes
associated with tethering containing helical rods
(CATCHR) and Class C vacuolar protein-sorting (Vps)
complexes. The exocyst, DSL1, Conserved Oligomeric Gol-
gi (COG), and Golgi-associated retrograde protein (GARP)
(Figure 3A,C,D) belong to the CATCHR family, which
exhibits low sequence identity but conserved helical-bun-
dle structures [45]. Homotypic fusion and protein sorting
(HOPS) (Figure 3B) and class C core vacuole/endosome
tethering (CORVET) belong to the Class C Vps complexes
and regulate transport from the late Golgi to endosomal/
lysosomal compartments [47]. The multisubunit Transport
Protein Particle (TRAPP) complexes (I, II, and III) have
also been considered as tethering factors. However, in
contrast to other tethering factors that function as small
GTPase effectors, TRAPPs act as guanine nucleotide ex-
change factors (GEFs) for the Rab small GTPases [48].
TRAPPI, which is required for ER–Golgi transport, inter-
acts directly with vesicle coats, thereby coupling Rab acti-
vation with coat recognition and vesicular tethering
[49,50].
Tethering factors are associated with different intracel-
lular membranes via distinct mechanisms, including specif-
ic targeting motifs (such as the GRIP domain in several
golgins), phospholipid binding, and/or interactions with
small GTPases [46,51]. Most tethering factors are Rab or
Arl effectors that interact directly with small GTPases in
their GTP-bound form [51]. CORVET and EEA1 are Rab5
effectors, whereas HOPS is a Rab7 effector. Rab5 (Vps21p in
yeast) functions at the early endosome, whereas Rab7
(Ypt7p in yeast) functions at late endosomes and lysosomes
[52]. COG is a Rab1 (Ypt1p) and Rab6 (Ypt6p) effector
[53–56], whereas GARP interacts with small GTPases of
the Rab and Arl families (Ypt6p and Arl1p, respectively, in
Saccharomyces cerevisiae) [57]. The exocyst interacts with
the GTP-bound form ofthe Rab small GTPase Sec4p in yeast
and with the Rab11, Rab8, and Ral small GTPases in
mammals. The exocyst also interacts with Rho and Cdc42
small GTPases [58]. Comprehensive description of these
tethers and their structural features and subcellular distri-
bution has been published previously [42,45,51,59,60].
Although both MTCs and long coiled-coil tethers have
been implicated in vesicular tethering, coiled-coil tethers
are presumed to be involved in the initial, highly dynamic
stages of tethering, because they can form transient, re-
versible, and low-affinity interactions [44]. By contrast,
MTCs may regulate later events and be actively involved
in SNAREpin assembly. MTCs, which capture vesicles at
relatively short distances from their target membranes,
can interact simultaneously with different transport com-
ponents through their multiple subunits. These multiva-
lent interactions could integrate different trafficking
components and consequently couple vesicular tethering,
docking, and fusion events. The structural and functional
differences between long coiled-coil tethers and MTCs
suggest that coiled-coil tethers and MTCs could have
complementary roles and potentially cooperate during
vesicular tethering, docking, and fusion. Consistent with
this hypothesis, previous studies have shown direct inter-
actions between long coiled-coil tethers such as p115 or
Golgin 84 and the COG complex [61,62].
In addition to their established roles in vesicular cap-
turing and docking, tethering factors also positively regu-
late SNAREpin assembly by direct interaction with
components of the fusion machinery – SNAREs and SM
proteins [12–15]. These interactions could influence: (i) the
stability of certain SNAREs and/or their corresponding
complexes; (ii) the assembly of SNARE subcomplex inter-
mediates; and (iii) the speed and specificity of SNAREpin
assembly. A brief description of these interactions and
their physiological roles is given below.
Tethering factors interact with SNAREs and SM
proteins
Both coiled-coil tethers and MTCs interact directly
with different SNAREs. The coiled-coil tether p115, for
Box 1. Genetic mutations in MTCs, SNAREpin assembly,
and inherited diseases
In recent years, multiple mutations in genes encoding different
subunits of MTCs have been identified as the cause of severe
inherited diseases. For example, a single amino acid substitution
(L967N) in the carboxyl-terminal region of the Vps54 subunit of the
GARP complex was identified as the cause of defective spermiogen-
esis and a motor neuron disease in the ‘wobbler’ mouse [92].
Homozygous wobbler mice display unsteady (‘wobbly’) gait with
progressive muscle weakness, a phenotype resembling the motor
neuron degeneration observed in amyotrophic lateral sclerosis
(ALS). This phenotype is accompanied by the formation of
ubiquitinated TDP-43 inclusions [93]. It was proposed that the
L967N mutation reduces the level of functional GARP and conse-
quently its associated autophagic activity, thereby attenuating the
clearance of TDP-43 aggregates and facilitating motor neuron death
[57]. Likewise, point mutations within the human VPS33B gene, a
subunit of the HOPS complex, cause arthrogryposis, renal dysfunc-
tion, and cholestasis (ARC) syndrome (Online Mendelian Inheritance
in Man [OMIM] 208085). ARC is an autosomal-recessive, multi-
system disorder characterized by renal tubular abnormalities,
cholestasis, hypotonia, and platelet storage-pool deficiency [94]. It
was proposed that the VPS33B mutations cause abnormal assembly
of the entire HOPS complex and consequently impair SNAREpin
assembly and biogenesis and/or fusion of lysosome-related orga-
nelles. Strikingly, a direct influence on the assembly of Golgi
SNAREpins has been demonstrated in Cog7- and Cog8-deficient
fibroblasts derived from patients with congenital disorders of
glycosylation (CDGs) [95]. CDGs are a heterogeneous group of
inherited disorders characterized by pleiotropic glycosylation de-
fects [96]. In recent years, multiple mutations in the genes encoding
different COG subunits have been identified as the cause of CDG
[97–101]. These mutations affect the assembly of the COG complex,
thereby impairing intra-Golgi retrograde transport and conse-
quently the proper localization of Golgi glycosylation enzymes
[102]. Overall, these findings demonstrate the importance of
tethering factors for normal cell function and their critical physio-
logical roles.
Review Trends in Cell Biology January 2014, Vol. 24, No. 1
38

5. example, interacts with several Golgi SNAREs including
Stx5, GS28, GS15, Ykt6, rSec22, and GS27/membrin [13]
and the endosomal EEA1 tether interacts with Stx6 [63].
Different subunits of all MTCs except the TRAPP com-
plexes physically interact with various SNAREs both in
yeast and mammals (Table 1 in [42]).
Mechanistically, tethering factors can interact with
multiple structural motifs in SNAREs. For example, the
Tip20 and Sec39 subunits of the DSL1 complex, which
regulates Golgi-to-ER retrograde transport, interact di-
rectly with the N-terminal regulatory domains of the t-
SNAREs Use1 and Sec20 at the ER membrane (Figure 3C)
[64,65]. The Vps51 subunit of the GARP complex, which
regulates endosome-to-TGN retrograde transport, inter-
acts with the N-terminal Habc domain of Stx6 (Tlg1p in
yeast) [66,67] and the Vps53 and Vps54 subunits of GARP
interact with the SNARE motifs of the Golgi SNAREs Stx6,
Stx16, and Vamp4 (Figure 3A) [68,69]. Several subunits of
the octameric COG complex (Cog1–8), which regulates
intra-Golgi and endosome-to-Golgi retrograde transport,
also interact with the SNARE motifs of multiple Golgi
SNAREs (Figure 3A) [12,70,71]. These interactions with
SNARE motifs are mediated by N-terminal coiled-coil
domains found in the GARP and COG subunits [43,72].
Subunits of the HOPS complex, which is required for
multiple fusion events at the late endosome and homotypic
fusion of vacuoles in yeast [73–75], also interact directly
with SNAREs. The Vps33 subunit of the HOPS complex
binds the SNARE motifs of the t-SNAREs Vam3 and Vam7
as well as the SNARE motif of the v-SNARE Nyv1. The
Vps11, Vps16, and Vps18 subunits of HOPS bind the N-
terminal Habc domain of Vam3 and the PX domain of
Vam7 (Figure 3B) [76–78]. Vps33, which is also an integral
subunit of the CORVET complex, interacts directly with
endosomal SNAREs, including the yeast syntaxin Pep12p
[74]. Remarkably, Vps33 is an SM protein that functions as
Vps52
Vps53
Vps54
Vps51
Golgi
Stx5
GS28Ykt6
Stx16
VƟ1a
Vam
p4
Stx6
Sly1
p
Exocyst
PM
Sec9
Sso1/2
Sec4 (Rab)
Vesicle
Snc1/2
Sec1 Sec15
Sec10Sec6
Sec8 Exo84
Exo70
Sec5
Sec3
DSL1
Sec39
Dsl1
Tip20
ER
COP-I
Vesicle
Sec22
Ufe1
Use1
Sec20
HOPS
16
33 41
18
11
39
Rab/Ypt7
Vam3
Vam7
Vam3
Vam7
Nyv1
Stx6
Stx16
Vamp4
Rab/Ypt7
Vam
3
VƟ1
Vam
7
Nyv1
SNARE
abc
abc
COG
4
1
8
2
3
7
5
6
Vps45
GARP
(D)
(B)(A)
(C)
PX
t-SNAREs
TRENDS in Cell Biology
Figure 3. Multisubunit tethering complexes (MTCs) interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to couple vesicular
tethering with fusion. (A) Conserved Oligomeric Golgi (COG) and Golgi-associated retrograde protein (GARP) MTCs interact with multiple SNAREs on the Golgi
membranes. The eight subunits of the COG complex are organized in two structurally and functionally distinct lobes (light and dark blue) [103]. The Cog4, Cog6, and Cog7
subunits bind the SNARE domains of the indicated SNAREs. Cog4 also binds the Sec1/Munc18 (SM) proteins Sly1 and Vps45 [12,82]. The Vps52, Vps53, and Vps54 subunits
of GARP (purple) can be assembled into a core complex and localize to the Golgi in the absence of Vps51 [67]. The Vps51 subunit (pink) interacts with the N-terminal
regulatory Habc domain of Syntaxin (Stx)6 (Tlg1p in yeast) [66,67]. The N-terminal regions of human Vps53 and Vps54 bind to the SNARE motifs of Stx6, Stx16, and Vamp4
[68]. (B) The yeast homotypic fusion and protein-sorting (HOPS) complex via its Vps33 subunit (pink) interacts with the SNARE motifs (pink) of target membrane SNAREs (t-
SNAREs) Vam3 (Stx7 in mammals) and Vam7 (Stx8 in mammals) and the transport vesicle SNARE (v-SNARE) Nyv1. The Vps11, Vps16, and Vps18 subunits (green) interact
with the regulatory Habc and PX domains of the t-SNAREs Vam3 and Vam7 on the vacuole or lysosome membrane (green). Vps39 and Vps41 subunits (yellow) bind the
small Rab GTPase Ypt7 [52]. (C) A schematic presentation of the yeast DSL1 complex according to its crystal structure [64]. DSL1 interacts with the t-SNAREs Sec20 and
Use1 via its Tip20 and Sec39 subunits and induces t-SNARE gathering on the endoplasmic reticulum (ER) membrane. The Dsl1p subunit interacts with subunits of the COP-I
coat and tethers COP-I vesicles to the ER membranes [90]. (D) The yeast exocyst complex tethers secretory vesicles to the plasma membrane (PM). The Sec3 and Exo70
(pink) subunits interact with the PM via their positively charged residues, whereas the Sec15 subunit interacts with the Rab GTPase Sec4, which localizes on a secretory
vesicle. These interactions are required for vesicular tethering [104]. The interactions of the Sec6 subunit with the t-SNARE Sec9 (SNAP23 in mammals) and the SM protein
Sec1 and of the Sec3 subunit with Sso1/2 (Stx1 in mammals) regulate the assembly the Sec9–Sso1 t-SNARE complex that binds the v-SNARE Snc1/2 (VAMP3 in mammals)
as described in the text.
Review Trends in Cell Biology January 2014, Vol. 24, No. 1
39

6. one subunit of the HOPS and CORVET heterohexameric
complexes and mediates multiple interactions with
SNAREs [52]. The incorporation of an SM protein into
MTCs demonstrates the coordinated functions of tethers,
SM proteins, and SNAREs in regulating intracellular
membrane fusion. Consistent with this mode of action,
both the exocyst and the COG complex interact directly
with SM proteins and SNAREs.
The exocyst, an evolutionarily conserved complex im-
plicated in the tethering of secretory vesicles to the plasma
membrane, comprises eight subunits (Sec3, Sec5, Sec6,
Sec8, Sec10, Sec15, Exo70, and Exo84) [58]. The complex
is directly involved in the assembly of the Sec9–Sso1–Snc2
SNAREpin at the plasma membrane in yeast (Figure 3D).
Its Sec6 subunit binds the SM protein Sec1 and the t-
SNARE Sec9 via overlapping binding sites that prevent
the formation of a ternary Sec6–Sec9–Sec1 complex. Fur-
thermore, Sec6 interacts with Sec9 in the absence of other
exocyst subunits, but interacts with Sec1 in the presence of
exocyst subunits [79,80]. When Sec6 is found outside the
exocyst complex, its interaction with Sec9 inhibits the
interaction between Sec9 and its syntaxin partner Sso1
[79], thereby preventing the formation of a premature t-
SNARE complex at the plasma membrane. However, the
assembly of the exocyst complex leads to the release of Sec6
from Sec9, opening of the autoinhibitory domain of Sso1,
and concomitant recruitment of Sec1 for regulating vesic-
ular tethering and fusion events [81]. These observations
suggest that the interactions between subunits of MTCs,
SNAREs, and SM proteins are crucial for spatial and
temporal regulation of SNAREpin assembly.
Consistent with these observations, previous studies of
the COG complex suggest that the interaction between its
Cog4 subunit and the SM protein Sly1 is crucial for SNAR-
Epin assembly and membrane fusion at the Golgi mem-
branes. However, in this case a ternary complex between
Cog4, Sly1, and Stx5 can form due to distinct adjacent
binding sites for Sly1 and Stx5 at the N-terminal coiled-coil
domain of Cog4 [82]. The interaction of Cog4 with both Sly1
and Stx5 is required for the assembly of Stx5–GS28–Ykt6–
GS15 SNAREpin at the Golgi complex and consequently for
intra-Golgi retrograde transport [70,82]. Strikingly, Cog4
also interacts with the SM protein Vps45 and Stx16, which
regulate endosome-to-Golgi retrograde transport. These
interactions are also mediated by the N-terminal coiled-coil
domain of Cog4 and compete with the Sly1–Stx5 interac-
tions [12]. These observations suggest that the COG com-
plex employs similar mechanisms to regulate the assembly
of the two different Golgi SNAREpins (Stx5–GS28–Ykt6–
GS15 and Stx6–Stx16–Vti1a–VAMP4) and thereby may
coordinate their corresponding transport routes.
It seems that both COG and exocyst complexes are in-
volved in recruiting and/or orienting the SM proteins to a
partially assembled SNARE complex, and possibly stabiliz-
ing SM–SNARE interactions. These effects might enhance
direct interaction between the SM proteins and both the t-
and v-SNAREs of SNARE complex intermediates, thereby
facilitating the progression of fusion events [11].
The roles of tethering factor–SNARE interactions
Functionally, the interactions between tethering factors
and SNAREs can influence the assembly of SNARE
SNAREpin assembly
tSNAREs gathering
Vesicle
UncoaƟng
Vesiclular
fusion
SNARE complex
disassembly
Golgi
Sly1
GS15 VAMP4
Vps45
COG
Vesicle capture
UncoaƟng
Ykt6
GS28
Stx5
VƟ1a
Stx6
Stx16
TRENDS in Cell Biology
Figure 4. Roles of the Conserved Oligomeric Golgi (COG) complex in vesicular tethering and SNAREpin assembly at the Golgi apparatus. The COG complex via its different
subunits interacts with distinct t-SNAREs and SM proteins on the Golgi membranes and gathers cognate t-SNAREs and SM protein, thereby promoting the assembly of
specific ternary t-SNARE complexes. The COG complex also interacts with vesicle coats [56] and consequently promotes vesicular tethering. It is currently unknown at
which step COG dissociates from the assembled SNARE complex, but it possibly stabilizes the assembled complex [70]. The COG complex possibly employs similar
mechanisms to assemble two distinct fusogenic Golgi SNARE complexes (Stx5–GS28–Ykt6–GS15 and Stx6–Stx16–Vtia–VAMP4), thereby coordinating their associated
transport routes. We propose that the assembly of fusogenic SNARE complexes prevents promiscuous assembly of non-fusogenic t-SNARE interactions on the Golgi
membranes [12].
Review Trends in Cell Biology January 2014, Vol. 24, No. 1
40

7. complex intermediates, stabilize SNARE complex assem-
bly, and spatially and temporally organize fusion events at
specific membrane domains.
Many MTCs can interact simultaneously through their
different subunits with multiple SNAREs and gather spe-
cific SNAREs at specific membrane compartments. Sub-
units of the GARP, DSL1, and COG complexes, for
example, can simultaneously interact with specific t-
SNAREs and enhance t-SNARE subcomplex assembly
on the Golgi or ER membranes (Figure 3) [12,64,68,69].
The formation of t-SNARE subcomplexes represents an
intermediate step in SNAREpin assembly [7]. Hence,
SNARE gathering appears to be the major role of MTCs
in SNAREpin assembly (Figure 4). This gathering mediat-
ed by multivalent interactions not only facilitates the
assembly of fusogenic SNARE complexes that functionally
drive membrane fusion, but can also prevent the assembly
of non-fusogenic SNARE interactions in intact cells. Re-
cently it was shown that the Cog4 subunit positively
regulates the assembly of fusogenic SNARE complexes
at the Golgi complex, thereby preventing non-fusogenic
t-SNARE interactions [12]. The interaction of the Sec6
subunit of the exocyst with Sec9 also prevents the forma-
tion of the Sec9–Sso1 t-SNARE complex before the assem-
bly of the exocyst complex and recruitment of Sec1. This
role of MTCs may be a general mechanism that inhibits
promiscuous assembly of non-functional or premature
SNARE complexes in biological membranes. In vitro stud-
ies suggest that SM proteins may also prevent the assem-
bly of non-fusogenic SNAREs; the interaction of Sed5p
(Stx5 in mammals) with Sly1p, for example, prevents its
interactions with non-physiological SNAREs [39].
In addition to SNARE gathering, tethering factors can
stabilize SNARE proteins or SNARE complexes and can
thereby influence SNAREpin assembly. The Cog6 subunit,
for example, interacts directly with and stabilizes Stx6.
Depletion of theCog6 subunit leads to abnormal proteasomal
degradation of Stx6, thus impairing the assembly of Stx6–
Stx16–Vti1a–VAMP4 SNARE complex and consequently
endosome-to-Golgiretrograde trafficking [71]. Abnormal pro-
teasomal degradation of the Golgi SNAREs GS28 and GS15
wasalsoobservedincellsdepletedofotherCOGsubunits[83]
anda directinteractionbetween GS28 andCog4 was recently
observed [12]. These direct interactions between MTCs and
SNAREs might induce conformational changes in the
SNAREs that could protect them from degradation as was
previously proposed for SM proteins. Sly1, for example,
directly interacts with the ER t-SNARE Ufe1p, a yeast
homolog of Stx18, and protects it from ERAD degradation
[84],whereasVps45pinteractswithTlg2p,theyeasthomolog
of Stx16, and protects it from proteasomal degradation [85].
The HOPS complex employs a different mechanism to
stabilize trans-SNARE complexes: it inhibits their disas-
sembly by Sec17p/Sec18p (SNAP/NSF) [86]. The HOPS
complex has affinity for multiple vacuolar SNAREs and
can facilitate the formation of trans-SNARE complexes. Its
ability to interact with both t- and v-SNAREs (Figure 3B)
possibly inhibits the access of Sec17p and/or Sec18p and
consequently SNAREpin disassembly [87].
As discussed, tethers can induce SNARE gathering
through multivalent interactions, stabilize SNAREs or
SNARE complexes, or activate SNARE complex assembly
possibly through interaction with SM proteins [11,40].
However, it appears that SNARE–tether interactions have
bidirectional effects and, in some cases, the SNAREs influ-
ence the localization of the tethers. SNAREs, for instance,
can enhance the binding of HOPS to liposomes and possi-
bly stabilize the interaction of HOPS with the vacuole [88].
Likewise, the interaction of the DSL1 complex with ER
SNAREs is possibly required for its membrane association,
because DSL1 does not interact with the Rab small
GTPases that generally mediate membrane recruitment
[64,89]. Strikingly, DSL1 also interacts directly with COP-
I subunits [90] and HOPS interacts with the Apl5 subunit
of the AP-3 coat [91]. These interactions with both coats
and SNAREs are possibly required for coupling tethering
with fusion events.
Concluding remarks
Studies over the past few years strongly suggest that
tethering factors do not simply bridge two membrane
compartments, but rather are actively involved in fusion
and influence fusion specificity, efficiency, and speed. A
network of protein–protein interactions between tethers,
SNAREs, and/or SM proteins is the molecular basis for
their critical roles in SNAREpin assembly and conse-
quently membrane fusion. The ability of tethers to inter-
act with multiple components of the transport machinery,
either simultaneously or sequentially, enables them spa-
tially to regulate fusion events at specific membrane
domains. Multiple tethering factors exist in eukaryotic
cells; some regulate similar transport routes and even the
assembly of the same SNARE complexes. It remains un-
clear how these tethers function coordinately and why
there are so many factors with potentially overlapping
functions. For example, both GARP and COG regulate the
assembly of the Stx6–Stx16–Vti1a–VAMP4 SNARE com-
plex and consequently endosome-to-TGN retrograde
transport. In light of the described studies, it could be
that different tethers regulate the assembly of similar
SNARE complexes but concomitantly prevent the assem-
bly of distinct SNARE complexes or vice versa. This might
depend on the temporal localization of the tethering fac-
tors and their ability to interact with other cellular
SNAREs. Other aspects including the affinity of tethers
to their interacting SNAREs, the avidity of binding, or
even post-translational modifications in tethers and/or
SNAREs can influence SNAREpin assembly and/or its
disassembly. Collectively, these different aspects could
dynamically influence SNAREpin assembly and tempo-
rally and spatially control membrane-fusion events. Fur-
ther in vitro and in vivo studies on tether–SNARE–SM
interactions will shed light on the molecular mechanisms
that regulate vesicular fusion.
Acknowledgments
S.L. is the incumbent of the Joyce and Ben B. Eisenberg Chair of
Molecular Biology and Cancer Research. This work was supported by the
Binational Science Foundation (BSF), Grant No. 2011404.
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