http://www.elsevier.com/locate/bba

Biochimica et Biophysica Ac

Review

Golgi tethering factors

Vladimir Lupashina,*, Elizabeth Sztulb

aDepartment of Physiology and Biophysics, University of Arkansas for Medical Sciences, Biomed 261-2, Slot 505,

200 South Cedar St, Little Rock, AR 72205, USAbDepartment of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

Received 20 January 2005; received in revised form 30 March 2005; accepted 31 March 2005

Available online 14 April 2005

Abstract

Transport of cargo to, through and from the Golgi complex is mediated by vesicular carriers and transient tubular connections. In this

review, we describe vesicle tethering events with the understanding that similar events occur during transport via larger structures. Tethering

factors can be generally divided into a group of coiled-coil proteins and a group of multi-subunit complexes. Current evidence suggests that

these factors function in a variety of membrane–membrane tethering events at the Golgi complex, interact with SNARE molecules, and are

regulated by small GTPases of the Rab and Arl families.

D 2005 Elsevier B.V. All rights reserved.

Keywords: ER; Golgi; Tethering; Protein traffic; Coiled-coil protein

1. Introduction

Biochemical, molecular and genetic analyses have

produced a general picture of the molecular mechanisms

involved in protein and lipid transport between organelles.

Transport occurs by means of membrane intermediates that

bud from a donor compartment, traverse a certain distance,

and fuse with an acceptor compartment. Vesicles and larger

pleomorphic structures appear to be the predominant trans-

port intermediates. In this review we describe vesicle

tethering, with the understanding that similar events occur

during transport via larger structures. Vesicle budding

0167-4889/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbamcr.2005.03.013

Abbreviations: IF, immunofluorescence; siRNA, short (or small)

interfering RNA; PM, plasma membrane; ER, endoplasmic reticulum;

COPI, vesicle coat protein complex I; COPII, vesicle coat protein complex

II; VTC, vesicular– tubular cluster; CCV, clathrin coated vesicle; GEF,

guanine nucleotide exchange factor; GAP, GTPase activating protein;

TRAPP, transport protein particle; COG, conserved oligomeric Golgi

complex; GRASP, Golgi re-assembly stacking protein; TGN, trans-Golgi

network; SM, Sec1/Munc proteins; ARF, ADP-ribosylation factor; Arl,

ARF-like protein; GRAB, GRIP-like ARF-binding domain; EM, electron

microscopy; GARP, Golgi-associated retrograde protein complex

* Corresponding author. Tel.: +1 501 603 1170; fax: +1 501 686 8167.

E-mail address: vvlupashin@uams.edu (V. Lupashin).

requires protein coats (for review see [1]) and small

GTPases of the ARF family [2], while vesicle targeting

and fusion depend on a large family of SNAREs (soluble N-

ethylmaleimide-sensitive factor attachment protein receptor)

[3], small GTPases of the Rab family [4], and a diverse

group of tethering factors. The SNARE protein family

contributes a v-SNARE on the vesicle and cognate t-

SNAREs on the target membrane: fusion involves the

formation of a 4-a-helix bundle, where one helix is

contributed by the v-SNARE and the remaining 3 are

donated by three SNAREs on the target membrane. The

SNARE bundle bridges the two membranes, and its

formation is thought to overcome the energy barrier

preventing the two membranes from fusing (for review

see [3]).

The term ‘‘tethering factors’’ describes a group of

proteins believed to mediate initial, loose Ftethering_ of

vesicles with their targets. This loose interaction is followed

by tighter, more stable, Fdocking_ interactions involving

SNAREs [5].The tethering proteins are believed to contrib-

ute to the fidelity of vesicle fusion. Tethering factors form

physical links between the vesicles and the acceptor

membrane before the engagement of SNAREs (Fig. 1).

Tethering may provide the initial level of recognition that is

ta 1744 (2005) 325 – 339

Fig. 1. A model for membrane tethering. (1) A transport intermediate approaches the target membrane. The movement can be by diffusion or by a motor-

mediated process. (2) The transport intermediate tethers to the target membrane by coiled-coil proteins or through multimeric tethering complexes. Tethering

can occur at distances of >200 nm. It has been proposed that tether assembly may precede vesicle budding, and thus couple vesicle production with targeting to

the appropriate target membrane. (3) The cognate v-SNARE on the transport intermediate and t-SNAREs on the target compartment pair to form trans-SNARE

complexes. This process is sometimes referred to as ‘‘docking’’. (4) The assembly of SNARE complexes drives membrane fusion. Transported cargo is

incorporated into the membrane of the target compartment or released into the lumen.

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339326

then amplified by SNARE pairings. Despite significant

progress, it is obvious that tethering is a complex process

that may involve multiple interactions and occur in

temporally and spatially distinct stages.

Tethering factors can be generally divided into a group of

coiled-coil proteins and a group of multi-subunit complexes.

The coiled-coil tethers Uso1p/p115, GM130, giantin, and

golgin84 appear involved in ER–Golgi and intra-Golgi

traffic, golgin97 participates in traffic from the endosome to

the trans-Golgi network (TGN) [6], and EEA1 is involved

in endosomal traffic. The multisubunit TRAPPI/II and COG

complexes participate in ER–Golgi and intra-Golgi traffic,

while the HOPS and GARP complexes are involved in

TGN-endosomal-vacuolar traffic, and the exocyst facilitates

fusion with the PM. Localization and postulated site of

action of each tether are depicted in Fig. 2. Detailed

discussions on the cellular roles of the endosomal tether

EEA1, the HOPS/Class C VPS complex and the exocyst

complex can be found elsewhere [7–21]. In this review we

will focus on the Golgi-operating tethers.

Fig. 2. Location of known tethering factors in the secretory and endosomal

pathways. Individual coiled-coil proteins and multi-subunit complexes are

placed next to the transport step they facilitate or adjacent to the

compartment to which they localize. The drawing is a composite of

tethering factors from yeast and mammalian cells.

2. Coiled-coil tethers

Coiled-coil proteins comprise more than 5% of the

eukaryotic proteome, implying an involvement in numerous

cellular processes [22]. Several long coiled-coil proteins

have been identified on the Golgi (Table 1). Some of these

proteins were identified by functional assays (Uso1, Rud3,

Imh1), but majority were found by indirect means, either as

interactors in yeast two-hybrid screens or as antigens

recognized by sera from patients with autoimmune diseases.

Potential role of these proteins in membrane traffic has often

been predicted from their Golgi location and their structure.

Long rod-like molecules are attractive candidates for factors

that link Golgi cisternae or capture transport vesicles in the

proximity of the cisternae prior to fusion. Molecular features

and potential role in membrane tethering of several well-

studied Golgi-localized coiled-coil tethers are discussed

below.

Table 1

Golgi-localized coiled-coil proteins proposed to play a role in membrane tethering

Names M.W. of human

protein (kD)

Other names Domains Ref.

Yeast Mammals

Bicaudal-D1/2 110/120 – [120]

CASP 80 COY1 CCAAT displacement protein isoform c [121,122]

GCC88 88 – GCC1 GRIP [58]

GCC185 185 – GCC2 GRIP [58]

GCP16 – GOLGA7 [123]

GCP60 60 ACBD3 [52]

Giantin ¨400 – GOLGB1, GCP372. Macrogolgin [41,47,48,57]

GM130 130 – GOLGA2, Golgin-95 [40,57,72,73, 115, 124–129]

GMAP210 210 RUD3, GRP1 CEV14, Trip11, Trip230 GRAB [65,66,69,71,130,131]

GOLGA6 – GLP [132]

Golgin-45 45 – BLZF1, JEM-1 [53,133]

Golgin-67 67 – [134]

Golgin-84 84 – GOLGA5 [56,135]

Golgin-97 97 – GOLGA1 GRIP [57,58,60,136]

Golgin-160 160 – GOLGA3, GCP170 [123,137,138]

Golgin-245 230 Imh1 GOLGA4, p230, tGolgin-1 GRIP [57,59,60,62,136,139–142]

GRASP55 55 – GORASP2, GOLPH6, GRS2 [53,116,143–146]

GRASP65 65 – GORASP1, P65; GOLPH5 [72,73,125,136]

P115 115 Uso1 VDP, TAP [23,24,26,31,33,34,36,50,117,

127,147–150]

TMF1 Sgm1 ARA160 [151]

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339 327

2.1. Uso1/p115

Uso1p (yUSOu means transport in Japanese) was

initially identified as a yeast ER–Golgi transport factor

by showing that the temperature-sensitive mutant, uso1,

blocks the transport of the secretory protein invertase prior

to its delivery to the Golgi [23]. The function of Uso1p at

the ER–Golgi stage was further supported by the finding

that Uso1 defects can be suppressed by overexpressing

each of the known ER-to-Golgi SNAREs (Bet1p, Bos1p,

Sec22p, and Ykt6p) [24] and of the small GTP-binding

protein Ypt1p known to function in ER–Golgi traffic [25].

In vitro studies provided direct evidence for the role of

Uso1p in tethering COPII vesicles to Golgi membranes.

COPII vesicles do not bind to Golgi membranes in the

absence of functional Uso1p [26], but the addition of

functional Uso1p to the assay reconstitutes binding. Uso1p-

mediated vesicle tethering was proposed to be SNARE

independent because normal levels of tethering were

observed when SNARE function was inhibited by adding

inhibitory anti-SNARE antibodies or by using inactive

SNARE mutants [27]. However, in both cases, SNARE

proteins were present and could theoretically participate in

Uso1p-mediated events, despite their inability to catalyze

fusion.

Uso1p is a soluble cytoplasmic protein that periph-

erally associates with membranes. The association of

Uso1p with membranes is maximal in the presence of

active GTP-bound form of yeast Rab protein Ypt1p [27].

Although Uso1p binding to membranes is reduced in

yeast strains depleted of Ypt1p, Uso1p can still be

recovered with membranes [28], suggesting that additional

factors may be involved. In agreement, the mammalian

homologue of Uso1p, p115, associates with membranes in

cells expressing a dominant negative inactive Rab1 mutant

[29].

p115 was initially identified as a cytosolic factor

required for intra-Golgi transport in an in vitro transport

assay [30,31]. Subsequent in vivo studies documented

p115 function in ER–Golgi transport [32], possibly by

promoting the fusion of COPII vesicles [33]. In agree-

ment, p115 has been detected on COPII vesicles

generated in vitro and is required for the binding of such

vesicles to Golgi membranes [34]. p115 is also required

for the reassembly of the Golgi complex after mitosis

[35].

Uso1p and p115 are parallel homo-dimers with two

globular heads and a long tail composed of multiple coiled-

coil domains. The overall structure is reminiscent of

myosin II. The heads of Uso1p and p115 are ¨9 nm.

The Uso1p tail is ¨150 nm and the p115 tail is ¨45 nm.

The tails of both proteins have internal hinges, and this has

been proposed to facilitate an ‘‘accordion-like’’ collapse of

the tether to bring the vesicle and acceptor membranes into

proximity [36].

p115 binds the active GTP-bound form of Rab1 and

acts as a Rab1 effector [34]. In addition to the Rab, p115

interacts directly with a set of COPII vesicles associated

SNAREs (syntaxin5, membrin and GS28) [34,37]. p115

interacts with GM130 and giantin (see below), and it has

been proposed that GM130 and/or giantin act as

membrane receptors for p115. However, p115 mutants

lacking the GM130/giantin binding domain show normal

localization, indicating that p115 is recruited to mem-

branes independently of its interaction with GM130/

giantin [38].

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339328

2.2. GM130

GM130 is a mammalian peripheral membrane protein

that is tightly bound to Golgi membranes via the Golgi re-

assembly stacking protein of 65kDa (GRASP65). GM130

is an extended rod-like protein with 6 coiled-coil domains.

The role of GM130 in tethering is suggested by in vitro

studies showing that GM130 interacts with activated Rab1-

GTP and is required for COPII vesicle targeting/fusion

with the cis-Golgi [39]. The tethering of COPII vesicles to

Golgi elements is proposed to be mediated by p115 on

COPII vesicles binding to GM130 on cis-Golgi membranes

[39,40]. The model for p115–GM130 tethering is sup-

ported by the findings that addition of anti-GM130

antibodies or an NH2-terminal GM130 peptide that

prevents p115–GM130 interaction, inhibits vesicle docking

[41].

However, multiple lines of evidence raise issue with

the requirement for p115–GM130 tethering in traffic.

First, the Krieger laboratory isolated mutant Chinese

hamster ovary (CHO) cells that are defective in traffick-

ing of the LDL receptor at the non-permissive temper-

ature of 39.5 -C (ldlG cells) [42]. ldlG cells lack

detectable GM130. At the permissive temperature, traffic

(measured by glycoprotein processing of cargo protein)

and secretion are normal. In addition, the ultrastructure of

the Golgi apparatus examined by immunofluorescence

(IF) and immunoelectron microscopy (EM) appears

normal. These findings suggest that at reduced temper-

ature, GM130 function in tethering COPII vesicles to the

Golgi is not necessary. However, GM130 function is

required at higher temperatures since incubation at 39.5

-C for 12 h causes disassembly of the Golgi into

dispersed vesicles. It is possible that GM130–or a

GM130-dependent protein(s)–plays a role in maintaining

Golgi structure at higher temperatures [43].

Second, the Linstedt laboratory showed that Golgi

structure is normal when p115–GM130 tethering is

inhibited [44]. Using short interfering RNA (siRNA),

coupled with microinjection of plasmid DNA, they reduced

the level of endogenous p115 and expressed p115 unable to

bind GM130 in the same cell. The mutant p115 does not

bind GM130 and does not participate in p115–GM130

tethering. Despite this, the mutant p115 could support

normal Golgi structure and traffic.

Third, we have shown that p115 is required in ER–

Golgi traffic at a pre-Golgi stage, much earlier than

the requirement for GM130 within the Golgi complex

[33].

Fourth, the depletion of the Drosophila homologue of

GM130 did not perturb the morphology of the secretory

compartments or secretory traffic [45]. Fifth, while p115

homologues exist in all eukaryotes so far examined, GM130

is only found in metazoans. Together, these findings suggest

that the p115–GM130 interaction may have functions other

than tethering.

2.3. Giantin

Giantin has been proposed to act in concert with p115

and GM130 to facilitate the tethering of COPI vesicles to

cis-Golgi membranes [41]. Giantin is a mammalian coiled-

coil, rod-like type II Golgi membrane protein, with most of

its mass projecting into the cytoplasm [46]. A COOH-

terminal sequence (residues 3059–3161) adjacent to the

transmembrane domain is required for Golgi localization of

giantin [47]. Giantin is found in in vitro generated COPI

vesicles, and has been postulated to bind p115 that then

would bind GM130 on the acceptor Golgi membrane [41].

This ‘‘bridging’’ model is supported by the finding that pre-

treatment of such vesicles with anti-giantin antibodies

inhibits both, the binding of p115 and the docking of these

vesicles to Golgi membranes. In agreement, a peptide

analogous to the NH(2)-terminal p115-binding domain of

giantin (and shown to bind p115 in vitro and in vivo) blocks

cell-free Golgi reassembly [48]. Such reassembly has been

shown to involve COPI vesicle events [49].

Further support for the GM130–p115–giantin tether

comes from experiments in which the binding of p115 to

Golgi membranes is inhibited by microinjection of an N-

terminal p115-binding peptide of GM130 or overexpression

of GM130 lacking the N-terminal p115-binding domain.

Electron microscopic analysis of microinjected or transfected

cells shows that the number of COPI-sized vesicles in the

Golgi region increases substantially, suggesting that COPI

vesicles continue to bud but are unable to tether and fuse.

However, recent findings raise concerns about such

interpretation. The ‘‘bridging’’ model for GM130–p115–

giantin tether would suggest that p115 binds to both,

GM130 and giantin at the same time. Surprisingly, the

GM130 and the giantin binding sites in p115 map to the

same C-terminal acidic domain, and the proteins compete

for p115 [50]. It is therefore possible that the p115–giantin

and the p115–GM130 interactions mediate independent

membrane tethering events. In addition, expression of p115

mutant devoid of the giantin-binding site in p115-depleted

cells is able to sustain traffic and does not lead to Golgi

disassembly [51]. Furthermore, giantin appears to be present

only in mammalian cells, arguing against a conserved role in

membrane tethering. Together, the results suggest that

giantin function in vivo may involve events other than

COPI vesicle tethering.

Giantin interacts with another coiled-coil Golgi protein,

GCP60 [52]. The exact function of GCP60 is unknown, but

it is likely to participate in ER–Golgi traffic or in the

maintenance of Golgi structure since its overexpression

inhibits ER-to-Golgi traffic and disrupts Golgi architecture

[52].

2.4. Golgin-45

Golgin-45 is a coiled-coil Golgi protein shown to interact

with GRASP55 via a C-terminal sequence [53]. Golgin-45

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339 329

binds the active Rab2-GTP through its coiled-coil region.

Overexpression of golgin-45 causes disruption of the Golgi,

with Golgi components localizing to punctate structures

dispersed throughout the cell. Depletion of golgin-45 by

siRNA causes a distinct type of Golgi disruption, similar to

that in cells treated with BFA. In both cases, Golgi enzymes

relocate to the ER, while Golgi matrix proteins localize to

punctate structures dispersed throughout the cell. Direct

analysis of VSV-G traffic in golgin-45 depleted cells shows

arrest in anterograde traffic at the level of the ER. The exact

function of golgin-45 in facilitating tethering events remains

to be defined.

2.5. Golgin-84

Golgin-84 is a mammalian integral membrane protein

with a single transmembrane domain close to its C terminus.

Cross-linking indicates that golgin-84 forms dimers [54].

Cryo-electron microscopy localizes golgin-84 to the cis-

Golgi network and shows that it is enriched on tubules

emanating from the lateral edges of, and often connecting,

Golgi stacks [55]. A tethering/stacking function for golgin-

84 is suggested by the finding that overexpression or

depletion of golgin-84 results in fragmentation of the Golgi

ribbon [56]. In agreement, antibodies to golgin-84 inhibit

stacking of cisternal membranes in a cell-free assay for

Golgi reassembly, whereas the cytoplasmic domain of

golgin-84 stimulates stacking and increases the length of

re-assembled stacks. Together, these data suggest that

golgin-84 is involved in generating and maintaining the

architecture of the Golgi apparatus [55]. Golgin-84 binds to

active Rab1 but not to cis-Golgi matrix proteins. The exact

mechanism of golgin-84 mediated tethering is unknown.

2.6. Golgin-97

Golgin-97 is characterized by the presence of a GRIP

domain at its C-terminus [57]. In addition, golgin-254,

GCC88 and GCC185 also contain GRIP domains [58,59].

GRIP domains are ¨45 amino acids long and poorly

conserved, except for an invariant tyrosine residue at

position 4, followed eight residues later by a phenylalanine

or a tyrosine. GRIP domain proteins have been found in all

eukaryotes examined so far, suggesting an essential func-

tion. The GRIP domain is required and sufficient for Golgi

targeting. The GRIP domains interact with small GTPase

Arl1 (ARF-like protein), and active Arl1-GTP recruits the

golgin to the membrane [60–63]. In turn, membrane

recruitment of Arl1 is likely to require the active form of

Arl3-GTP since yeast Imh1p (golgin-245 homologue) and

human golgin-97 expressed in yeast associate with mem-

branes by a mechanism requiring activated Arl3p-GTP [64].

Structural studies of the GRIP domain of golgin-245 in

complex with Arl1-GTP show that the GRIP domain forms

a homodimer that binds two Arl1-GTPgs [61]. The complex

structure suggests that the C-termini of golgins are

immobilized at the membrane by Arl1, while the majority

of the protein either extends from the membrane or aligns on

its surface. The bivalent nature of the interactions between

GRIP-containing golgins and Arl1 may provide additional

control of the location and duration of golgin residency at

the membrane, and hence its function.

Golgin-97 appears to facilitate traffic from the endosome

to the TGN [6]. Using semi-intact cells supplemented with

anti-golgin-97 antibodies or depleted of golgin-97, and

intact cells microinjected with antibodies or depleted of

golgin-97 by siRNA, the Hong laboratory established a

clear requirement for golgin-97 in traffic from the endo-

somes to the TGN. Golgin-97 is required at a step preceding

the requirement for syntaxin16, consistent with a tethering

function for golgin-97. However, the exact mechanism of

golgin-97 action remains to be defined.

2.7. GMAP210

GMAP210 (Golgi microtubule-associated protein of

210kD) is a human cis-Golgi protein with a GRAB

(GRIP-related Arf-binding) domain at its C-terminus [65].

Both, GRAB and GRIP domains interact with small

GTPases, Arl in case of GRIP and ARF in case of GRAB,

and both mediate binding of the corresponding golgin to the

membrane. The conserved tyrosine residue found in all

GRIP domains is replaced by a leucine residue in the GRAB

domain. This leucine is essential for GRAB domain

function since mutations lead to loss of membrane targeting

of the yeast homologue of GMAP210, Rud3p [65]. The

correct association of Rud3p with membranes appears

complex, since in addition to ARF interactions, Rud3p

localization to the Golgi also requires the ER cargo receptor

Erv14p [65]. The functional implication of this requirement

remains to be defined.

GMAP210 was initially proposed to facilitate Golgi

localization around the centrosomes [66] by simultaneously

linking to g-tubulin via the GRAB domain and Golgi

membranes by an N-terminal domain [67]. Recent studies

on GMAP210 indicate that the C-terminal GRAB domain

interacts with Golgi membranes, suggesting that GMAP210

is unlikely to link Golgi to centrosomes and g-tubulin [65].

Overexpression of GMAP210 blocks anterograde transport

of both a soluble form of alkaline phosphatase and the

transmembrane hemagglutamin of the influenza virus at a

pre-cis-Golgi stage [68]. In addition, retrograde transport of

Shiga toxin B-subunit is also inhibited at a stage between

the Golgi and the ER. In cells expressing high levels of

GMAP210, the Golgi is disrupted into ¨10–20 clusters

composed of hundreds of small 50-nm vesicles. The data are

consistent with a possible tethering function for GMAP210

since increased tethering may prevent efficient fusion and

lead to massive accumulation of tethered vesicles.

RUD3 (also called GRP1 [69,70]) was identified through

a genetic screen for multicopy suppressors of a mutation in

USO1 [71]. A large portion of Rud3p is predicted to form a

Table 2

Multisubunit golgi tethering complexes

Complex Components Other names

Yeast Mammals

COG COG1 [88] Cod3, Sec36 [89],

Tfi1 [85]

LdlB [152]

COG2 [91] Sec35 LdlC [96]

COG3 [71] Sec34, Grd20 [93] hSec34 [86]

COG4 [88] Cod1, Sgf1,

Sec38 [89]

hCod1 [88]

COG5 [88] Tfi3 [85] GTC-90 [153]

COG6 [88] Cod4 hCod2 [88]

COG7 [88] Cod2, Sec37 [89],

Tfi2 [85]

hCod5 [88]

COG8 [88] Cod5 hDor1 [88]

Dor1

Dsl1 Dsl1 [105] Dsl1 ZW10 [108]

Tip20 [154] Tip1 RINT-1 [108]

Sec20 [154] Sec20 BNIP1 [155]

GARP Vps51 [103,156] API3, VPS67, WHI6 ?

Vps52 [101] SAC2 ARE1, SAC2,

SACM2L

Vps53 [101] Vps53 FLJ10979,

hVps53L

Vps54 [101] CGP1, LUV1, TCS3 HCC8, SLP-8p,

VPS54L,

hVps54L [157]

TRAPP I and

TRAPP II

Bet3 [158] Bet3 TRAPPC3

Bet5 [159] Bet5 TRAPPC1

Trs85 [76] Trs85, GSG1 TRAPPC8

Trs65 [76] Trs65, Kre11 TRAPPC7

Trs20 [74] Trs20 TRAPPC2,

SEDL [83]

Trs23 [74] Trs23 TRAPPC4,

synbindin [160]

Trs31 [76] Trs31 TRAPPC5

Trs33 [74] Trs33 TRAPPC6A

Trs120 [76] Trs120 TRAPPC9

Trs130 [76] Trs130 TRAPPC10

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339330

coiled-coil structure. Although RUD3 is not essential for

growth, the loss of RUD3 results in a growth defect at high

temperature. This phenotype is similar to that observed in

ldlG mammalian cells that lack GM130 [43]. Rud3p-

depleted cells efficiently secrete underglycosylated inver-

tase, suggesting a disturbance in Golgi function. Rud3p-

GFP predominantly co-localizes with the cis-Golgi marker

Och1p [69]. Deletion of RUD3 is lethal in the absence of the

Golgi Rab GTPase Ypt6p [65]. The exact molecular

mechanism of Rud3p function is unknown.

2.8. GRASP65

GRASP65 is a mammalian cis-Golgi protein, highly

conserved from yeast to mammals. GRASP65 is acylated

and is stably associated with Golgi membranes. GRASP65

has been implicated in the stacking of cis- and medial-

cisterna since antibodies to GRASP65, and a truncated

GRASP65, block cisternal stacking in a cell-free system

[72]. GRASP65 and GM130 interact in detergent extracts of

Golgi membranes under both interphase and mitotic

conditions, and this complex can bind the p115 tether

[73]. It is therefore possible that in addition to cisternal

stacking, GRASP65 participates in tethering COPII vesicles

(containing p115) to cis-Golgi by acting as a membrane

receptor for GM130.

2.9. GRASP55

GRASP55 (Golgi re-assembly stacking protein of 55

kDa) is homologous to GRASP65. Cryo-electron micro-

scopy localizes GRASP55 to the medial-Golgi. Recombi-

nant GRASP55 and anti-GRASP55 antibodies block the

stacking of Golgi cisternae, which is similar to the

observations made for GRASP65. These results suggest

that GRASP55 and GRASP65 may function in distinct

stages of stacking of Golgi cisternae [35]. The exact

mechanism of their action is unknown.

3. Multi-subunit complexes

3.1. TRAPP complexes

The TRAPP I (transport protein particle) complex

contains seven subunits (Bet5p, Trs20p, Bet3p, Trs23p,

Trs33p, Trs31p and Trs85p; Table 2) [74]. Additional three

subunits (Trs65p, Trs120p and Trs130p) are present in the

TRAPPII complex [75]. Biochemical characterization of

both yeast and human TRAPPs suggests that these

complexes are anchored to the Golgi [76]. The nature of

the TRAPP receptor(s) remains to be determined, but

recent structural studies provide insight into the mechanism

for TRAPP association with membranes. The crystal

structure of mouse Bet3 reveals a dimeric structure with

hydrophobic channels [77]. The channel entrances are

located on a putative membrane-interacting surface that is

distinctively flat, wide and decorated with positively

charged residues. Both, a channel-blocking mutation and

a charge-inversion mutation on the flat surface of the yeast

Bet3p lead to conditional lethality, incorrect localization

and membrane trafficking defects. These data suggest a

molecular mechanism for Golgi targeting and anchoring of

Bet3 that involves the charged surface and insertion of

Golgi-specific hydrophobic moieties into the channels. The

essential Bet3 subunit could then direct other TRAPP

components to the Golgi. The stable association of the

TRAPPs with Golgi membranes has been proposed to mark

these membranes for incoming COPII and COPI vesicles

[75].

Yeast TRAPPI has guanine nucleotide exchange factor

(GEF) activity and accelerates GDP/GTP exchange on the

cis-Golgi small Rab GTPase Ypt1p [78]. Mutants with

defects in several TRAPPI subunits are temperature

sensitive in their ability to displace GDP from Ypt1p, and

block secretion. In addition to acting as a GEF for Ypt1, co-

precipitation and overexpression studies suggest that

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339 331

TRAPPI also facilitates GDP/GTP exchange on trans-Golgi

localized Ypt31/32 [79].

Using chemically pure TRAPPI and COPII vesicles,

Ferro-Novick and colleagues reconstituted vesicle tethering

in vitro [80]. The binding of COPII vesicles to Golgi-

associated TRAPP I is specific, blocked by GTPgS, and,

surprisingly, does not require other tethering factors. These

findings have been interpreted as TRAPPI being the initial

receptor on the Golgi for COPII vesicles. Once the vesicle

binds to TRAPP I, the TRAPP I GEF activity activates

Ypt1p, leading to the recruitment of other tethering factors

[75]. The TRAPPII complex is proposed to mediate intra-

Golgi membrane trafficking [75].

Mammalian TRAPPI homologues have been identified

[81]. Semi-intact cell transport assays were used to explore

the function of mammalian Bet3 in ER–Golgi traffic [82].

Staging experiments with cytosols depleted of various

components suggest a sequential action of COPII > Bet3 >

Rab1 > a-SNAP > EGTA-sensitive step > GS28 SNARE

during ER-to-Golgi traffic of cargo proteins. This position-

ing of Bet3 is consistent with its function as a GEF for

Rab1.

Human wild-type SEDL protein (homolog of the Trs20

subunit) functionally complements yeast Trs20p [83]. A 2.4

A resolution structure of SEDL reveals an unexpected

similarity to the structure of the N-terminal regulatory

domain of two SNAREs, Ykt6p and Sec22b, despite no

sequence homology to these proteins [84]. This finding

suggests a possible interaction between subunits of the

TRAPP complexes and SNAREs (see below). However,

direct binding of TRAPPs to SNAREs has not been

documented.

3.2. COG (Conserved oligomeric Golgi) complex

The COG (conserved oligomeric Golgi) complex con-

sists of eight subunits (Table 2) [42,85–90]. The network of

inter-molecular interactions of the mammalian COG com-

plex, revealed by in vitro translation and co-immunopreci-

pitation approaches, was reported recently [90]. According

to these in vitro approaches, COG4 may serve as a core

component of the complex by interacting directly with

COG1, COG2, COG5 and COG7. A role for COG in

membrane trafficking was suggested by biochemical and

genetic studies in yeast that have identified a large number

of COG-interacting genes that encode proteins implicated in

Golgi trafficking [71,85,91]. The COG complex interacts

genetically and physically with Ypt1p, intra-Golgi SNARE

molecules, as well as with the COPI coat complex. In

addition, electron microscopy revealed that cog2 and cog3

temperature-sensitive yeast mutants accumulate vesicles at

the non-permissive temperature [92]. These findings led to

the hypothesis that the COG complex acts as a tether that

connects COPI vesicles with cis-Golgi membranes during

retrograde traffic [85]. In addition, the yeast COG has been

proposed to function as a vesicle tether in anterograde ER-

to-Golgi traffic [71,91,92]. COG is also involved in proper

localization of yeast enzymes in the trans-Golgi network

[93], and possibly in cargo sorting during exit from the ER

[94]. Whether all these functions are related to a tethering

role remains unknown.

COG3, COG4, COG5, COG6, and COG8 from yeast and

mammalian cells are structurally homologous. The remain-

ing COG1/LdlBp, COG2/LdlCp, and COG7 from yeast and

mammals are not structurally related, but may represent

functional counterparts [88]. As in yeast, mutations in COG

subunits (Cog1–8) have been shown to affect the structure

and function of the Golgi in Drosophila melanogaster

sperm, and in mammalian somatic cells [85,87,89,95,96].

Compromising COG function causes defects in glycosyla-

tion, intracellular protein sorting, protein secretion and, in

some cases, cell growth. For example, in CHO cells mutant

for Cog1 (ldlB) or Cog2 (ldlC), multiple Golgi cisternae are

dilated [87]. Such cells exhibit pleiotropic defects in a

number of medial- and trans-Golgi-associated glycosylation

reactions affecting virtually all N-linked, O-linked, and

lipid-linked glycoconjugates [42,85]. The diversity and

heterogeneity of protein glycosylation defects suggests that

the COG mutations affect the compartmentalization or

activity of multiple Golgi glycosylation enzymes without

substantially disrupting secretion or endocytosis. The

activities of glycosylating enzymes depend on their proper

intra-Golgi localization [97,98]. Thus, COG may play a role

directly or indirectly in the transport, retention, or retrieval

to appropriate cisternae of resident Golgi proteins. Alter-

natively, COG may play a role in maintaining Golgi

structure and/or lumenal environment.

Wu et al. [99] have recently described two human siblings

with congenital disorders of glycosylation caused by a

mutation in the gene encoding COG7. The mutation impairs

the integrity of the COG complex and alters Golgi trafficking,

resulting in disruption of multiple glycosylation pathways.

We have recently used siRNA strategy to achieve an

efficient knock-down of Cog3p in HeLa cells [100].

Cog3p depletion was accompanied by a reduction in

Cog1, 2 and 4 protein levels and by the accumulation of

COG complex-dependent (CCD) vesicles carrying v-

SNAREs GS15 and GS28 and cis-Golgi glycoprotein

GPP130. A prolonged block in CCD vesicles tethering is

accompanied by extensive fragmentation of the Golgi

ribbon. Fragmented Golgi membranes maintain their

juxtanuclear localization and cisternal organization, and

are competent for anterograde trafficking of VSVG protein

to the PM. In contrast, Cog3p knock-down resulted in the

inhibition of retrograde trafficking of the Shiga toxin.

Further, the mammalian COG complex physically interacts

with GS28 and COPI and specifically binds to isolated

CCD vesicles [100].

The COG complex appears structurally related to other

tethering complexes [88]. Iterative searches of databases

using the N-terminal domains of several COG components

revealed similarities in the N-terminal domains of compo-

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339332

nents of the exocyst and the GARP (Golgi-associated

retrograde protein) complex. It seems likely that the COG,

the exocyst and the GARP complexes are distantly related

multimeric assemblies evolved to tether membranes at

distinct stages of the secretory pathway.

3.3. GARP (Golgi-associated retrograde protein) complex

Vps51p, Vps52p, Vps53p, and Vps54p form a multi-

subunit complex (Table 2) required for protein sorting at

the yeast late Golgi. Mutation of VPS52, VPS53, or VPS54

results in the missorting of 70% of the vacuolar

carboxypeptidase Y, as well as the mislocalization of late

Golgi membrane proteins to the vacuole. These mutations

do not affect protein traffic through the early part of the

Golgi complex [101]. GARP subunits show distant

homology to components of two other tethering com-

plexes, the exocyst and the COG complex, suggesting that

tethering factors involved in different membrane traffic

steps may be structurally related [88].

GARP binds two trans-Golgi localized small GTPases,

Ypt6-GTP [102] and Arl1-GTP [62]. Vps51p mediates the

association of the GARP complex with the late Golgi t-

SNARE Tlg1p. As for other tethering proteins and

complexes, the binding of this small, coiled-coil protein to

the conserved N-terminal domain of the t-SNARE may

provide a functional link between components of the

tethering and the fusion machinery [103].

3.4. Dsl1 complex

DSL1 encodes an essential yeast 88 kDa ER-localized

peripheral membrane protein that can be extracted from the

membrane in a multi-protein complex. Immunoisolation of

the complex yielded Dsl1p and two additional proteins, one

of which was identified as Tip20p. Tip20p exists in a tight

complex with Sec20p [104]. Both Sec20p and Tip20p

function in retrograde Golgi-to-ER traffic are ER-localized

and bind to the ER t-SNARE Ufe1p. These findings suggest

that an ER-localized complex of Dsl1p, Sec20p, and Tip20p

may function in retrograde traffic, perhaps upstream of the

Ufe1p SNARE-mediated fusion.

The inviability of strains bearing several mutant alleles of

DSL1 can be suppressed by the expression of either Erv14p

(a protein required for the transport of specific proteins from

the ER to the Golgi), Sec21p (the gamma-subunit of the

COPI coat complex), or Sly1-20p (a SNARE-interacting

member of the Sec1/Munc family). Because the strongest

suppressor is SEC21, it suggests that Dsl1p functions

primarily in retrograde Golgi-to-ER traffic [105]. In support,

the dsl1-22 mutation causes severe defects in Golgi-to-ER

retrieval of ER-resident SNARE proteins and integral

membrane proteins harboring a C-terminal KKXX retrieval

motif, as well as of the soluble ER protein BiP/Kar2p, which

utilizes the HDEL receptor, Erd2p, for its recycling to the

ER. Furthermore, Dsl1p specifically binds to COPI vesicle

coat in vitro [106]. A highly acidic region in the center of

Dsl1p and containing crucial tryptophan residues is required

for binding to delta-COP1 [104] and to alpha-COP1 [107].

An additional N-terminal Tip20p binding region and an

evolutionarily well-conserved C-terminal domain have been

identified in Dsl1p, but their function remains to be

elucidated.

The Tagaya laboratory has recently found Dsl1-like

mammalian complex (ZW10, RINT-1 and p31) that interacts

with syntaxin 18, an ER-localized t-SNARE implicated in

membrane trafficking [108].

4. Function of tethering proteins in traffic

4.1. Static linking of membranes

The key unresolved question is how tethers promote

correct membrane pairings. Based on the morphological

observations of long proteinacous connections between

vesicles and the Golgi and between Golgi cisternae [109],

a model of tethers as static bridges spanning two membranes

has been proposed. The tether would ensure that the vesicle

remains within the vicinity of its target membrane, thus

increasing the possibility of fusion with that membrane.

Since tethers appear compartment-specific, each tether

would provide specificity to the bridging reaction and

impose membrane selectivity on the process.

Despite the wide acceptance of this mechanism of action,

there is limited information on the molecular details of this

process. Specifically, the identity of tether receptors on the

membranes is still unclear. Membrane association of

tethering factors has been shown to require active GTP-

bound forms of a Rabs, ARF or Arl. However, the exact

mechanisms by which active GTPases generate tether-

binding sites and tether assembly are unknown. We propose

that active Rabs (and perhaps other GTPases) participate in

generating a tether-binding site by coordinating tether

binding to SNAREs. This differs from previous models in

which tether formation is independent of and precedes

SNARE-mediated events (Figs. 3A versus B). Our model is

supported by recent evidence linking tether function with

SNARE assembly (see below).

It appears that many (if not all) tethers interact with

GTPases (Table 3). However, the nature of that interaction

may vary. For example, the coiled-coil tethers interact

exclusively with active Rabs in GTP-bound form [85,103]

and function as Rab effectors. In contrast, the multimeric

tethers TRAPP I complex and the HOPS complex involved

in endosomal/vacuolar tethering bind to non-activated Rabs

and participate in their activation [78,79,110]. Interestingly,

in addition to acting as a GEF for Rabs, the HOPS complex

also interacts with the GTP-bound Rab. Thus, the HOPS

complex is both a Rab GEF and a Rab effector.

It is possible that multiple tethering complexes facilitate a

single vesicular targeting step. For example, the TRAPP I

Fig. 3. Speculative models for tether function. (A) A tether is bound to

specific receptors on the vesicle and target membranes, creating a bridge.

The initial tethering is SNARE-independent. The tether may remain

ignorant of the downstream SNAREs or the tether may subsequently

interact with the SNAREs and facilitate their pairing. In this model, the

specificity of tethering is due to the restricted distribution of the tether

receptors. (B) A tether is bound to specific receptor on the vesicle and to the

t-SNARE on the target membranes, creating a bridge. All known tethers

have been shown to interact with a t-SNARE. The interaction of the tether

with the t-SNARE actively facilitates trans-SNARE pairing. In this model,

the specificity of tethering is determined by the position of the t-SNARE.

(C) A tether is bound to a v-SNARE on the vesicle and to a t-SNARE on

the target membranes, creating a bridge. The p115 coiled-coil tether has

been shown to interact with v- and t-SNAREs. The binding of the tether

actively facilitates trans-SNARE pairing. In this model, the specificity of

tethering is imposed by the dual proof-reading of v- and t-SNAREs. For

simplicity, other regulatory molecules that influence SNARE pairing are not

diagrammed. The experimental findings addressing each of these models

are discussed in the text.

Table 3

Interaction of Golgi tethering components with GTPases and SNAREs

Tethering factor GTPase SNARE

CASP/COY1

GCC88 Arl1/3 Sec22p, Gos1p [121]

GCC185 ARL1/3

GM130 Rab1, Rab2

GMAP210/RUD3 ARF [65]

Golgin-45 Rab2 [53]

Golgin-84 Rab1 [56]

Golgin-97 Arl1/3 Syntaxin16 [6]

Golgin-245 Arl1/3

p115/USO1 Rab1 [34]/Ypt1 [27] GS28, membrin, Syntaxin5

[37]

COG complex Ypt1 [85] Gos1, Sed5, Ykt6 [85]

Dsl1/ZW10

complex

Ufe1 [161,162]/Syntaxin18

[108]

GARP complex Ypt6 [102] Tlg1 [103,156]

TRAPP Ypt1, Ypt1 GEF [78,79],

YPT31/32 GEF [79]

Ykt6, Sec22b [74,82]

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339 333

complex and the coiled-coil tether p115/Uso1p appear

involved in ER–Golgi trafficking [24,30–32]. The TRAPP

complex activates Ypt1p/Rab1 and is therefore likely to

initiate the tethering cascade. A model can be proposed in

which the TRAPP complex acts upstream to activate Rabs

and thereby facilitate the recruitment of Rab effectors such

as the coiled-coil tethers (p115/Uso1p). Together, the two

classes of tethers would provide increasingly more stringent

layers of selectivity to membrane fusion. Thus, unique sets

of tethering factors together with step-specific Rabs and

SNARE complexes may control high fidelity of intracellular

membrane trafficking.

4.2. SNARE complex assembly

The majority of tethering coiled-coil proteins and multi-

subunit complexes have been shown to directly interact with

SNAREs (Table 3). Specifically, p115 interacts with a select

set of COPII vesicle-associated SNAREs (Syntaxin5,

membrin, GOS28) involved in ER–Golgi trafficking [34].

The COG complex interacts with the intra-Golgi SNARE

Sed5, Gos1 and Ykt6 [85], and the exocyst complex co-

immunoprecipitates with Syntaxin 1, a PM SNARE protein

critical for neurotransmission [111]. Within the endosomal/

TGN system, the GARP complex binds to the Tlg1p

SNARE [103].

The tether–SNARE interactions may reflect a ‘‘bridg-

ing’’ association in which the tether does not influence the

activity of the SNARE. Alternatively, tether binding may

actively promote SNARE complex assembly. A direct role

in SNARE complex assembly is suggested by abundant

genetic data in yeast. In all tested cases, the overexpression

of the SNAREs suppresses defects in tethering [85]. For

example, the overexpression of the ER–Golgi SNAREs

Bet1p and Sec22p suppresses the lethality of USO1 deletion

[24], indicating that Uso1p function is upstream of

SNAREs.

Experimental evidence for tethers directly promoting

SNARE complex assembly has been reported only for p115.

Shorter and colleagues have recently shown that the first

coiled-coil motif of p115 shares similarities with the

SNARE motif, suggesting a possible role of p115 in

SNARE dynamics. Using the SNARE-related domain from

p115, they showed that the addition of this p115 region

stimulates the specific assembly of endogenous Golgi

SNAREpins containing the t-SNARE syntaxin 5 in vitro

[37]. In agreement, experiments in vivo in mammalian cells

indicate that the p115 SNARE-interacting domain (rather

than its GM130/giantin binding domain assumed to mediate

tethering) is required for Golgi biogenesis. This suggests

that p115 acts directly, rather than via other tethers, to

catalyze trans-SNARE complex formation preceding mem-

brane fusion [44]. A direct role for p115 in SNARE

assembly also is in agreement with the finding that lack of

functional Uso1p prevents SNARE complex formation in

vivo [24]. It is likely that tethers other than p115/Uso1p also

facilitate SNARE complex assembly since similar suppres-

sion phenotypes by SNARE overexpression are observed

for other tethers.

The exact mechanism by which tethers facilitate SNARE

complex assembly is unknown. The activating event is

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339334

likely to involve a conformational change that exposes the

SNARE motif and facilitates the formation of the 4-helix

bundle. At least two scenarios may be proposed for such

activation. First, some t-SNAREs contain an auto-inhibitory

N-terminal domain that interacts with the SNARE domain

and thereby masks its availability for SNARE–SNARE

interactions. It is possible that tethering proteins bind to

such N-terminal regions to ‘‘open’’ them and thus alleviate

the inhibition. Experimental results document that the N-

terminal domain of Tlg1p binds the GARP complex [102],

and may increase the SNARE availability. Similarly, the N-

terminal domain of Vam2p binds HOPS, and such inter-

action facilitates fusion [112].

An alternative possibility is that tethers regulate the

activity of the Sec1/Munc (SM) family of proteins. The

manner in which SM proteins regulate SNAREs differ

[113], but several findings suggest that SM proteins

coordinate Rab and SNARE activities [114]. It is possible

that tethers may also participate in this process. For

example, the structural similarity between the Vps33

component of the HOPS complex and Sec1 proteins

suggests that Vps33 may facilitate similar events in SNARE

trans-complex formation.

4.3. Cargo selection

Recent findings suggest an unexpected role for tethering

factors in the organization of ER exit sites and cargo

selection at ER exit sites. The depletion of the Drosophila

homologue of p115 (dp115) by siRNA leads to the

predicted morphological changes in the Golgi stack [45].

In addition, dp115 depletion also influences the morphol-

ogy of ER exit sites. Using conventional and EM

microscopy, the Rabouille’s laboratory showed that Golgi

stacks are converted into clusters of vesicles and tubules,

and that ER exit sites (marked by the Sec23p component

of the COPII coat) lose their focused organization.

Surprisingly, this morphologically altered exocytic path-

way is nevertheless largely competent in anterograde

protein transport. These studies suggest that dp115 could

modulate the architecture of both the Golgi stacks and ER

exit sites.

Studies from the Riezman laboratory have shown that

GPI-anchored proteins exit the ER in distinct vesicles from

other secretory proteins [94]. When this sorting event is

analyzed in an in vitro assay, it requires functional Uso1p. In

addition to Uso1p, the COG complex also appears involved

since the Cog2p and Cog3p components are required for

sorting of GPI-anchored proteins from other secretory

proteins. The sorting also requires the Rab GTPase Ypt1p.

The sorting defect observed in vitro with uso1 and ypt1

mutants was reproduced in intact cells.

Golgin-45 also appears relevant for the correct function

of ER exit sites. In cells depleted of golgin-45 by siRNA,

resident Golgi proteins and anterograde cargo VSV-G

protein do not exit the ER [53].

In addition, tethers may directly ‘‘transit’’ specific cargo

proteins through the secretory pathway. For example,

GM130 specifically interacts with the human ether-a-go-

go-related gene (HERG)-encoded potassium channel. The

overexpression of GM130 suppressed HERG current

amplitude in Xenopus oocytes, suggesting retention prior

to delivery to the PM. These findings indicate that GM130

plays a previously undefined role in cargo selection exit

from Golgi [115].

GRASP55 at endogenous levels has been shown to

associate with the transmembrane TGF-alpha. C-terminal

mutations of TGF-alpha that decrease or abolish its

interaction with GRASP55 strongly impair the cell surface

expression of TGF-alpha. These observations suggest a role

for GRASP55 in escorting transmembrane proteins, includ-

ing TGF-alpha, during their transport to the cell surface

[116].

4.4. Coat events

Perhaps related to cargo selection is the described

interaction between p115 and GBF1 (Golgi-specific brefel-

din-A-resistant factor 1), a GEF for ARF [117]. GBF1 was

initially identified in a yeast two-hybrid screen with full-

length p115 as bait, and confirmed biochemically, using in

vitro and in vivo assays. p115 and GBF1 colocalize

extensively in the Golgi and in peripheral ERGIC. The

interaction occurs through the C-terminal proline-rich

region of GBF1 and the N-terminal head region of p115.

Mutagenesis analysis indicates that the interaction is not

required for targeting GBF1 or p115 to membranes. The

expression of the p115-binding region of GBF1 in cells

leads to Golgi disruption, suggesting that the interaction

between p115 and GBF1 is functionally relevant. A model

can be postulated in which the p115 tether acts as a

multifunctional platform: p115 facilitates anterograde fusion

events by stimulating SNARE complex assembly, and

simultaneously initiates recycling events by recruiting

GBF1 and initiating ARF activation and COPI recruitment

to form retrograde COPI vesicles. Whether other tethers

have a similar mechanism of action remains to be explored.

4.5. Cytoskeletal events

Rather than tethering membranes together, some Golgi-

localized coil-coiled proteins link membranes to the

cytoskeleton, thus adding a new function for this class of

proteins. GMAP210 binds to microtubules and has been

postulated to link Golgi membranes to centrosomes [66].

Similarly, bicaudal-D1 (BICD1) and -D2 (BICD2) link

vesicles to the cytoskeleton [118]. BICD1 and BICD2 bind

to Rab6, colocalize with Rab6a on the TGN and on

cytoplasmic vesicles, and associate with membranes in a

Rab6-dependent manner [119,120]. The overexpression of

BICD1 enhances the recruitment of dynein–dynactin to

Rab6a-containing vesicles. Conversely, the overexpression

V. Lupashin, E. Sztul / Biochimica et Biophysica Acta 1744 (2005) 325–339 335

of the carboxy-terminal domain of BICD, which can interact

with Rab6a but not with cytoplasmic dynein, inhibits

microtubule minus-end-directed movement of green fluo-

rescent protein (GFP)-Rab6a vesicles [120]. Whether other

golgins also participate in tethering vesicles to microtubules

remains to be defined.

5. Conclusion

Despite remarkable progress, the details of the temporal

and spatial relationships between the distinct types of

tethering complexes, SNAREs, and Rabs remain to be

defined. Based on their relationship to Rab activation, the

various tethering complexes can be ordered as those that

facilitate Rab activation and those that act as Rab effectors.

Within the ER–Golgi leg of the secretory pathway, the

TRAPP complexes act as Rab1/Ypt1 activators and are likely

to initiate the tethering cascade. Subsequently, Rab1/Ypt1-

dependent tethers, namely p115/Uso1p and COG, can be

assembled and impose additional level of pre-fusion selec-

tivity. Based on the current information, it appears that unlike

Rabs and SNAREs that appear to share mechanisms of

action, distinct tethers utilize distinct mechanisms to facilitate

membrane traffic. A sub-class of coiled-coil tethers contain

the Golgi targeting GRIP domain and, rather than binding

Rabs, interact with and are recruited tomembranes by another

class of GTPases, the Arls. Current evidence therefore

suggests that Golgi coiled-coil proteins function in a variety

of membrane–membrane and membrane–cytoskeleton teth-

ering events at the Golgi apparatus, and that all these are

regulated by small GTPases of the Rab and Arl families. It is

very likely that further investigation of the role of different

tethering factors in membrane traffic will reveal much that is

interesting and surprising.

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