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Organization of SNAREs within the Golgi Stack

Jo¨rg Malsam and Thomas H. So¨llner

Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany Correspondence: [email protected]

Antero- and retrograde cargo transport through the Golgi requires a series of membrane fusion events. Fusion occurs at the cis- and trans-side and along the rims of the Golgi stack. Four functional SNARE complexes have been identified mediating merger in the Golgi. Their function is tightly controlled by a series of reactions involving vesicle tethering and SM . This network of interactions spatially and tem- porally determines the specificity of transport vesicle targeting and fusion within the Golgi.

t steady state, the Golgi maintains its struc- compartments along the secretory and endo- Atural and functional organization despite cytic pathway (Jahn and Scheller 2006; Malsam a massive lipid and protein flow. A balanced et al. 2008). On a functional/topological basis, anterograde and retrograde membrane flow SNAREs can be classified as v- and t-SNAREs, are required to constantly recycle the transport because they are usually active on vesicles and machinery and cargo containers (vesicles). In target membranes (So¨llner et al. 1993b). In the absence of efficient recycling, directional the case of homotypic fusion, a clear distinction net cargo transport would cease and the Golgi between the two compartments is not possible, would collapse. Thus, transport vesicles con- however, the reaction mechanisms driving fu- stantly leave and enter at both sides of the Golgi sion are comparable to those of heterotypic fu- stack and bud and fuse along the rims of the sion. On a structural basis, SNARE motifs, the cisternae. To maintain the compartmental hallmark signature characterizing all SNARE identity, vesicle fusion occurs in a specific and family members, provide the underlying prin- orchestrated manner. These fusion events are ciple to understand SNARE complex assembly mediated by a cascade of reactions centered and regulation (Fasshauer et al. 1998). Upon around the membrane fusion proteins SNAREs pairing of a v-SNARE with its cognate t-SNARE (SNAP receptors) (So¨llner et al. 1993b). on opposing membranes, the SNARE motifs assemble to form trans-v/t-SNARE com- plexes, also termed SNAREpins, a process that SNARE COMPLEX ASSEMBLY AND occurs in a zipper-like manner starting at DISASSEMBLY the amino-terminal membrane-distal ends and SNAREs represent a family of tail-anchored progressing toward the carboxy-terminal mem- membrane proteins, whose individual mem- brane-proximal regions of the SNAREs (Melia bers are localized to distinct intracellular et al. 2002; Pobbati et al. 2006). This assembly

Editors: Graham Warren and James Rothman Additional Perspectives on The Golgi available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a005249 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a005249

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J. Malsam and T.H. So¨llner

reaction pulls the two opposing membranes (Fig. 1). With the exception of a central “0” together to overcome the repulsive forces of layer, which usually contains either a hydro- the surrounding aqueous environment, which philic glutamine or an arginine residue, all provides an energy barrier for lipid bilayer other layers are characterized predominantly merger. Several in vitro and in vivo studies by hydrophobic side chains. Based on the have established that SNAREs are sufficient to composition of the central layer drive membrane merger, and thus SNAREs and other structural features, the classification: have been considered to be the minimal mem- Qa-, Qb-, Qc-, and R-SNAREs were introduced brane fusion machinery (Weber et al. 1998; in addition to the topological/functional Hu et al. 2003). The fully assembled postfusion v- and t-SNAREs definition (Fasshauer et al. cis-SNARE complex, now located in a single 1998). Functional SNARE complexes always lipid bilayer, becomes a substrate for SNAPs contain one member of these four SNARE (soluble NSF attachment factors), which in subfamilies. turn recruit the hexameric ATPase NSF For fusion to occur, the SNARE motif and (N-ethylmaleimide sensitive factor) (Block et al. the trans-membrane domain need to be directly 1988; Whiteheart et al. 1993). Both SNAPs coupled, indicating that the folding of the cyto- and NSF are cytosolic proteins acting on all solic SNARE motifs provides direct physical cis-SNARE complexes at the various transport work on the lipid bilayer (Grote et al. 2000; steps. ATP hydrolysis by NSF dissociates the McNew et al. 2000b). Indeed, the crystal SNARE complexes resulting in largely unstruc- structure analysis of the assembled neuronal tured SNARE proteins, which now can be v-/t-SNARE complex, containing the trans- recycled for another round of transport (So¨llner membrane regions, revealed a helical extension et al. 1993a; Mayer et al. 1996). Thus, membrane of the SNARE motifs into the membrane (Stein fusion is driven by protein folding (v-/t- et al. 2009). SNARE complex assembly), and a net input of Remarkably, most fusogenic SNARE com- energy (ATP hydrolysis) is required to unfold plexes show a strict topological restriction. the SNAREs. Thus, when a SNARE complex is built from its cognate Qabc- and R-SNAREs, the SNAREs need to be distributed in a defined manner SNARE STRUCTURE between the two membranes to be fused. In The SNARE motif is located in the cytosolic most cases, only one out of eight possible topo- domain of the SNAREs and in close proximity logical distributions appears to be functional as to the trans-membrane domain (Fasshauer has been shown by in vitro reconstitution et al. 1998). An individual SNARE protein usu- experiments (McNew et al. 2000a; Parlati et al. ally contains a single SNARE motif. Exceptions 2000). However, in the endosomal system, are members of the SNAP-25 subfamily, which mediating homotypic fusion, other topological contain two SNARE motifs joined in a single combinations have been found to fuse recon- polypetide chain. The SNARE motif consists stituted liposomes (Zwilling et al. 2007). Bio- of a stretch of about 60–70 amino acids, which chemical studies have shown that t-SNAREs contains heptad repeats that have a propensity form stable subcomplexes, which function as to form coiled-coil structures (Fig. 1). However, templates for v-SNARE binding (Fasshauer in the absence of a binding partner, SNARE and Margittai 2004). A functional t-SNARE motifs are largely unstructured (Fasshauer complex always contains one molecule of the et al. 1997). When a functional (fusogenic) /Qa-SNARE subfamily and usually v-/t-SNARE complex forms, four SNARE comprises three partially assembled SNARE motifs assemble into a stable four-helix bundle motifs, which in many cases match the Qabc- (Sutton et al. 1998). Based on the presence of composition. (The syntaxin/Qa-SNARE and the heptad repeats, 16 stacked layers of amino the nonsyntaxin t-SNARE components have acid side chains stabilize the four-helix bundle also been called t-SNARE heavy and light

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Organization of SNAREs within the Golgi Stack

A

TM B –7 –6 –5 –4 –3 –2 –10 +1 +2 +3 +4 +5 +6 +7 +8 Stx 18 DEVRQIEGRVVEISRLQEIFTEKVLQQEAEIDSIHQLVVGATENIKEGNEDIREAIKNNAGFR–VWILFFLVMCSFSLLFLDWYDS– Stx 5 DTMQNIESTIVELGSIFQQLAHMVKEQEETIQRIDENVLGAQLDVEAAHSEILKYFQSVTSNRWLMVKIFLILIVFFIIFVVFLA––– Qa Stx 16 REIRQIVQSISDLNEIFRDLGAMIVEQGTVLDRIDYNVEQSCIKTEDGLKQLHKAEQYQKKNRKMLVILILFVIIIVLIVVLVGVKSR

SEC20 ESLMGISRMMAQQVQQSEEAMQSLVTSSRTILDANEEFKSMSGTIQLGRKLITKYNRRELTDKLLIFLALALFLATVLYIVKKRLFPFL Membrin SSLQKVHNGMDDLILDGHNILDGLRTQRLTLKGTQKKILDIANMLGLSNTVMRLIEKRAFQDKYFMIGGMLLTCVVMFLVVQYLT–––– Qb GOS28 DHLRNSDRLIEETISIAMATKENMTSQRGMLKSIHSKMNTLANRFPAVNSLIQRINLRKRRDS–LILGGVIGICTILLLLYAFH––––– VTIla RRLEAGYQIAVETEQIGQEMLENLSHDREKIQRARERLRETDANLGKSSRILTGMLRRIIQNRILLVILGIIVVITILMAITFSVRRH–

USE1 KLAEEMLGLARSLKTNTLAAQSVIKKDNQTLSHSLKMADQNLEKLKTESERLEQHTQKSVNWLLWAMLIIVCFIFISMILFIRIMPKLK BET1 RLTESLRSKVTAIKSLSIEIGHEVKTQNKLLAEMDSQFDSTTGFLGKTMGKLKILSRGSQT––KLLCYMMLFSLFVFFII–YWIIKLR– RMADSLASKVTRLKSLALEIDRDAEDQNRYLDGMDSDFTSMTSLLTGSVKRFSTMARSGQDNRKLLCGMAVGLIVAFFILSYFLSRART Qc GS15 Stx 6 EQLELVSGSIGVLKNMSQRIGGELEEQAVMLEDFSHELESTQSRLDNVMKKLAKVSHMTSDRRQWCAIAILFAVLLVVLILFLVL––––– Stx 10 QQLEMVSGSIQVLKHMSGRVGEELDEQGIMLDAFAQEMDHTQSRMDGMLRKLAKVSHMTSDRRQWCAIAVLVGVLLLVLILLFSL––––

Sec22b RNLGSINTELQDVQRIMVANIEEVLQRGEALSALDSKANNLSSLSKKYRQDAKYLNMHSTY––AKLAAVAVFFIMLIVYVRFWWL–––––– YKT6 DPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAI–––––––––––––––––––––––––––––– R VAMP3 RRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS––– VAMP4 DKIKNVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT

Figure 1. Structural organization of the SNARE four-helix bundle and SNARE motifs of ER and Golgi SNAREs. (A) Skeleton diagram of the synaptic SNARE four-helix bundle showing interacting layers (image is modified from Fasshauer et al. [1998] and reprinted, with permission, from the National Academy of Sciences #1998). (B) SNARE motifs and membrane anchors of human SNAREs involved in ER and Golgi transport were aligned using the MULTALIN program (Corpet 1988). Interacting layers are shown in gray; amino acids in “0” layer and trans-membrane regions are highlighted in color. Ykt6 contains a CAXX box, which is farnesy- lated and palmitoylated. The Qa-, Qb-, Qc-, and R-SNARE classification is shown and SNAREs are listed accord- ing to their position in the secretory pathway: SEC20 (NP_053583.2), USE1 (NP_060937.1), BET1 (NP_005859.1), GS15 (AFF37877.1), syntaxin 6 (syx6) (CAA05177.1), syntaxin 10 (syx10) (AAC05087.1), SEC22b (NP_004883.2), YKT6 (NP_006546.1), VAMP3(NP_004772.1), VAMP4(NP_003753.2), syntaxin 18 (BAA95213.1), syntaxin 5 (AAC71078.1), syntaxin 16 (syx16) (AAC05647.1).

chains, respectively.) Other SNARE topologies they contain regulatory domains, which are might form transient SNARE complexes, which found at their amino-termini and control however are not stable enough to allow SNARE- SNARE motif accessibility and structure. The pin formation across membranes, or provide type of regulatory domains differs between not enough energy to drive membrane fusion. SNARE subfamilies (Malsam et al. 2008). This topological restriction has important Based on a comparative analysis of several implications for the regulation of membrane model organisms, the four subfamilies can be fusion, because a functional t-SNARE complex further divided into 20 distinct conserved func- will only form when all three t-SNARE compo- tional subgroups, which might represent the nents reside within the same membrane. Fur- minimal repertoire of a proto-eukaryotic cell thermore, even when all three cognate SNARE (Kloepper et al. 2007). During evolution, the components are within the same membrane, SNARE sets were modified by duplication and regulatory components still control the assem- diversification (Dacks and Field 2007; Kloepper bly of the t-SNARE complex and subsequent et al. 2007). In particular, the SNARE repertoire v-t-SNARE complex formation. /Qa- in the endosomal and exocytic transport steps SNAREs are key control sites for SNARE- has undergone a substantial expansion, most complex assembly. Like many other SNAREs, likely to adapt to the rising complexity of these

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J. Malsam and T.H. So¨llner

trafficking routes in metazoa. By contrast, a very could theoretically form at the Golgi, only two limited number of different SNAREs operates at distinct SNARE complexes are capable of driv- the and Golgi complex, ing membrane fusion (McNew et al. 2000a; Par- suggesting that the initial fusion steps of the lati et al. 2000; Parlati et al. 2002). Remarkably, secretory route are highly conserved through these studies also revealed that one particular evolution (Kienle et al. 2009). SNARE protein can operate in two different complexes, indicating that nature uses a combi- natorial code to make efficient use of a limited GOLGI SNAREs number of genetically encoded SNAREs (Parlati The Golgi of mammals and plants is a highly et al. 2002; Shorter et al. 2002). Both of these polarized compartment, which is comprised Golgi SNARE complexes share the same syn- of at least three distinct cisternae: cis, medial, taxin/Qa molecule: syntaxin 5 in mammals, and trans (Farquhar and Palade 1998). On Sed5p in yeast (Parlati et al. 2002). One complex the cis-side, the Golgi is preceded by the in- consists of syntaxin 5/Sed5p, membrin/Bos1p termediate compartment (IC), (also named (also known as GS27) and ERS24/Sec22p, ER-Golgi intermediate compartment [ERGIC] which assemble to form the t-SNARE, and or cis-Golgi network [CGN]), which receives rBet1/Bet1p functioning as the cognate v- membrane transport intermediates from the SNARE (Fig. 2) (Xu et al. 2000). In the second endoplasmic reticulum (ER) (Appenzeller- complex, syntaxin 5/Sed5p forms a different Herzog and Hauri 2006). On the trans-side, t-SNARE with GOS28/Gos1p (also known as the Golgi is succeeded by the trans-Golgi Net- GS28) and Ykt6/Ykt6p to create a binding site work (TGN), which obtains transport vesicles for the cognate v-SNARE Gs15/Sft1p. Whereas from two distinct endosomal compartments: the former complex is implicated in transport the early and late endosomes. Thus, in the sim- from the ER to the Golgi, and possibly in intra- plest model, these three membrane trafficking Golgi transport between subsequent cisternae steps that deliver material to the Golgi require within the Golgi stack, the latter complex might three different v-/t-SNARE complexes. Addi- exclusively function in COPI-dependent intra- tional SNARE complexes might be required to Golgi transport (Newman and Ferro-Novick mediate vesicular transport at the rims of the 1987; Newman et al. 1990; Shim et al. 1991; cisternae, functioning in the retrieval of Golgi Hardwick and Pelham 1992; Subramaniam resident proteins and the anterograde transport et al. 1996; Zhang et al. 1997; Cao and Barlowe of cargo in a percolating fashion within the 2000; Zhang and Hong 2001; Xu et al. 2002; Golgi stack. Because of variable numbers of Volchuk et al. 2004). The distribution of these cisternae within different cell types, and the SNAREs within the Golgi in budding profiles genetic restriction of SNARE diversity, it natu- and COPI-coated vesicles was also analyzed rally follows that one or a few SNARE complexes by electron microscopy (Nagahama et al. 1996; mediate inter-cisterna transport. Todate, a total Paek et al. 1997; Orci et al. 2000; Volchuk et al. of four functional SNARE complexes have been 2004). By quantitative analysis, syntaxin 5 has identified in association with the Golgi (Fig. 2). been shown to distribute evenly throughout A fifth complex, which mediates retrograde the stack, confirming its role as the general Golgi transport to the ER is mentioned here, because syntaxin/Qa SNARE. By contrast, a comparison the Golgi contains SNAREs involved in this of the two specific v-SNAREs revealed that they transport step. have opposite distributions within the stack. The majority of Golgi-localized SNARE Whereas the v-SNARE GS15 is present in a gra- complexes and their individual components dient of increasing concentration toward the have been analyzed in great detail in in vitro trans-face of the stack, its counterpart Bet1 pro- and in vivo studies. Reconstituted membrane gressively decreases in concentration from the fusion assays have provided evidence that CGN toward the trans-face (Fig. 2) (Volchuk among 147 tested SNARE complexes, which et al. 2004). In fact, the distribution of both

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Organization of SNAREs within the Golgi Stack

ER-Golgi/intra-Golgi intra-Golgi

? ? trans Ykt6 ERS24 Ykt6p

Sec22p Golgi Golgi medial ? ? TRAPPII/ COG/ GOS28 Gos1p membrin ? Bos1p ? cis ? ? Golgins Gs15 Sft1p Sedp5 TRAPPI/ Sed5p IC IC IC ? ? ? syntaxin 5 ?

Golgins syntaxin 5

Bet1p rBet1

ER ER Q -SNARE Yeast Metazoa a Yeast Metazoa Qb-SNARE

Qc-SNARE Golgi-ERR -SNARE Vacuole/Endosome-Golgi

Vacuole Early Endosome Late Endosome Golgi Snc1p VAMP4 VAMP3

Tlg2p syntaxin 16 syntaxin 16 GARP/ IC Vti1p TGN Vti1a Vti1a syntaxin 10 VFT Tlg1p syntaxin 6 Sec22p ERS24

Ufe1p syntaxin 18 Golgi Dsl1 Sec20p ER SEC20 Use1p USE1 Yeast Metazoa Yeast Metazoa

Figure 2. The localization and assignment of SNAREs and tethers to distinct membrane-trafficking steps at the Golgi of mammals and yeast. Four different sets, each containing one member of Qa-, Qb-, Qc-, and R-SNAREs mediate delivery to and transport within the Golgi. According to a morphological and quantitative analysis, the local distribution of some SNAREs involved in intra-Golgi transport is displayed. Tethering components are highlighted in gray. (ER: endoplasmic reticulum, IC: intermediate compartment, TGN: trans-Golgi network.)

v-SNAREs together with their cognate Qb- complexes might also become substrates for SNAREs across the Golgi appears as a mirror SNAP and NSF, resulting in complex disassem- image and supports the concept that these bly. Nevertheless, it has been shown in in vitro SNARE complexes mediate membrane traffick- studies that noncognate SNARE complexes ing in opposite directions. In immunoprecipi- can affect membrane fusion (Varlamov et al. tation studies, syntaxin 5 has been detected in 2004). A reconstituted liposome fusion assay a variety of other noncognate complexes, but revealed that the cis-Golgi SNAREpin was these might form spontaneously in an unspe- inhibited by the addition of SNAREs that local- cific manner only after detergent lysis (Tsui ize to the late-Golgi. Conversely, the late-Golgi et al. 2001). When reconstituted into liposomes, SNAREpin was inhibited by the addition of these noncognate complexes are not fusogenic cis-Golgi SNAREs. Because both SNARE com- (Parlati et al. 2002). If such noncognate SNARE plexes form gradients of opposite distributions complexes form in significant amounts in vivo within the stack, cognate SNAREs greatly out- is unclear. A spatial separation into membrane number SNAREs that are concentrated at the subcompartments, in combination with a other end of the stack. Although this situation proteinaceous machinery controlling SNARE would favor the formation of cognate SNARE- complex assembly, might efficiently prevent the pins, SNAREs meant to function at the other formation of nonfunctional complexes. Such end of the Golgi would become sequestered.

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J. Malsam and T.H. So¨llner

Thus, such inhibitory i-SNAREs may be an syntaxin 16/Vti1a/syntaxin 10/VAMP3)are lo- effective tool to sharpen the continuous gra- calized to the TGN and share two components dient of SNAREs within the Golgi stack. The in the corresponding t-SNARE subcomplexes physiological relevance of this concept remains (syntaxin 16/Vti1a) (Mallard et al. 2002; Gan- to be established. A systematic study analyzing ley et al. 2008). Thus, the presence of either syn- the presence of SNARE (sub) complexes at taxin 6 or syntaxin 10 in these two t-SNARE steady state within the Golgi stack in living cells complexes apparently provides enough infor- has been difficult because of the limited spatial mation to specify by itself or in connection resolution of light microscopy. However, ad- with other factors, if either VAMP4 or VAMP3 vances in microscopic techniques in combi- liposomes will fuse with the TGN. The syntaxin nation with the introduction of site-specific 16/Vti1a/syntaxin 6/VAMP4 complex is in- fluorescent labels showing FRET will make volved in retrograde transport from early endo- such an endeavor possible in the near future. somes and the syntaxin 16/Vti1a/syntaxin 10/ The Qa-SNARE syntaxin 5/Sed5p is not the VAMP3 complex in retrograde transport from only Golgi SNARE that is found in different late endosomes (Ganley et al. 2008). In yeast, functional complexes. As already mentioned, the Tlg2p/Vti1p/Tlg1p/Snc1p complex is the R-SNARE Sec22p functions together with implicated in retrograde transport to the TGN syntaxin 5/Sed5p and membrin/Bos1p as (Holthuis et al. 1998; Paumet et al. 2001). t-SNARE in anterograde transport from the ER to the Golgi. Sec22p is also part of a SNARE SNARE LOCALIZATION complex, which mediates retrograde transport from the Golgi to the ER. This complex con- Because SNAREs show compartmental specific- tains the syntaxin 18/Ufe1p, SEC20/Sec20p, ity and provide the road map for membrane and USE1/Use1p as cognate SNARE compo- trafficking, mechanisms must exist that target nents (Lewis et al. 1997; Hatsuzawa et al. 2000; SNAREs to their final destinations and retain Burri et al. 2003; Dilcher et al. 2003). Although, them in place. Two types of localization signals USE1 does not contain an R or a Q in the central are involved in this process. layer of the SNARE motif, it has been classified First, the membrane anchors of SNAREs, as a Q-SNARE family member based on other in particular the length and the degree of hy- criteria (Fasshauer et al. 1998; Dilcher et al. drophobicity of the trans-membrane domain 2003). If Sec22p functions in this complex as a (TMD) play a role. Distinct compartments v- or t-SNARE remains to be shown. This issue along the secretory pathway differ in their lipid could be directly resolved with liposome recon- bilayer thickness, which increases toward the stitution experiments testing which topological plasma membrane (Bretscher and Munro SNARE combination is fusogenic. Nevertheless, 1993; Munro 1995; Reggiori et al. 2000). In an in vitro assay measuring retrograde transport the case of the ER syntaxin/Qa-SNARE Ufe1p, from the Golgi to the ER already indicates that it has been shown that alterations in the amino Sec22p and potentially Bet1 can function as retro- acid composition, or an increased length of the grade-directed v-SNAREs (Spang and Schekman TMD result in mislocalization of this t-SNARE 1998). A study analyzing the interaction of component (Rayner and Pelham 1997). Ykt6, regulatory proteins with components of the functioning in intra-Golgi transport, lacks a retrograde Golgi–ER SNARE complex further trans-membrane region but it is palmitoylated supports that Sec22p operates as a v-SNARE and farnesylated and contains a longin do- (Kraynack et al. 2005). Thus, a single SNARE pro- main, which together determine the intracellu- tein might function as part of a t-SNARE in one lar distribution of Ykt6 (Fukasawa et al. 2004; direction and as v-SNARE binding to a distinct Meiringer et al. 2008). In addition, membrane t-SNARE in the opposite transport reaction. subdomains and lipid interactions might play The remaining two SNARE complexes a role in SNARE localization. In the case of (syntaxin 16/Vti1a/syntaxin 6/VAMP4 and syntaxin 1, a syntaxin/Qa-SNARE involved

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Organization of SNAREs within the Golgi Stack

in regulated exocytosis, cholesterol-dependent Whereas the sorting sequence of the v-SNARE clusters have been observed in the plasma mem- Bet1p is exposed in the free v-SNARE but brane (Lang et al. 2001; Sieber et al. 2007). The sequestered in the four-helix SNARE bundle, partitioning of Golgi SNAREs into membrane the sorting signal of Sed5p is hidden in an auto- microdomains has not been determined yet. inhibitory Sed5p conformation, but is accessi- The second type of SNARE sorting signals ble in the Sed5p/Bos1p/Sec22p t-SNARE mediates the direct and specific binding to dis- complex (Mossessova et al. 2003). This data tinct vesicle coat components, which drive indicates that the vesicle coat not only selects vesicle budding, cargo sorting, and determine but also sequesters cognate v- and t-SNAREs the vesicle size (see Popoff et al. 2011). This in their active conformation and thus primes type of interaction becomes of particular im- these vesicles for homotypic fusion. portance, when SNAREs have already reached Such structural information is not available their final destination and now function as for COPI-vesicles. Nevertheless, morphological fusion machinery, e.g., as a v-SNARE at a and biochemical studies show that SNAREs are defined trafficking step. In this case, a budding also recruited into COPI vesicles. Interactions of vesicle needs to incorporate a “functional” Bet1p and Sec22p with Glo3p and Arf1 have v-SNARE to allow the fusion with the target been reported (Rein et al. 2002). In addition compartment, because vesicles lacking SNAREs to SNARE interactions, Glo3p also binds cargo would become dead end transport intermedi- proteins, COPI coat proteins, and functions as a ates. In the early secretory pathway, SNARE- GTPase-activating protein for Arf1p, which in coat interactions have been intensively studied its GTP-bound state recruits COPI coats (coat- in the anterograde ER–Golgi transport step. omer) to the Golgi. These interactions provide In this case, COPII coated vesicles bud at the an interesting analogy to the recruitment of ER and can fuse either with each other in a SNAREs into COPII vesicles, in which Sec23 homotypic fashion generating the cis-Golgi functions as GTPase-activating protein for network (CGN) or with the CGN (Xu and Sar1p and together with Sec24 recruits SNAREs, Hay 2004). Indeed, COPII vesicles contain all cargo, and the COPII proteins (Springer and SNAREs required to form the functional com- Schekman 1998; Mossessova et al. 2003). plex (Sed5p, Sec22p, Bos1p, and Bet1p) for Furthermore, Arf1 itself in its activated GTP- this fusion step. In vitro reconstituted budding bound state is capable of selectively binding assays revealed that Sec22p and Bos1p were the SNAREs GS15, Ykt6, and membrin, but enriched in COPII vesicles (Matsuoka et al. not GOS28 (Honda et al. 2005; Lee et al. 1998). By contrast, the syntaxin/Qa-SNARE 2005). Nevertheless, GOS28 is enriched at Golgi Ufe1p, which forms a functional SNARE com- rims and in COPI vesicles, but it is unclear plex with Sec20p, Sec22p, and Use1p for retro- whether GOS28 is recruited into COPI vesicles grade Golgi–ER transport, was excluded from by direct coat protein interactions or by a sto- these vesicles. Bet1p and Bos1p interact with chastic process (Nagahama et al. 1996). In con- the COPII subunits Sec23/Sec24 and in the trast, the membrane surface concentration of presence of either GTPor GDP with the GTPase syntaxin 5 in COPI vesicles is twofold lower Sar1p, which in its GTP-bound state recruits than in the Golgi stack, which is consistent COPII-coats onto the ER-membrane (Springer with a requirement for t-SNAREs and in partic- and Schekman 1998). Remarkably, structural ular syntaxin/Qa-SNAREs in the cisternae analyses suggest that COPII coats selectively (Fig. 2) (Volchuk et al. 2004). Furthermore, in package the active v- and t-SNAREs into bud- COPI vesicles, the concentrations of v-SNAREs ding COPII vesicles. The sorting signals are and t-SNARE light chains exceed the concentra- short peptides, which provide low affinity inter- tion of syntaxin 5 at least by a factor of 2. This actions with the coat components, and their ratio might ensure that excess free v-SNAREs availability depends on the conformational are available for membrane fusion, whereas state of the SNARE (Mossessova et al. 2003). the t-SNARE components found in the same

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J. Malsam and T.H. So¨llner

vesicle might cycle as cargo through the Golgi members associated with the Golgi are named stack. In the cisternal maturation/progression Golgins (see Munro 2011). The other family model, in which cisternae form at the cis-Golgi, contains heterogeneous assemblies of multisu- progress along the Golgi, and finally disassem- bunit protein complexes, which operate not ble at the trans-Golgi, t-SNARE components exclusively at the Golgi, but also on other organ- need to be present in retrograde-directed COPI elles. The relevant multisubunit complexes vesicles to ensure fusion machinery recycling associated with the ER and the Golgi are: (see Glick and Luini 2011). Dsl1, TRAPPI/II, COG, and GARP/VFT (see Likewise, some of the three potential Table 1). Many of these proteins directly bind v-SNAREs (Sec22p, Bet1p, GS15p) present in specific t-SNARE components, either COPI a single COPI vesicle might travel as inactive or COPII vesicle coats, and interact in a GTP- cargo proteins in the process of being recycled dependent manner with distinct compart- between compartments. Alternatively, the indi- ment-specific small GTP binding proteins vidual v-SNAREs might be located to separate called Rabs (Stenmark 2009). COPI vesicle populations, fusing either with The TRAPPI and TRAPPII complexes differ the ER, the CGN/cis-Golgi, or at the rims of significantly from the other multisubunit teth- the Golgi stack. This question still remains to ers, because they function as guanine nucleo- be resolved and will require the isolation of tide exchange factors (GEF) for Rabs and do COPI vesicle using antibodies directed against not directly bind SNAREs. The TRAPPI com- individual v-SNAREs. plex is localized at the cis-side of the Golgi Altogether, SNAREs seem to be recruited and is implicated in anterograde transport into both COPII- and COPI-coated vesicles by from the ER to the Golgi and COPII vesicle teth- a similar mechanism that requires interactions ering (Sacher et al. 2001; Kim et al. 2006). It with coat components, Arfs and Arf-GAPs. functions as a GEF for the protein Ypt1 Interestingly, the cold-sensitive phenotype of a (Jones et al. 2000; Wang et al. 2000; Sacher Glo3 deletion mutant can be rescued by the et al. 2001). TRAPPII is implicated in intra overexpression of Bet1p, Bos1p, and Sec22p Golgi and retrograde endosome-to-TGN trans- indicating that in the absence of a selective port (Cai et al. 2005). It interacts with COPI- SNARE uptake mechanism, the stochastic coats and functions as a GEF for Ypt1 and incorporation of SNAREs into transport Ypt31p/Ypt32p (Morozova et al. 2006). Thus, vesicles can suffice to ensure fusion competence the GEF activities of TRAPPI/II complexes and cell survival (Poon et al. 1999). recruit specific Rab proteins to budding COPII Following vesicle budding, pinching off, and and COPI vesicles, respectively. uncoating, a vesicle fuses with its target com- The GARP or Vps fifty-three (VFT) com- partment requiring the presence of all three plex is implicated in retrograde transport from t-SNARE components in a functionally active early and late endosomes to the TGN (Siniosso- conformation. The assembly of a t-SNARE com- glou and Pelham 2001; Conibear et al. 2003; plex is mediated by regulatory proteins, which Quenneville et al. 2006). It consists of four also function as transport step-specific vesicle proteins (Vps51/52/53/54). In yeast, Vps51 tethering components. Thus, the location and directly binds the amino-terminal domain of type of tether make an important contribution, Tlg1p and may release the putative autoinhibi- where a functional t-SNARE complex forms. tion of the amino-terminal domain of Tlg1p thus favoring t-SNARE complex formation (Siniossoglou and Pelham 2001; Conibear SNARE INTERACTIONS WITH TETHERS et al. 2003; Fridmann-Sirkis et al. 2006). In Prior to SNAREpin formation, transport ves- mammals, the GARP/VFT complex interacts icles are captured by two types of mem- with the SNARE motifs of syntaxin 6, syntaxin brane tethers (Sztul and Lupashin 2009). One 16, and VAMP4(Perez-Victoria and Bonifacino family consists of coiled-coil proteins, and the 2009). In addition, it binds the Rab protein

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Organization of SNAREs within the Golgi Stack

Table 1. Multisubunit tethering proteins and interacting partners Coiled-coil Multisubunit tether SNARE Rab Coat SM tether References Dsl1/ZW10 (Dsl1, Ufe1/Sec20/Use1, COPI Lewis et al. 1997; Tip20, Dsl3) syntaxin 18 (a-COP, Frigerio 1998; d-COP) Reilly et al. 2001; Andag and Schmitt 2003; Hirose et al. 2004; Arasaki et al. 2006 COG (Cog1–8) Sed5/Ykt6/Gos1, Ypt1, COPI Sly1 p115, Ram et al. 2002; Sed5, Ypt6, (b-COP, GM130 Suvorova et al. syntaxin 5, Rab6, g-COP) 2002; Zolov and GOS28 Rab30, Lupashin 2005; Rab41 Shestakova et al. 2007; Sohda et al. 2007; Fukuda et al. 2008; Laufman et al. 2009 GARP/VFT Tlg1, Ypt6 Siniossoglou and (Vps51, Vps52, syntaxin 10, Pelham 2001, Vps53, Vps54) syntaxin 16, 2002; Conibear syntaxin 6, et al. 2003; Liewen VAMP4 et al. 2005; Perez-Victoria and Bonifacino 2009 TRAPP I Ypt1 COPII Wang et al. 2000; (Bet3, Bet5, (Sec23) Cai et al. 2007 Trs20, Trs23, Trs31, Trs33) TRAPPII Ypt1, Rab1, COPI Jones et al. 2000; (TRAPPIþTrs65, Ypt31/32 (g-COP) Yamasaki et al. Trs120, Trs130) 2009

Ypt6p (Siniossoglou and Pelham 2001). Thus, Reilly et al. 2001). The Dsl1 complex stabilizes the GARP complex could tether vesicles con- a SNARE complex containing Ufe1p/syntaxin taining Rab6/Ypt6p to preassembled t-SNARE 18, Sec20p/BNIP1, and Use1p/p31 in the ER complexes at the TGN. (Kraynack et al. 2005; Aoki et al. 2008). Interact- The Dsl1 complex is implicated in the ing Rab proteins have not been identified. Thus, tethering of retrograde COPI vesicles directed the Dsl1 complex could tether COPI-coated from the Golgi to the ER (Andag et al. 2001; vesicles to preassembled t-SNARE complexes Reilly et al. 2001). It consists of three subunits on the ER. It has been suggested that Dsl1 might (yeast: Dsl1, Tip20, Dsl3; mammals: ZW10, also favor COPI vesicle uncoating, exposing the RINT-1, NAG) (Ren et al. 2009; Tripathi et al. cognate v-SNARE (Zink et al. 2009). 2009). A central acidic domain binds d- and The COG complex is localized to the Golgi, a-subunits of the COPI coat that function and implicated in retrograde trafficking from in analogy to the adaptor and cage the trans-Golgi or an endosomal compartment components, respectively (Andag et al. 2001; to the cis-Golgi (Whyte and Munro 2002; Vasile

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et al. 2006; Shestakova et al. 2007). It consists of further controlled by another protein family eight subunits (Cog1–8) and forms a bilobe called SM proteins. stucture (Ungar et al. 2002). Cog subunits spe- cifically interact with COPI coat components SM PROTEINS OF THE GOLGI: Sly1, Vps45 (Suvorova et al. 2002; Zolov and Lupashin 2005). The depletion of the Cog3 subunit causes SM (Sec1p/Munc18) proteins represent a fam- the accumulation of Golgi-derived vesicles con- ily of arch-shaped cytosolic proteins, whose taining Golgi residents and SNAREs (GS15 and individual members affect SNARE complex GOS28) and blocks the retrograde traffic of assembly at distinct transport steps (Toonen Shiga toxin (Zolov and Lupashin 2005). Cog4 and Verhage 2003). In addition, SM proteins and Cog6 directly bind syntaxin 5 and the seem to be involved in the proper localization COG complex enhances the stability of syntaxin and stabilization of syntaxin/Qa-SNAREs. SM 5-containing SNARE complexes (Zolov and proteins are essential for membrane trafficking Lupashin 2005; Shestakova et al. 2007). In addi- and their inactivation results in cell death, dem- tion, several Rab proteins seem to bind to Cogs: onstrating the importance of their interaction Rab1/Ypt1p, Rab6/Ypt6p, Rab30, and Rab41 with SNAREs. Consequently, SNAREs together (Suvorova et al. 2002; Fukuda et al. 2008). with SM proteins have been declared as the uni- Remarkably, Cog2 binds the coiled-coil tether versal fusion machinery (Su¨dhof and Rothman protein p115 and Cog4 binds the SM protein 2009). A single SM member can bind several Sly1 (Sohda et al. 2007; Laufman et al. 2009). SNARE complexes and serves a subset of trans- Both Sly1/Sly1p and p115/Uso1p directly port steps. The SM proteins functioning at the bind to syntaxin 5/Sed5p and regulate its activ- Golgi are Sly1 and Vps45. Sly1/Sly1p binds syn- ity (Sapperstein et al. 1996; Grabowski and Gall- taxin 5/Sed5p in the Golgi and syntaxin 18/ witz 1997). Furthermore, temperature-sensitive Ufe1p in the ER and controls anterograde and mutants in the yeast p115 (-1) and cog2-ts retrograde transport between the ER and Golgi are synthetically lethal supporting the hypothe- and within the Golgi stack (Lupashin et al. 1996; sis that multisubunit and coiled-coil tethers Li et al. 2005). Vps45 binds syntaxin 16/Tlg2p work together in controlling vesicle tethering, at the TGN and directs transport from early/ docking, and SNARE complex assembly (Van- recycling endosomes and late endosomes to Rheenen et al. 1998). One possibility would be the TGN (Simonsen et al. 1998; Tang et al. that one of the tethers is incorporated during 1998; Struthers et al. 2009). In addition, it binds budding and thus ensures that the emerging Pep12 in early endosomes controlling endoso- vesicle does not leave the rims of the Golgi mal trafficking (Tellam et al. 1997). SM pro- restricting its movement. The second tether tein-Qa-SNARE interactions can stabilize the then directly affects SNARE complex assembly Qa-SNARE and vice versa. Vps45p stabilizes on the neighboring target membrane. However, Tlg2p, and Sly1p protects Ufe1p from degrada- the cascade of reactions and the exact functions tion (Bryant and James 2001; Braun and Jentsch of these two distinct types of tethers still need to 2007). With the exception of the Sly1p-Sed5p be further established. interaction, which seems not to affect Sed5p In summary, the following reaction path- stability, similar cross stabilizations have been way emerges. During vesicle budding, distinct observed for SM proteins and Qa-SNAREs coat proteins recruit specific cargo proteins, functioning in regulated exocytosis (Toonen SNAREs, and Rab-GEFs to the budding site. and Verhage 2003; Braun and Jentsch 2007). Rab-GEFs activate specific Rabs, which now Thus, it appears that SM proteins can control bind their effectors, multisubunit and coiled- the amount of different Qa-SNAREs. Remark- coil tethers, which in turn activate specific ably, the overexpression of SM proteins can t-SNARE proteins on the target membrane. boost protein secretion by increasing the size Then, t-SNAREs are substrates for v-SNARE of secretory organelles in certain cells (Peng binding and SNAREpin formation, which is and Fussenegger 2009).

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Organization of SNAREs within the Golgi Stack

In addition to the syntaxin/Qa-SNARE Sec20p, which are part of the Ufe1p complex interaction, SM proteins bind partially as- (Li et al. 2005). It also shows low affinity inter- sembled SNARE complexes and stimulate actions with the Sed5p t-SNARE components membrane fusion. One of the best-studied Bos1p and Gos1p and with the v-SNAREs systems to understand how SM proteins direct Bet1p and Sft1p, and enhances SNAREpin SNARE complex assembly has been regulated formation (Dascher et al. 1991; Dascher and exocytosis (Toonen and Verhage 2007). In this Balch 1996; Kosodo et al. 2002; Peng and case, the neuronal SM protein Munc18-1 has Gallwitz 2002; Peng and Gallwitz 2004). both an inhibitory and stimulatory function Remarkably, Sly1p restricts the interactions of (Hata et al. 1993; Misura et al. 2000; Verhage Sed5p with noncognate SNAREs (Peng and et al. 2000; Shen et al. 2007). Munc18-1 binds Gallwitz 2002). Thus, Sly1 could also act as a the Qa-SNARE syntaxin 1 in a closed conforma- molecular shield to prevent the formation of tion and inhibits t-SNARE (syntaxin 1/SNAP- unproductive (nonfusogenic) SNARE com- 25) and v-/t-SNARE (syntaxin1/SNAP-25/ plexes. This might be of particular importance VAMP2) complex assembly (Dulubova et al. in the early secretory pathway, because most 1999; Misura et al. 2000). Remarkably, the cog- SNAREs are tail-anchored proteins, which as nate v-SNARE VAMP2 directly binds Munc18-1 newly synthesized proteins are initially inserted and relieves the inhibition (Xu et al. 2010). In into the ER membrane (Kutay et al. 1995; addition, the specific binding of the SM protein Rabu et al. 2009). Thus, the presence of various to both the v- and t-SNARE stimulates mem- SNAREs, which travel through the ER and brane fusion (Shen et al. 2007). The existence Golgi to their final destinations, would increase of an inhibitory and a stimulatory binding the possibility that noncognate SNARE com- mode requires distinct binding sites between plexes form. Sly1 could block such adverse the SM protein and the syntaxin/Qa-SNARE interactions. (Su¨dhof and Rothman 2009). Recent data indi- In summary, SM proteins contribute a layer cate that the stimulatory SM function in the of specificity to SNARE complex formation and neuronal system depends on the binding of a favor membrane fusion, likely by specifically helical amino-terminal peptide of the Qa- stabilizing cognate SNAREpins. In this context, SNARE into a hydrophobic pocket in domain it is also of note that SM proteins are function- 1 of the SM protein (Hu et al. 2007; Khvotchev ally coupled to tethering proteins. Sly1 directly et al. 2007, Shen et al. 2007; Shen et al. 2010). interacts with the COG tethering complex and This binding mode seems to be conserved and both bind syntaxin 5 with distinct binding sites has been shown for the Sly1p-Ufe1p, Slyp1- (Laufman et al. 2009). Obliteration of Sly1-Cog Sed5p, and the Vps45p-Tlg2p interactions binding impairs SNARE pairing and attenuates (Bracher and Weissenhorn 2002; Dulubova retrograde Golgi-to-ER transport (Laufman et al. 2002; Yamaguchi et al. 2002). It also has et al. 2009). Thus, the concerted action of SM been established that the presence of the Tlg2p and tethering proteins controls SNAREpin N-peptide can change the Vps45p-Tlg2p inter- assembly. action modus (Carpp et al. 2006; Furgason et al. 2009). However, it is still debated whether CONCLUDING REMARKS all SM protein-Qa-SNARE pairs in the Golgi show the same properties as their neuronal For productive fusion to occur, several require- counterpart (MacDonald et al. 2010). Neverthe- ments need to be fulfilled on opposite mem- less, it is reasonable to assume that SM protein- branes (Fig. 3). First, during vesicle budding, SNARE interactions are functionally conserved transport step-specific coat proteins select and that modest changes represent transport cargo proteins, specific SNAREs, and a partic- step-specific adaptations. In any case, SM pro- ular Rab-GEF, which activates and recruits a teins bind cognate SNARE complexes. Sly1p specific Rab protein onto the budding vesicle. binds the non Qa-SNAREs Use1p/pSLT1p and Second, the Rab protein binds a specific tether,

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t-SNAREs v-SNARE

ADP + Pi 4. after vesicle fusion, ATP cis-SNAREpins are disassembled NSF/a-SNAP Rab by the action of a-SNAP and NSF Tether

v-SNARE

1. vesicle coat proteins bind tethers and SNAREs for uptake into a budding vesicle

Rab SM Protein Tether 3. SM proteins support Rab the formation of cognate trans-SNAREpins Tether Rab Tether Tether SM Protein

2. tethers capture vesicles at the target membrane and trigger t-SNARE complex formation

Figure 3. Basic machinery controlling membrane fusion and SNARE recycling. SNAREs and tethers are recruited by vesicle coat components for uptake into a budding vesicle. The initial interaction of a vesicle with its target membrane is mediated by tethering proteins and a small Rab GTPase. Components of the tethering machinery trigger the formation of t-SNARE complexes, which assemble with their cognate v-SNAREs, resulting in the for- mation of SNAREpins. These events involve SM proteins and finally culminate in membrane fusion, which results in cis-v/t-SNARE complexes. In the presence of SNAPs, ATP-hydrolysis by NSF segregates v- and t-SNAREs for another round of fusion.

which together with the specific SM protein Thus, various proteins and a potential cas- controls t-SNARE complex assembly on the cade of reactions controlling SNARE complex opposite membrane. Third, if a cognate v- assembly have been identified, accompanied SNARE is present, it will interact with the by a morphological map showing the distri- t-SNARE while the SM protein provides an bution of these components within the Golgi additional control level and stimulates SNARE- stack. However, a functional map at high spatial pin assembly, resulting in membrane merger. In resolution, resolving transport steps between the absence of a cognate v-SNARE, stable individual cisternae, is still missing. Because, SNAREpins will not be established and GTP by its very nature, the transport machinery hydrolysis by Rab will eventually release the cycles between individual compartments, it tether, likely allowing another round of tether- will be necessary to distinguish between inac- ing to be initiated. Depending on the length, tive and active components on distinct vesicle number, and type of tethers, which link a given subpopulation. The identification of various vesicle to the Golgi, the vesicle could probe dif- COPI coats and distinct vesicle tethers clearly ferent areas of the Golgi stack, without ever leav- supports the existence of distinct vesicle pop- ing the Golgi, until productive SNAREpins have ulations within the Golgi (Moelleken et al. been established and fusion proceeds. 2007). Such subpopulations can be isolated

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Organization of SNAREs within the Golgi Stack

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Organization of SNAREs within the Golgi Stack

Jörg Malsam and Thomas H. Söllner

Cold Spring Harb Perspect Biol 2011; doi: 10.1101/cshperspect.a005249 originally published online July 18, 2011

Subject Collection The Golgi

Structure of Golgi Transport Proteins Golgi and Related Vesicle Proteomics: Simplify to Daniel Kümmel and Karin M. Reinisch Identify Joan Gannon, John J.M. Bergeron and Tommy Nilsson Golgi Biogenesis Organization of SNAREs within the Golgi Stack Yanzhuang Wang and Joachim Seemann Jörg Malsam and Thomas H. Söllner Golgi Glycosylation and Human Inherited Golgi during Development Diseases Weimin Zhong Hudson H. Freeze and Bobby G. Ng Models for Golgi Traffic: A Critical Assessment Entry and Exit Mechanisms at the cis-Face of the Benjamin S. Glick and Alberto Luini Golgi Complex Andrés Lorente-Rodríguez and Charles Barlowe Architecture of the Mammalian Golgi COPI Budding within the Golgi Stack Judith Klumperman Vincent Popoff, Frank Adolf, Britta Brügger, et al. Evolution and Diversity of the Golgi Mechanisms of Protein Retention in the Golgi Mary J. Klute, Paul Melançon and Joel B. Dacks David K. Banfield Evolutionary Forces Shaping the Golgi The Golgin Coiled-Coil Proteins of the Golgi Glycosylation Machinery: Why Cell Surface Apparatus Glycans Are Universal to Living Cells Sean Munro Ajit Varki Golgi Positioning Signaling at the Golgi Smita Yadav and Adam D. Linstedt Peter Mayinger

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