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SNAREs * 43 SNAREs David K. Banfield and Wanjin Hong Introduction Eukaryotic cells contain multiple membrane-bound compartments between which proteins and lipid molecules are continually shuttled via membranebound vesicular carriers. Despite the constant flux of proteins and lipid through these compartments their functional and composition integrity is maintained. While the molecular machinery involved in vesicle recognition and fusion can often be transport-step/fusion-event specific, one group of proteins – the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) play a common and central role in this process. Transport-step-specific combinations of SNARE proteins, localized to the vesicle and the target organelle, form complexes that facilitate the final step leading to the fusion of vesicles with their cognate target organelles. In general, the role of SNAREs appears to be conserved irrespective of their location of function in the cell, and much of what has been established for SNAREs in a particular trafficking pathway or organelle, is broadly applicable to SNAREs that function in the Golgi. Here we review Golgi SNAREs and the role they play in membrane and protein trafficking in the Golgi apparatus with, a particular emphasis on their functions in yeast and human cells. General features of Golgi SNAREs The majority of SNARE proteins that function in the Golgi are type II integral membrane proteins anchored in the lipid bilayer by virtue of their single C-terminal transmembrane domain (TMD) see Fig. 1. The TMDs of SNAREs are presumably crucial for the stable association of SNARE proteins with membranes, but also play a role in establishing the steady-state distribution of SNAREs in the Golgi (Banfield et al. 1994; Rayner and Pelham 1997; Watson and Pessin 2001). In addition, in vitro fusion assays have established that the transmembrane domains of v-SNAREs (Xu et al. 2005) and of Qa-SNAREs (Han et al. 2004) are important for the formation of the hemi-fusion intermediates that precede membrane fusion and vesicle content mixing with the target compartment. Adjacent to the TMD is a short stretch of amimo acids (10 in length) referred to as the membrane proximal region (MPR). The amino acid sequence and length of this region is not evolutionarily conserved. The MPR 44 * D. K. Banfield and W. Hong Figure 1. General features of Golgi SNAREs. Based on their structural and functional features Golgi SNAREs are grouped into four categories. Category I is comprised of the Qa-SNAREs and some Qb- and Qc-SNAREs. Category II is mainly comprised of the Qc-SNAREs. Category III the R-SNARE Sec22p/Sec22b and category IV the R-SNARE Ykt6. The filled rectangles denote the location of the membrane proximal region (MPR) see Table 1 and the text for further details. serves to separate the TMD from the SNARE-motif, which precedes it. The length of the MPR appears to be important for the function of some SNAREs, at least in vitro (McNew et al. 1999, 2000; Melia et al. 2002) however, whether these observations extend to Golgi-localized SNAREs is presently not known. The SNARE-motif is comprised of a number of heptad-repeats, typically 7–8, which are responsible for the formation of the amphipathic helical bundles characteristic of SNARE complexes. An evolutionarily conserved amino acid residue that occupies a central position in the SNARE-motif, and which contributes to the zero ionic layer of SNARE complexes, is the basis of a SNARE protein family classification scheme (see below). In addition to the so-called SNARE-motif or core domain, SNARE proteins also contain N-terminal extensions (N-terminal domain (NTD)) of varying length and folds (see Figs. 1, 2 and Table 1). Golgi Qa- and Qb-SNAREs contain a domain which adopts a three-helix fold, termed an Habc domain, whereas Golgi R-SNAREs, with the exception of VAMP4, contain a longin fold. Golgi Qc-SNAREs typically contain short N-terminal regions that are predicted to be unstructured, although the NTDs of the Qc-SNAREs Tlg1p and Syntaxin 6, likely adopt an Habc fold. The longin fold, found in Golgi R-SNAREs, is also present in several sub-units of the Golgi-localized vesicle tethering complexes TRAPPI and TRAPPII (Kim et al. 2006) and is predicted to be present in two sub-units of the Golgi vesicle coat complex – coatomer, although the significance of this is not presently understood (Schlenker et al. 2006). In some cases the N-terminal domains of Golgi SNAREs are capable of binding to their respective SNARE-motif, in which case the SNARE is said to adopt a closed or folded-back conformation. Folded-back conformations are known to occur for the R-SNAREs Ykt6p (Tochio et al. 2001) and Sec22p SNAREs * 45 Figure 2. The N-terminal domain folds of Golgi SNAREs. (A) Cartoon representation of the crystral structure of the human Vti1b Habc domain (Miller et al. 2007; pdb accession number 2qyw) viewed from the side. The three helices of the domain are labelled from N – to C a, b and c. (B) The same structure is in (A) but viewed from the N-terminus down the three helix bundle. (C) The NMR-derived solution structure of the longin domain of yeast Ykt6p (Tochio et al. 2001; pdb accession number 1h8m). The cartoons represent 180 rotations of one another. (Mancias and Goldberg 2007). For, Ykt6p this conformation appears to be important for the proteins stability and likely plays a key role in the targeting of this protein by regulating the association of the cytoplasmic prenylated form of Ykt6 with membranes (Tochio et al. 2001; Fukasawa et al. 2004; 46 * D. K. Banfield and W. Hong Table 1. Yeast and human Golgi-resident SNAREs Type Human Yeast homolog TMD N-terminal extention N-terminal fold Qa Syntaxin 5 Sed5p Yes Yes Habc Syntaxin 16 Tlg2p Yes Yes Habc (predicted) Syntaxin 10 – Yes Yes Habc (predicted) Syntaxin 11 – No Yes Habc (predicted) GS27 (membrin, GOS-27) Bos1p Yes Yes Habc (predicted) Vti1a (Vti1-rp2) Vti1p Yes Yes Habc GS28 (GOS-28) Gos1p Yes Yes Habc (predicted) Syntaxin 6 Tlg1p Yes Yes Habc (predicted) Bet1 Bet1p Yes No Random coil GS15 Sft1p Yes No Random coil Sec22b (ERS-24) Sec22p Yes Yes Longin Ykt6 Ykt6p No (prenyl) Yes Longin VAMP4 – Yes Yes Unstructured SNAP-29 (GS32) – No No – Qb Qc R Qb þ Qc Hasegawa et al. 2004). For Sec22p, a folded-back conformation appears to be a prerequisite for this SNAREs efficient incorporation into COPII-coated vesicles (Liu et al. 2004; Mancias and Goldberg 2007). The N-terminal domain of the Golgi syntaxins Sed5p (yeast)/Syn5p (mammals) are known to bind to the Sec1–Munc18 (SM) family member protein Sly1 (Yamaguchi et al. 2002; Dulubova et al. 2003; Arac et al. 2005). The association of Sly1p with Sed5p is important for the specificity of Golgi SNARE complex assembly (Peng and Gallwitz 2002) whereas the association of Sly1 with Syntaxin 5 is important for ER–Golgi transport (Williams et al. 2004). A folded-back conformation of Sed5p may be involved in the efficient packaging of this SNARE into COPIIcoated vesicles (Mossessova et al. 2003). Apparently, COPII preferentially binds Sed5p when the protein is part of the Sed5p–Bos1p–Sec22p SNARE SNAREs * 47 Figure 3. The topological arrangements of v- and t-SNAREs. complex because t-SNARE assembly presumably removes the auto-inhibitory contacts of the closed conformation of the protein, exposing its COPII sorting signal (Mossessova et al. 2003). SNARE protein classification and nomenclature Functionally, SNAREs can be classified as either v-SNAREs or t-SNAREs. v-SNAREs are localized to the transport vesicle, whereas t-SNAREs are predominantly localized to the vesicles target compartment. Currently, the generally accepted view is that a single membrane-anchored v-SNARE forms a SNARE-complex in trans with a heterotrimeric t-SNARE. See Fig. 3 and Table 1 for a description of yeast and human Golgi-resident SNARE proteins. SNARE proteins can be further sub-divided based on their amino acid sequence similarities and the position their homologs occupy in SNARE complex macromolecular structures (Fasshauer et al. 1998; Bock et al. 2001). The macromolecular structures of the exocytic and endocytic SNARE complexes revealed that they are parallel four-helical bundles (Sutton et al. 1998; Antonin et al. 2002). In the case of the endocytic SNARE complex, four different SNARE proteins contribute a single helix each to the complex (Fig. 4), this arrangement is also very likely to be the case for Golgi SNARE-complexes. Thus, the syntaxin sub-family has been termed the Qa-SNAREs whereas SNAREs that share the greatest degree of amino acid similarity with the Nterminal SNARE-motif of SNAP-25 (SNAP-25N) are referred to as Qb-SNAREs. Similarly, SNAREs that are most similar to the C-terminal SNARE-motif of SNAP-25 (SNAP-25C) are referred to as Qc-SNAREs. Members of the so-called VAMP family are collectively referred to as R-SNAREs. The Qa-, Qb-, Qc- and R-SNARE nomenclature refers to the presence of a highly evolutionarily conserved amino acid residue at the so-called zero ionic layer of the four-helical bundle – a glutamine for the Q-SNAREs and an arginine for the R-SNAREs (Fig. 4). The general expectation is that members of each family will occupy the equivalent position in their respective SNARE complexes as the corresponding SNARE in the exocytic and endocytic SNARE complexes – adopting a Qa:Qb:Qc:R stoichiometry often referred to as the 3Q:1R rule. 48 * D. K. Banfield and W. Hong Figure 4. The SNARE complex is a four-helical bundle. (A) An elongated side view cartoon representation of the macromolecular structure of the endocytic SNARE complex (Zwilling et al. 2007). The position of the zero ionic layer is indicated by the arrow. Syntaxin 6 is represented by the yellow helix, whereas Syntaxin 13, Vti1a and VAMP4 are represented by the blue, magenta and green helices, respectively. (B) An enlarged and skewed side view of the SNARE complex cartoon. The colour scheme used is as in (A). (C) A view down the helical bundle of a cartoon representation of the SNARE complex in which the amino acid residue side-chains defining the zero ionic layer are indicated. The colour scheme used is as in (A). (D) The zero ionic layer residues of the endocytic SNARE complex. Note that while Vti1a is classified as a Q-SNARE, it contributes an aspartic acid, rather than glutamine to the layer. Cartoons where generated using MacPyMOL and the pdb file 2nps. SNAREs * 49 Although the 3Q:1R rule is crucial for the formation of properly functioning SNARE complexes, amino acid substitution experiments have shown that it is not necessary, for example, that arginine be contributed by an R-SNARE per se (Katz and Brennwald 2000; Graf et al. 2005). However, exceptions to these general rules exist. For example, Sft1p and Bet1p, two yeast Golgi resident Qc-SNAREs, contain an aspartic acid and a serine (respectively) at the zero layer position, however the biological significance of this variability is presently unknown. The general mode of SNARE protein function It is now generally accepted that the predominate function of SNARE proteins is to act as facilitators of intra-cellular membrane fusion events within the endomembrane system, through the formation of complexes between SNAREs on vesicles and SNAREs on organellar membranes. This association of SNAREs in trans is thought to be important in bringing the vesicle and organellar membranes close enough together to facilitate membrane fusion. In general, the SNARE complex that forms conforms to the 3Q:1R rule (Katz and Brennwald 2000). How are the individual SNARE proteins contributed to the SNARE complex? In vitro fusion assays with yeast Golgi SNAREs revealed that the heterotrimeric t-SNARE is comprised of a heavy chain – a Qa-SNARE (a syntaxin such as Sed5p or Syn5) and two different SNAREs which comprise the two t-SNARE light chains. Thus the t-SNARE consists of one Qa-SNARE together with either a Qb þ Qc, Qb þ R or Qc þ R pair defining the t-SNARE light chains. Employing this scheme the v-SNARE would be contributed by the remaining SNARE, i.e., either a Qb-, Qc- or R-SNARE, depending on the composition of the t-SNARE complex (Fig. 2). The v-SNARE is often an RSNARE, but this may not be so for SNAREs in the Golgi, as liposome fusion assays have established that the Qc-SNAREs, Bet1p and Sft1p, function as vSNAREs in this context (McNew et al. 2000; Parlati et al. 2002). However, an in vitro transport employing yeast Golgi SNAREs revealed that, in addition to Bet1p, the Qb-SNARE Bos1p and the R-SNARE Sec22p may also function as vSNAREs in transport between the ER and Golgi (Spang and Schekman 1998). Although the composition of the t-SNARE complex and its respective vSNARE appears to be quite rigid in vitro (Parlati et al. 2000, 2002) it seems likely that greater compositional flexibility exists in cells (Tsui and Banfield 2000; Tsui et al. 2001; Banfield 2001). In yeast, for example, some Golgi SNAREs interact with Qa-SNAREs other than Sed5p and in so doing participate in multiple transport steps (e.g. Vti1p, Ykt6p and Tlg1p). In addition, several yeast Golgi SNAREs are not essential for yeast cell growth. Given the importance of SNAREs in membrane fusion these observations have been viewed as being consistent with a functionally redundant role of SNAREs and reflexing a lack of selectivity in the composition of SNARE complexes. Despite the apparent flexibility in SNARE pairing interactions in cells, adherence to 50 * D. K. Banfield and W. Hong Figure 5. The Golgi SNARE cycle. the 3Q:1R rule remains important. This requirement has been successfully exploited as a means to identify functionally interacting SNARE complexes (Graf et al. 2005). While the importance of residues in the immediate vicinity of the zero ionic layer have been documented (Stone et al. 1997; Graf et al. 2005) a comprehensive examination of the relative importance of other regions of the SNARE-motif in Golgi SNARE function is lacking. What directs the specificity of SNARE complex formation? Prior to SNARE complex formation, the v-SNARE and t-SNAREs encounter each other in cis (Fig. 5) and SNARE complex formation appears to proceed from the N- to Cterminus (Sorensen et al. 2006; Pobbati et al. 2006). The close opposition of the v- and t-SNAREs is mediated by a variety of factors, so-called tethering factors, which presumably function to ensure that only the correct SNAREs form biologically meaningful trans-complexes. The formation of cognate, fusogenic SNARE complexes between opposing membranes drives fusion. Although cartoons, such as the one depicted in Fig. 5, often show the formation of trans-SNARE complexes comprised of 1–2 complexes (for the sake of simplicity) the average number of complexes participating in one fusion reaction, based on studies on the neuronal exocytic SNARE complex, is likely to be on the order of 3–8 (Han et al. 2004; Rickman et al. 2005; Montecucco et al. 2005). The rosette-like structures that are observed to form from the association of multiple SNARE complexes may be important for mediating membrane fusion, perhaps via the transmembrane domain of SNAREs (Han et al. 2004). Following fusion of the vesicle with the Golgi, SNAREs remain bound to one another in trans. Trans-SNARE complexes are dissociated through the combined actions of a-SNAP/Sec17p and NSF/Sec18p after which, SNAREs are free to be recycled and reused in another round of vesicular transport and membrane fusion. Several Golgi SNAREs have been shown to cycle between SNAREs * 51 the Golgi and the ER (Ballensiefen et al. 1998; Wooding and Pelham 1998; Ossipov et al. 1999; Cosson et al. 2004), reflecting the requirement for SNAREs in anterograde as well as retrograde vesicle-mediated transport (Spang and Schekman 1998). In addition, apart from the requirement that cells reuse SNARE proteins in successive rounds of transport, it seems likely that this recycling process is intimately linked to the establishment and dynamic nature of the Golgi apparatus itself (Cosson et al. 2004). The specificity of SNARE complex formation Table 2 lists SNARE complexes known to function in transport to the Golgi in yeast and mammalian cells. The complexes that mediate such traffic in mammalian cells have predominantly been identified by co-immune precipitation experiments. In contrast, in budding yeast this information has been obtained from a variety of approaches, including co-immune precipitation, genetic studies and in vitro mixing and fusion assays. An observation that has Table 2. SNARE complexes known to function in transport to the Golgi Mammals Yeast Complex Transport step (s) Complex Transport step (s) Syntaxin 5 (Qa) GS28 (Qb) GS15 (Qc) Ykt6 (R) Recyling endosome–TGN Sed5p Gos1p Sft1p Ykt6p Intra-Golgi Syntaxin 5 GS28 Bet1 Ykt6 ERGIC – Golgi Sed5p Bos1p Bet1p Sec22p ER–Golgi Syntaxin 5 GS27 Bet1 Sec22p ER–ERGIC Sed5p Bos1p Bet1p Ykt6pa ER–Golgi Syntaxin 16 Vti1a Syn6 VAMP4 Early endosome–TGN Sed5p Gos1p Bet1pa Ykt6p Intra-Golgi Syntaxin 16 Vti1a Syntaxin 10 VAMP3 Late endosome–TGN a Assumed on the basis of over-expression experiments in sec22D (Liu and Barlowe 2004) and sft1D cells (Tsui et al. 2001). SNAREs in bold, italicized font are encoded by non-essential genes. ERGIC (ER-Golgi intermediate compartment). 52 * D. K. Banfield and W. Hong dogged the role of SNAREs in the specificity of membrane fusion events has been the apparent lack of specificity among many SNARE–SNARE associations. This lack of specificity is particularly apparent in in vitro mixing experiments using bacterially expressed mammalian as well as yeast SNARE proteins (Yang et al. 1999; Fasshauser et al. 1999; Tsui and Banfield 2000). Recent evidence suggests that non-cognate SNARE complexes form in cells, but that cells have some, as yet to be identified mechanism, which selects only the physiologically relevant complexes for use in membrane fusion reactions (Bethani et al. 2007). Thus, the identification of SNARE–SNARE interactions by co-immune precipitation may not be sufficient to assign particular SNAREs to a functional complex (Bethani et al. 2007). In in vitro fusion assays using the theoretical maximum tetrameirc combinations of SNAREs encoded by the yeast genome, only 9/275 were found to be fusogenic (McNew et al. 2000; Parlati et al. 2000, 2002; Paumet et al. 2004). Two of the nine fusogenic complexes contained the Golgi Qa-SNARE Sed5p: Sed5p/Bos1p/Sec22p (t-SNARE) þ Bet1p (v-SNARE) and Sed5p/Gos1p/Ykt6p (t-SNARE) þ Sft1p (v-SNARE), complexes which mediate fusion of vesicles with the cis- and trans-Golgi, respectively. These two complexes correspond to the mammalian Syntaxin 5-containing complexes: Syntaxin 5/membrin/ Sec22b (t-SNARE) þ Bet1 (v-SNARE) (Hay et al. 1998), although some studies suggest that the v-SNARE may be Sec22b (Xu et al. 2000; Joglekar et al. 2003) and to Syntaxin 5/GS28/Ykt6 (t-SNARE) þ GS15 (v-SNARE) (Xu et al. 2002). These in vitro fusion assay data suggest that SNARE proteins encode the necessary information to direct the formation of fusogenic SNARE complexes. In yeast, Sed5p is the only syntaxin required for transport through the Golgi, however, Sec22p and Gos1p are encoded by non-essential genes. Thus in cells lacking either the SEC22 or GOS1 genes (presumably) only a single Sed5p-containing SNARE complex would remain. Additional Sed5pcontaining fusogenic SNARE complexes have been proposed to form in cells on the basis of biochemical as well as genetic studies (Tsui and Banfield 2000; Liu and Barlowe 2002) see Table 2. In cells, Ykt6p appears to be able to substitute for Sec22p (Liu and Barlowe 2002). Similarly, under conditions when the Qc-SNARE Bet1p is ectopically over-expressed, cells can survive without the Qc-SNARE Sft1p (Tsui and Banfield 2000). Thus, with the exception of the QaSNARE, yeast Golgi Qb-, Qc- and R-SNAREs display varying degrees of presumptive functional redundancy. Whether these additional complexes constitute functionally overlapping SNARE complexes, redundant complexes or complexes that form as a result of the absence of the cognate SNARE, requires further investigation. The observation that some Sed5p interacting SNAREs also form complexes with other Qa-SNAREs functioning on other organelles suggests that a single SNARE is likely to be insufficient to direct complex specificity. For example, Vti1p (Lupashin et al. 1997; Von Mollard et al. 1997) and Ykt6p (Kweon et al. 2003) bind to multiple Qa-SNAREs and function in multiple transport pathways. Combinatorial binding interactions may therefore influence the specificity of SNAREs * 53 SNARE complex formation (Banfield 2001) in vivo. Finally, the extent to which regions of the SNARE-motif, other than the zero ionic layer, contribute to the specificity of Golgi SNARE complex assembly is an important issue which remains to be addressed. i-SNAREs In vitro mixing studies with the soluble forms of Sed5p and its Golgi SNARE binding partners revealed far more ternary complexes than were identified on the basis of SNARE-mediated liposome fusion assays (Tsui et al. 2001; McNew et al. 2000; Parlati et al. 2002). The presence of more than two fusion competent Sed5p-containing SNARE complexes would help to reconcile conceptual problems arising from the fact that one SNARE from each of these complexes is non-essential – Sec22p and Gos1p, respectively (see Table 2) (McNew et al. 2000; Parlati et al. 2002). However, another explanation has been proposed to account for the additional Sed5p-containing SNARE complexes observed in in vitro mixing studies with soluble SNAREs. Using their well established SNARE-mediated liposome fusion assay Varlamov et al. (2004) established that certain sub-units of the cis-Golgi SNARE complex could inhibit fusion mediated by the trans-Golgi SNARE complex and vice versa (Varlamov et al. 2004) – the authors termed these SNAREs as i-SNAREs. While the opposing distribution of cis- and trans-Golgi SNAREs (Volchuk et al. 2004) could in principle account for the distribution of the fusogenic SNARE complexes, the authors argue that i-SNAREs would enhance this phenomena – essentially fine-tuning the specificity of membrane fusion events in the Golgi. While this is a particularly attractive notion, the concept of i-SNAREs functioning in the Golgi still awaits in vivo validation. Localization of SNAREs to the Golgi In general SNAREs are predominantly localized to the vesicles and compartments on which they function and are absent from those on which they do not. How are SNAREs localized to the Golgi? Accumulating evidence suggests that both the transmembrane domain of SNAREs as well as signals in their cytoplasmic domains accounts for their steady-state distributions. A requirement of the transmembrane domain in the localization of Golgi enzymes is well established in mammalian cells. Such studies have led to the proposals that (1) the length of the transmembrane is an important factor in Golgi localization and that sorting/or localization is the result of Golgi membrane bilayer thickness (Bretcher and Munro 1993) or (2) that the TMDs of Golgi residents oligomerize and are prevented from exiting the Golgi (Nilsson et al. 1993). The transmembrane domains of the yeast SNAREs Sed5p and Sft1p contribute to their Golgi localization (Banfield et al. 1994; Rayner and Pelham 1997) and TMD length has been shown to be important for the Golgi localization of Syn5 in mammalian cells (Watson and Pessin 2001). However 54 * D. K. Banfield and W. Hong in yeast, TMD length alone is not sufficient to ensure exclusive Golgi localization of SNARE protein chimeras (Rayner and Pelham 1997). The steady-state distribution of SNAREs proteins in the Golgi appears be dynamic – relying on the active retrieval of SNAREs from distal cisternae and their retrieval to earlier sub-compartments or to the ER, from where they return to Golgi. In yeast, Sed5p (Wooding and Pelham 1998), Sec22p (Ballensiefen et al. 1998) and Bos1p (Ossipov et al. 1999) have been shown to cycle between the Golgi and the ER. Although yeast Bet1p does not undergo such cycling (Ossipov et al. 1999), in mammalian cells Bet1 and Sec22b have been shown to continually cycle between the Golgi and the ER (Hay et al. 1998). The recycling of Bet1 does not appear to require interactions with its cognate SNAREs (Joglekar et al. 2003). In the case of yeast Sec22p and Bos1p, the retrieval of these SNAREs from the Golgi requires a functional COPI coat (Ballensiefen et al. 1998; Ossipov et al. 1999). An analysis of the lateral distribution and vesicle incorporation of SNAREs in the mammalian Golgi using electron microscopy is also consistent with a dynamic localization mechanism (Cosson et al. 2005). The Golgi SNARE Ykt6 does not contain a TMD, but rather is dually lipid modified at its C-terminus (Fukasawa et al. 2004). The farnesylated form of Ykt6 resides in the cytoplasm whereas farnesylated, palmitoylated Ykt6 is found predominantly on Golgi membranes in non-neuronal mammalian cells (Fukasawa et al. 2004; Hasegawa et al. 2004). How Ykt6 is targeted to Golgi membranes remains to be determined. SNAREs and COPI interactions Several SNAREs have been shown cycle within or from the Golgi in a COPIdependent manner, data that implies an interaction between these SNAREs and the coat protein complex. The COPI coat is comprised of the heptameric complex termed coatomer, together with the GTPase Arf1. Arf1 cycles on and off Golgi membranes as a function of its nucleotide-bound state. GTP-bound Arf localizes to membranes whereas GDP-bound Arf is found in the cytoplasm. The nucleotide status on Arf1 is controlled through the action of its exchange factor (Arf GEF) and its activating protein (Arf GAP). In vitro studies using yeast Golgi SNAREs revealed that the Arf1p GAPs, Glo3p and Gsc1p, act catalytically on Golgi SNAREs promoting a conformational change that facilitates stoichiometric recruitment of Arf1p to SNAREs (Rein et al. 2002). In agreement with these findings, studies in mammalian cells have identified a motif on Arf that is required for the recruitment of Arf to Golgi membranes by the Qb-SNARE membrin (Honda et al. 2005). Schindler and Spang (2007) have shown that Gcs1p accelerates the formation of SNARE complexes in vitro and suggested that Arf GAPs may function as folding chaperones for SNAREs. Such mechanisms may function to couple SNARE recruitment to vesicle formation in cells, thus ensuring that each vesicle carries sufficient SNAREs capable of forming cognate SNARE complexes at its target compartment. SNAREs * 55 The role of SNAREs in the morphological and functional organization of the Golgi Presumably recycling Golgi SNAREs is important for ensuring efficient vesiclemediated transport, as this would allow these proteins to be employed in successive rounds of trafficking. Mathematical modeling using a minimal system, in which the variables were restricted to cytoplasmic coat protein complexes and SNAREs, was sufficient to generate stable non-identical compartments (Heinrich and Rapoport 2005). A requirement of Heinrich and Rapoports (2005) model was that each vesicle generating coat complex preferentially bound and packaged a characteristic set of SNAREs. The lateral distribution of Golgi SNAREs observed by Cosson et al. (2005) may similarly reflect differential affinity of Golgi SNAREs for the COPI coat in vivo. Thus, the affinity of vesicle coats, or their cargo sorting affiliated partners, may function to promote and maintain the compositional integrity of Golgi cisternia through their intrinsic ability to bind different SNAREs with varying affinities. Regulators of Golgi SNARE function The activity of SNAREs is regulated at various stages of their action including the assembly post-translational of the t-SNARE and the assembly of the trans-SNARE complex (Fig. 5). A variety of proteins have been identified that modulate the activity of SNAREs. In addition, post-translational modifications such prenylation, palmitoylation and phosphorylation also influence the activity and or localization of SNAREs. NSF/Sec18p and a-SNAP/Sec17p NSF and a-SNAP represent two co-operating core regulators of SNARE protein activity. These proteins are responsible for the disassembly of cis-SNARE complexes (Fig. 5), an activity that frees-up SNAREs to be used in successive rounds of vesicle fusion. Three molecules of a-SNAP link the cis-SNARE complex with a hexamer of the ATPase NSF/Sec18p and together this complex is referred to as the 20 S complex (Hohl et al. 1998; Wimmer et al. 2001; Furst et al. 2003; Brunger and DeLaBarre 2003). NSF contains two ATPase domains termed D1 and D2. The D2 ATPase domain mediates the formation of the NSF hexamer whereas the D1 ATPase domain effects the dissociation of the cisSNARE complex. The association of NSF with a-SNAP into the 20 S complex stimulates the ATPase activity of NSF (Marz et al. 2003). The Sec1/Munc-18 like (SM) proteins Sec1/Munc-18 (SM) proteins bind directly to SNAREs and act downstream of vesicle tethering events. Sly1p, the yeast SM protein which binds to Sed5p, was identified because a mutant of this protein (sly1-20p) could suppress the loss of the essential Rab/Ypt GTPase Ypt1p (Dascher et al. 1991). The association of Sly1p with Sed5p has been shown to enhance Sed5p-containing transSNARE complexes (Kosodo et al. 2002), and to be important for the specificity 56 * D. K. Banfield and W. Hong of Golgi SNARE complex assembly (Peng and Gallwitz 2002) whereas the assocation of Sly1 with Syntaxin 5 has been demonstrated to be important for ER–Golgi transport (Williams et al. 2004). However, the interaction between Sed5p and Sly1p is dispensible for transport (Peng and Gallwitz 2004). Unlike the exocytic and neuronal syntaxins and their requisite SM protein interactions, the interaction between Sly1p and Sed5p is mediated by a short stretch of amino acids at the N-terminus of the protein which does not involve either the Habc or SNARE-motif domains (Yamaguchi et al. 2002; Bracher and Weissenhorn 2002; Dulubova et al. 2003) and thus this association does not promote a folded-back conformation for Sed5p. A similar mode of binding is also evident between the SM protein Vps45/Vps45p and the Qa-SNARE Syntaxin 16/Tlg2p (Dulubova et al. 2002). It is now apparent that Sly1p is capable of binding to non-syntaxin SNAREs as well as to SNARE complexes and that this SNARE binding property of Sly1p is important in the specificity of cognate SNARE complex formation (Peng and Gallwitz 2002, 2004; Li et al. 2005). The Golgins The Golgins are a class of large coiled-coil containing Golgi localized proteins with roles in tethering vesicles to the Golgi. Some golgins contain a single Cterminal transmembrane domain whereas other members of the family are peripherally associated with Golgi membranes. The peripheral membrane protein p115 has been shown to bind directly to SNAREs involved in ERGolgi intermediate compartment (ERGIC) as well as ERGIC-Golgi transport (Allan et al. 2000; Shorter et al. 2002). A SNARE-related coiled-coil region of p115 interacts with many Golgi SNAREs and such interactions likely promote the formation of trans-SNARE complexes (Shorter et al. 2002). The functional consequences of such interactions may be to ensure a direct connection between the tethering machinery and SNAREs as well as to facilitate the recruitment of p115 to membrane sites where unassembled SNAREs are located (Brandon et al. 2006; Bentley et al. 2006). Mutational analysis of p115 suggests that the SNARE-modulating activity of the protein is more important than its tethering activity in maintaining the structure and function of the Golgi (Puthenveedu and Lindstedt 2004). Uso1p is the yeast homologue of p115 (Sapperstein et al. 1995, 1996; Cao et al. 1998). Other Golgins have also been shown to bind to Golgi SNAREs, including GM130 which interacts directly with Syntaxin 5 (Diao et al. 2007) and GCC185, which binds directly to Syntaxin 16 (Ganley et al. 2008). The emerging picture of the role of Golgins is one in which these proteins sequester Rab GTPases and Qa-SNAREs/syntaxins, and in so doing keep these two key proteins in close proximity to the tether. Conserved oligomeric Golgi (COG) The COG complex is a member of the oligomeric vesicle tethering factor family which comprise a structurally diverse group of peripheral membrane SNAREs * 57 protein complexes involved in vesicle-organellar tethering events prior to SNARE complex assembly (Oka and Krieger 2005; Stzul and Lupashin 2006). The COG complex is involved in retrograde trafficking of Golgi-resident proteins and extensive genetic interactions have been documented between the COG complex and SNAREs. In addition the yeast and mammalian COG complexes co-immune precipitate with Golgi SNAREs (Suvorova et al. 2002; Zolov and Lupashin 2005) and the localization and stability of Golgi SNAREs is altered in cells with defective COG complex components (Oka et al. 2004; Fotso et al. 2005; Zolov and Luphasin 2005; Shestakova et al. 2007). More recently, the yeast COG complex has been shown to interact with the SNAREmotif of Sed5p and to preferentially bind to Sed5p-containing SNARE complexes, leading the authors to propose that one function of the COG complex is to stabilize intra-Golgi SNARE complexes (Shestakova et al. 2007). GATE-16 GATE-16, a member of the ubiquitin-fold (UF) protein family, is localized to the Golgi and interacts with NSF as well as GS28 (Sagiv et al. 2000). NSF/a-SNAP facilitates the interaction of GATE-16 with GS28 in a manner that requires ATP-binding but not ATP hydrolysis. Interestingly, GATE-16 binding prevents GS28 from interacting with Syntaxin 5 and in so doing prevents the formation of a functional t-SNARE (Muller et al. 2002). In addition, the yeast GATE-16 homologue, Aut7p, interacts with Bet1p, a Qc-SNARE involved in ER-to-Golgi transport and shows genetic interactions with BET1 and the ER-Golgi RSNARE SEC22 (Legesse-Miller et al. 2000). FIG FIG (also known as CAL, PIST and GOPC) localizes to the TGN where it interacts with the Qc-SNARE Syntaxin 6 (Charest et al. 2001). FIG contains two coiledcoil regions and a single PDZ domain and the proteins interaction with Syntaxin 6 is mediated via the second coiled-coil region and its C-terminal flanking region. Although the biological significance of this interaction remains to be determined, knock-out of the FIG gene in mice results in selective ablation of acrosome formation during spermatogenesis (Yao et al. 2002). The acrosome is believed to form from the Golgi apparatus and the absence of FIG leads to fragmented acrosomal vesicles suggestive of a role for FIG in the fusion of vesicles into the acrosome. Curiously, FIG also interacts with Golgin-160 (Hicks and Machamer 2005). Phosphorylation Many SNAREs and their regulatory proteins are known to be phosphorylated by a variety of kinases (Gerst 2003; Snyder et al. 2006). The yeast Golgi Qa-SNARE Sed5p is a phosphoprotein and Weinberger et al. (2005) have shown that amino acid substitutions to an evolutionarily conserved protein kinase A phosphorylation site adjacent to the transmembrane domain of the protein has dramatic effects on Golgi morphology. While expression of the 58 * D. K. Banfield and W. Hong pseudophosphorylated form of Sed5p (Ser317Asp) results in the accumulation of ER membranes and vesicles, expression of the non-phosphorylatable form of the protein (Ser317Ala) results in the accumulation of Golgi membranes reminiscent of the mammalian cell Golgi (Weinberger et al. 2005). The Ser317Ala mutant also shows an increased affinity for the COPI coat, suggesting that phoshorylation status of Sed5p in cells may play a role in regulating Golgi morphology. Palmitoylation Several SNAREs are known to be palmitoylated. Some of these SNAREs lack transmembrane domains and are anchored to the membrane by their palmitate moieties, such is the case for SNAP-25, SNAP-23 and Syntaxin 11 (Vogel and Roche 1999; Veit 2000; Prekeris et al. 2000). In contrast, the Golgi SNARE Ykt6p is anchored by a combination of prenylation and palmitoylation (Fukasawa et al. 2004). In addition, it is now apparent that several SNAREs bearing TMDs are also palmitoylated (Valdez-Taubas and Pelham 2005) including the TGN/ endosomal Qc-SNARE, Tlg1p. The yeast DHCC-CDR family member Swf1p is required for palmitoylation of Tlg1p and prevention of Tlg1p palmitoylation results in its ubiquitination and transportation, via the multivesicular body, to the vacuole for degradation. While palymitoyation of TMD-anchored SNAREs does not appear to be essential for their function, this modification may play a role in the membrane partitioning of these SNAREs and or in dissociation of these modified proteins from other SNAREs, following fusion (Valdez-Taubas and Pelham 2005). Based on amino acid sequence similarities with Tlg1p, the mammalian Golgi resident SNAREs Syntaxin 6, Syntaxin 10 and VAMP4 may also be substrates for palymitoylation (Valdez-Taubas and Pelham 2005). Unlike the TMD-anchored SNAREs, which are modified via DHHC-CDR palmitoyltransferases, Ykt6 appears to be capable of mediating its own palymitoylation (Veit 2004) via its longin fold (Dietrich et al. 2004). Ykt6 is found in two pools in cells–a cytoplasmic pool and a membrane associated pool. Ykt6 lacks a proteinaceous membrane anchor but contains a prenylation consensus sequence (a so-called CAAX box) at its C-terminus. The cytoplasmic pool of Ykt6 has been shown to farnesylated and the farnesylation of Ykt6 is prerequiste for the subsequent palymitoylation and membrane association of the protein (Fukasawa et al. 2004). Fukasawa et al. (2004) propose a cycle of membrane association of Ykt6 in which the farnesylated fold-back conformation of Ykt6p (mediated by an interaction between the longin domain and SNARE-motif, Tochio et al. 2001) is targeted to membranes, whereupon the protein is palymitoylated. This dual lipid modification may be required for stable membrane association of Ykt6 (Fukasawa et al. 2004). Golgi SNAREs and apoptosis During programmed cells death (apoptosis) the Golgi loses its cisternal organization and is fragmented into clusters of tubulovesicular elements SNAREs * 59 (Lane et al. 2001) and the early secretory pathway is blocked (Lane et al. 2002). While this phenotype is associated with the proteolytic cleavage of members of the Golgin family (Mancini et al. 2000; Chiu et al. 2002; Lane et al. 2002) Lowe et al. (2004) have shown that Syntaxin 5 is cleaved by caspase during apoptosis. Caspase-3 cleaves Syntaxin 5 at Asp 263, separating the SNARE-motif from the N-terminal Habc domain. Syntaxin 5 participates in several SNARE complexes in the Golgi, including partnerships with Bet1 (Qc-), membrin/GS27 (Qb-) and Sec22b (R-); Bet1 (Qc-), GS28 (Qb-) and Ykt6 (R-) as well as with GS15 (Qc-), GS28 (Qb-) and Ykt6 (R-) (Hay et al. 1998; Zhang et al. 2001; Xu et al. 2002). Thus, cleavage of Syntaxin 5 by caspase-3 is likely to affect several trafficking steps to and within the Golgi. Future perspectives While much has been learned about the role of SNAREs in the Golgi many important questions remain to be addressed. 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