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© The Rockefeller University Press,
0021-9525/1998//1489 $5.00
The Journal of Cell Biology, Volume 141, Number 7,
, 1998 1489-1502
Articles |
Localization, Dynamics, and Protein Interactions Reveal Distinct Roles for ER and Golgi SNAREs
Medical School, University of Utrecht, 3584CX Utrecht, The Netherlands
ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.
Abbreviations used in this paper: IC, intermediate compartment; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor; VTC, vesicular tubular cluster.
Address correspondence to Richard H. Scheller, Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5428. Tel.: 650-723-9075; FAX: 650-725-4436; E-mail: scheller{at}cmgm.stanford.edu
MAINTENANCE of the differentiated membrane compartments in cells requires precise control of intracellular membrane fusion reactions. Fusion of transport vesicles with target membranes allows directed movement of proteins, lipids, and cellular messengers between organelles while maintaining the metabolic integrity of both the donor and acceptor compartments. Several classes of proteins have been identified that mediate aspects of cargo recruitment and vesicle formation, targeting, and fusion. One class of vesicle-trafficking proteins, referred to as soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs),1 proteins of the vesicle-associated membrane protein (VAMP) and syntaxin families, may impart a component of the specificity to membrane fusion reactions through their compartment-specific localizations and protein interactions. Vesicle and target membrane SNAREs form tight oligomeric protein complexes proposed to direct membrane fusion (Söllner et al., 1993; Bennet and Scheller, 1993; Pevsner et al., 1994; Weber et al., 1998). Formation of SNARE complexes appears to be highly cooperative, since binding of one protein can greatly potentiate binding of another (Pevsner et al., 1994; Hayashi et al., 1994; Stone et al., 1997).
The mechanism of ER-to-Golgi transport has remained particularly elusive since specific roles have not yet been assigned to the five or more interacting ER/Golgi SNARE proteins. Our study (Hay et al., 1997) showed that immunoprecipitation of one ER/Golgi SNARE from rat liver membrane extracts, syntaxin 5 (Hardwick and Pelham, 1992; Bennet et al., 1993), resulted in coprecipitation of rbet1 (Hay et al., 1996; Zhang et al., 1997), rsec22b (Hay et al., 1996; Paek et al., 1997), membrin (Hay et al., 1997; Lowe et al., 1997), and Golgi SNARE of 28 kD (GOS-28; Subramaniam et al., 1996; Nagahama et al., 1996). Although these five proteins are coisolated from detergent extracts, little is known about the pattern of interactions among them, since coisolations from cell extracts (Hay et al., 1997; Søgaard et al., 1994) may involve a series of partially overlapping subcomplexes rather than a single complex containing all of these SNAREs. In addition, it is not known which proteins are included in the complex(es) by virtue of direct interactions with syntaxin 5, as opposed to indirect interactions mediated through other proteins. Several direct interactions have been observed among the potential yeast homologs of the ER/Golgi SNAREs (Stone et al., 1997; Sacher et al., 1997), but these have not yet been correlated with specific vesicle types or dynamics.
Progress has been made in identifying vesicle pathways in ER/Golgi transport through characterization of the vesicle coat complexes that mediate aspects of cargo selection and vesicle budding (Kuehn and Schekman, 1997; Cosson et al., 1997; Schekman and Mellman, 1997). COP II coats mediate budding from the ER and at least the initial stage of transport to the Golgi (Kuehn and Schekman, 1997; Rowe et al., 1996). COP I vesicles, on the other hand, are involved in retrograde retrieval from the intermediate compartment (IC) and Golgi back to the ER (Letourneur et al., 1994). Other data support an additional role for COPI in anterograde movement (Rowe et al., 1996, Orci et al., 1997), possibly acting after COPII (Rowe et al., 1996; Scales et al., 1997). ER/Golgi transport may also involve other uncharacterized coat complexes, since COP II-homologous genes are apparent in the yeast genome (Kuehn and Schekman, 1997). Studies on transport from the ER to the Golgi emphasize the importance of movement of larger polymorphic elements of the IC referred to as vesicular tubular clusters (VTCs). By electron microscopy, VTCs are observed to be either Golgi-adjacent or peripheral (Bannykh et al., 1996). Golgi-adjacent VTCs form a dense cluster some have referred to as the cis-Golgi network in the juxtanuclear region, making them difficult to resolve from Golgi by light microscopy. Peripheral VTCs, on the other hand, are scattered throughout the cell, and are readily differentiated from Golgi by light microscopy. These peripheral clusters are surrounded by budding, or exit sites located on rough ER tubules. From these peripheral sites, VTCs appeared to mediate transport of vesicular stomatitis virus (VSV) G protein through large expanses of cytoplasm to the Golgi complex (Presley et al., 1997; Scales et al., 1997), perhaps traveling along microtubule tracks. Mobile VTCs contain both COP I and COP II coats, blurring the functional distinction between these complex membrane structures and small coated transport vesicles. Incorporation of specific SNARE proteins and protein complexes into models of coat protein function would provide a firm mechanistic basis for the functional proposals. In fact, these two families of vesicle-trafficking proteins must act in concert to form and deliver vesicles, as illustrated by their coprecipitation from extracts of forming ER-derived transport vesicles (Kuehn et al., 1998).
Further characterization of ER/Golgi SNARE complexes between more precisely known subsets of proteins must be accompanied by a morphological understanding of the subcellular localization and dynamics of each of the participant SNAREs. What are the steady-state distributions of each SNARE in the ER, IC, and Golgi? Which of the ER/Golgi SNAREs cycle rapidly between ER and Golgi, and which remain more or less stationary? Do distinct subsets of SNARE proteins reside on COPI and COPII vesicles, thus specifying distinct destinations? By combining immunoelectron microscopy with immunofluorescence, subcellular fractionation, immunoprecipitation and yeast two-hybrid analysis, we here begin to define the specific compartmental localizations, dynamics, and interactions of the five ER/Golgi SNAREs: syntaxin 5, rbet1, rsec22b, membrin, and GOS-28. In contrast to preliminary EM analyses (Paek et al., 1997; Banfield et al., 1994), we use specific antibodies to detect endogenous SNARE proteins at steady state, without recombinant or tagged expression. We establish distinct yet overlapping trafficking patterns for rsec22b, rbet1, membrin, and syntaxin 5, and distinct protein interactions for the two isoforms of syntaxin 5. The localizations and pattern of protein interactions among these proteins suggest that functionally distinct protein complexes are spatially and temporally segregated in ER and Golgi transport.
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Materials and Methods |
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Immunogold Labeling of Ultrathin Cryosections
Subcellular localizations of the endogenous SNARE proteins were studied on ultrathin cryosections of PC12 and HepG2 cells (see Figs. 2–5) and exocrine pancreas, COS, and NRK cells (not shown). Cells and tissue were fixed in 2% formaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and prepared for cryosectioning and double immunogold labeling as described (Slot et al., 1991) with the slight modification that sections were picked up in a 1:1 mixture of methyl cellulose and 2.3 M sucrose, which results in an improved ultrastructure (Liou et al., 1996). The best results were obtained with affinity-purified polyclonal rabbit antibodies against rsec22b, membrin, and syntaxin 5, and with culture medium from the mouse hybridoma 16G6 against rbet1, all at a dilution of 1:10. For GOS-28 (monoclonal HFD9), no specific labeling was obtained. Labeling density for a specific SNARE differed considerably between cell types. In PC12 cells, all SNARE proteins exhibited a high yield of labeling, and therefore all quantitative and double-labeling studies were carried out in these cells. For a quantitative analysis of the subcellular distributions of the ER/Golgi SNAREs (Table I), a high-quality section was selected at low magnification. Subsequently, the section was scanned systematically at 25,000x. All gold particles within a distance of 30 nm of a membrane were allocated to that type of membrane. About 90–98% of all gold particles were found over membranes of the ER, IC, and Golgi complex. The number of gold particles found over a specific compartment was then expressed as the percentage of total gold. For each SNARE protein, at least three EM grids were analyzed in this manner, and all quantitations were carried out in cells double-labeled for COPII. Since in ultrathin sections putative connections of COPII-coated buds with the ER are not always visible, for the quantitative analyses all COPII-coated membranes were considered part of the IC. The data in Table I summarizes the results of the different counting sessions, with about 150–200 gold particles counted per session.
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trans polarity were selected. The number of gold particles found over the individual cisternae, numbered G1 G5 from cis to trans, was expressed as a percentage of the total number of gold particles present on the Golgi complexes.
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Fluorescence Microscopy
Cell lines were maintained in a 5% CO2 incubator using routine media formulations. Before 15°C incubations, tissue culture medium was adjusted to 40 mM Hepes, pH 7.4. For 15°C incubations, cells cultured in microscope slide growth chambers (Nunc Inc., Naperville, IL) were placed below the water surface in a 15°C bath. Regardless of incubation temperature, cells were fixed with 4% paraformaldehyde at room temperature for 30 min. Immunostaining and fluorescence microscopy were carried out as described (Hay et al., 1996).
Subcellular Fractionation
Two 15-cm plates of NRK cells growing at 37°C were buffered to pH 7.4 with 40 mM Hepes and then returned to 37°C or incubated below the surface of a 15°C water bath for 2 h. Cells were chilled on ice and then swollen in 10 mM ice-cold Hepes, pH 7.4, for 3 min. The Hepes was removed and replaced with 0.5 ml of homogenization buffer (Hay et al., 1996), and the cells were scraped from the plate with a no. 7 rubber stopper (sliced in half) and homogenized by five strokes in a Dounce homogenizer (Kontes Glass Co., Vineland, NJ). Nuclei and unbroken cells were removed by centrifugation at 1,000 g for 10 min. For the gradients in Fig. 8, A, D, and G, the postnuclear supernatant was adjusted to 0.75 M sucrose and used as the top layer of a 1.2-ml 0.75–1.75 M sucrose gradient in 20 mM Hepes, pH 7.2, 0.1 M KCl, 2 mM EGTA, 2 mM EDTA. The gradients were initially made with seven discrete layers, but diffusion during the overnight spin produced continuous density profiles. After an 18-h centrifugation at 135,000 g (rav) in a TLS55 rotor (Beckman Instruments, Inc., Fullerton, CA), gradients were fractionated by hand from the top. Each fraction was supplemented with 2 µg BSA before precipitation with ice-cold methanol. The concentrated fractions were then electrophoresed, immunoblotted, and quantitated by autoradiography and densitometry. For gradients in Fig. 8, B, C, E, F, H, and I, 300 µl of postnuclear supernatant was adjusted to 600 µl of 0.75 M sucrose and layered on top of 700 µl of 1.125 M sucrose. Centrifugation and fractionation were as above.
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We estimate that the radioactive intensities of the bands detected by phosphor imaging in Fig. 9 are approximately indicative of the molar quantities of the proteins present. When scaled up, precipitations similar to that shown in the first column of Fig. 9 were electrophoresed and stained by Coomassie blue (Hay et al., 1997), and the relative intensities of the stained SNARE bands closely corresponded to the relative Western signals analyzed in Fig. 9.
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Additional combinations of binding partners tested include pGBT9/ membrin in combination with pGAD424/membrin, msec22b, GOS-28, and rbet1. pGBT9/rsec22a as bait was tested in combination with pGAD424/msec22b, GOS-28, and rbet1. pGBT9/msec22b was tested for binding with msec22b, GOS-28, and rbet1. pGBT9/GOS-28 was tested for binding with pGAD424/GOS-28 and rbet1. Finally, pGBT9/rbet1 was tested with pGAD424/rbet1. None of these combinations resulted in sufficient binding to produce β-galactosidase activity above background.
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Subcellular localization of rsec22b was studied in detail in HepG2 (Fig. 2) and PC12 cells (Fig. 3 A), which gave identical labeling patterns. The majority of label was associated with the IC (Table I, Fig. 2 A). At the ultrastructural level, the IC consists of an elaborate network of vesicular and tubular membrane profiles that appear at distinct sites in the cell both near the Golgi and at the periphery. Many IC clusters, or VTCs, were found to be closely associated with RER cisternae exhibiting COPII-coated membrane buds representing export sites for anterograde cargo. Double-immunogold labeling revealed that rsec22b was often confined to these COPII-coated membranes of the ER and early IC (Fig. 2, A and B).
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To determine whether the ER/Golgi SNAREs displayed specificity for the characterized COPI and COPII vesicle pathways in the IC, we performed double-immunogold labelings with COPI (Figs. 2 B, 3 C, and 4 C; COPI is represented by β-COP) and COPII (Fig. 2, A and C, Fig. 3 B, and Fig. 5 A, COPII is represented by msec13). A quantitation of these analyses is presented in Table III. For this analysis we scored as positive only individual membrane profiles, including vesicles as well as larger tubular membranes, to which both sizes of gold particles colocalized. If a membrane was only partially coated, the structure was scored positive for colocalization only if the SNARE-representing gold particle was within a distance of 40 nm of the COP-representing gold particle. rbet1 and rsec22b were particularly enriched in COPII-coated structures (25% colocalization: Fig. 2, A and C, Fig. 3 B, Table III), consistent with their playing an important role on COPII-coated vesicles that are known to bud from the ER (Kuehn and Schekman, 1997), and are involved in the formation of peripheral VTCs (Rowe et al., 1998). All four SNAREs tested were present on COPI-coated membranes (Figs. 2 B, 3 C, and 4 C, Table III).
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Low-temperature Incubations Distinguish Static from Rapidly Cycling ER/Golgi SNAREs
Immunofluorescence subcellular localization of this set of proteins in NRK cells revealed that all five proteins most intensively stained the juxtanuclear Golgi area under control steady-state conditions (Fig. 6, first column; 37°C). This finding is consistent with the ultrastructural studies (Table I) since IC elements, which contain the majority of syntaxin 5, membrin, rsec22b, and rbet1, are most concentrated in the Golgi area and are in fact indistinguishable from the Golgi at the light level. GOS-28 appeared perhaps most tightly Golgi-localized, consistent with previous ultrastructural studies (Subramaniam et al., 1995; Nagahama et al., 1996). rsec22b and rbet1 occasionally displayed a distinctly spotty localization reminiscent of peripheral VTCs (Saraste and Svensson, 1991); however, most staining was juxtanuclear.
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Do the rapidly cycling proteins follow the same pathway? To look for potential differences in their recycling pathways, we performed double-label immunofluorescence comparisons among rbet1, rsec22b, and membrin, when arrested in peripheral VTCs. As shown in the top two panels of Fig. 7, the distributions of rbet1 and rsec22b at 15°C were very similar in double-labeled cells, with intense staining of VTCs. Significantly, in almost all instances both proteins were present in the same individual VTCs (Fig. 7, arrows), implying that rsec22b and rbet1 share a very similar recycling pathway. Notably, both proteins exhibited weak tubular/reticular ER staining (Fig. 7, arrowhead). Membrin and rbet1 antibodies also clearly labeled the same individual peripheral VTCs (Fig. 7, bottom two panels), indicating that these three dynamic SNAREs recycle together on the same VTCs. However, the membrin staining was very focused in the perinuclear area and peripheral VTCs, but was not apparent in the ER. Hence, unlike rsec22b and rbet1, membrin apparently does not cycle fully back through the ER. Alternatively, membrin may recycle through the ER very rapidly, making detection there difficult.
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Shallower sucrose gradients were able to resolve membranes of the Golgi and IC partially while pelleting ER. An analysis of membrin and GOS-28 on these gradients confirmed the dramatic difference in these SNAREs' dynamics observed by immunofluorescence. At 37°C, the two proteins resided on IC and Golgi membranes that substantially overlapped in their densities (Fig. 8 B). At 15°C, however, membrin and GOS-28 are noticeably better resolved (Fig. 8 C), presumably since membrin accumulates in peripheral VTCs, whereas GOS-28 remains statically in the Golgi (Fig. 6). We speculate that peripheral VTCs unable to migrate from the periphery at 15°C undergo shape, size, and/or content changes that reduce their density, resulting in a shift to lighter gradient fractions. Likewise, the Golgi at 15°C may be altered by the change in trafficking and shifts to fractions of slightly greater density. Other studies have identified accumulated VTCs in a light fraction approximately comigrating with TGN (J. Klumperman, A. Schweizer, H. Clausen, B.L. Tang, W. Hong, V. Oorschot, and H.-P. Hauri, manuscript submitted for publication). Interestingly, at 15°C, syntaxin 5 shifted to IC membranes of lesser density, as did membrin, but also retained a very significant presence on denser membranes containing GOS-28 (Fig. 8, E and F). This dual trafficking of syntaxin 5 would explain the protein's apparently slower and less complete recruitment to peripheral VTCs observed by immunofluorescence (Fig. 6). Perhaps a pool of Golgi syntaxin 5 is retained in the Golgi by its interactions with GOS-28 (see below). rbet1, rsec22b, and the IC marker p58 (called ERGIC-53 in primates) all displayed shifting distributions consistent with membrin at 37°C and 15°C (Fig. 8, H and I). Hence, the immunofluorescence (Figs. 6 and 7) and density gradient (Fig. 8) temperature shift experiments support itinerant cycling functions for rbet1, rsec22b, membrin, and syntaxin 5. In contrast, the relatively static localization of GOS-28 and a portion of syntaxin 5 is consistent with a function that is limited to the Golgi.
Syntaxin 5 Isoforms Participate in Distinct Protein Complexes
We previously found that two syntaxin 5–associated proteins—rbet1 and GOS-28—were not tightly associated with each other in detergent extracts (Hay et al., 1997), suggesting their presence in distinct mutually exclusive subcomplexes containing syntaxin 5 in common. To help clarify whether other exclusive interactions occur among this group of proteins, we performed immunoprecipitations with eight different polyclonal and monoclonal antisera against ER/Golgi SNAREs, and quantitated coprecipitating proteins by Western blotting using 125I-labeled secondary reagents and phosphor imaging. As shown in Fig. 9 and Table IV, syntaxin 5, as expected, coprecipitated at least four ER/Golgi SNAREs, including GOS-28, rsec22b, membrin, and rbet1. All of these syntaxin 5–associated proteins reciprocally coprecipitated syntaxin 5, except for rsec22b, which unfortunately was primarily present as a partially degraded form that apparently does not participate in the protein–protein interactions. Hence, rsec22b precipitations did not provide information about protein complexes containing rsec22b.
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Immunoprecipitation of rbet1 and membrin resulted in coprecipitation of stoichiometrically balanced ratios of associated SNAREs other than GOS-28, indicating that major exclusive relationships do not exist among syntaxin 5, rsec22b, membrin, and rbet1. These proteins may associate together simultaneously in a single quaternary complex.
Several ER/Golgi SNAREs Interact Directly with Syntaxin 5
Although four SNARE-like proteins coprecipitate with syntaxin 5 (Hay et al., 1997), the pattern of direct interactions among this group is unknown. We sought to determine which members of the complex may be included in the complex by virtue of direct interactions with syntaxin 5, as opposed to indirect interactions mediated through other proteins. The yeast two-hybrid system was used to detect and quantify interactions between syntaxin 5 (the 34-kD isoform) and seven potential syntaxin 5–binding proteins (Fig. 10). We used two-hybrid constructs containing the full-length SNAREs, including transmembrane domains. Two proteins presumably irrelevant to the syntaxin 5 complex, SNAP-25 and rsec22a, failed to stimulate β-galactosidase activity, consistent with the expectation that these proteins do not bind syntaxin 5. One of the proteins that coprecipitates with syntaxin 5, mouse sec22b, consistently stimulated a small amount of β-galactosidase activity, indicating that although this protein appears to bind syntaxin 5 weakly, other protein interactions may be critical for its inclusion in the syntaxin 5 complex. Four other members of the syntaxin 5 complex: rsly1 (Peterson et al., 1997; Dascher et al., 1996), GOS-28, membrin, and rbet1, all stimulated a relatively large amount of β-galactosidase activity, consistent with the possibility that each of these proteins can directly interact with syntaxin 5 independently of other protein interactions. Combinations of membrin, rsec22a, msec22b, GOS-28, and rbet1 (see Materials and Methods) were also tested for their ability to interact with each other in the two-hybrid system. No combinations were found to stimulate β-galactosidase activity above background levels. It is possible that pairwise interactions between these proteins are not of sufficient affinity to give a positive signal in the two-hybrid assay; however, the proteins may bind to each other in the context of higher order complexes. The two-hybrid studies delineating the pattern of interactions among this set of proteins limit the possible configurations of SNAREs that determine trafficking pathways in ER/Golgi transport, and help suggest supramolecular models of transport. However, since many of these proteins appear to interact directly with syntaxin 5, the number of possible distinct protein complexes is large, and more information is needed about precise individual complexes.
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Membrin is significantly less abundant in the ER (Figs. 7 and 8), less enriched in early VTCs, more enriched in Golgi-adjacent VTCs, and extends significantly further in the secretory pathway than do rsec22b and rbet1, with representation throughout the cis and medial Golgi (Tables I and II). Although membrin does not appear enriched in COPII-coated membranes (Table III), it does share residence with rsec22b and rbet1 on the peripheral VTCs that accumulate at 15°C (Figs. 6 and 7). Hence, our data is consistent with a cycling role for membrin where it would participate in fusion events between VTCs and ER-derived vesicles, as well as in later fusion of VTCs to form nascent Golgi cisternae. Membrin's extension through the Golgi suggests a role in Golgi trafficking, perhaps in addition to its presumed role in ER/Golgi transport (see Fig. 11). In fact, a recent study found functional evidence for membrin's involvement in intraGolgi trafficking (Lowe et al., 1997). Although this study did not demonstrate a role for membrin in ER-to-Golgi trafficking, the presence of membrin on rapidly cycling VTCs and its participation in protein complexes containing rsec22b and rbet1 argue for a role in ER/Golgi trafficking. In addition, there is good evidence for involvement in ER/Golgi transport of BOS1p (Lian and Ferro-Novick, 1993), a yeast sequence homolog of membrin. Perhaps membrin's role at earlier steps is less susceptible to antibody inhibition. Although the antibody inhibition clearly demonstrated a role for membrin in Golgi trafficking, an anterograde vs. retrograde role cannot be distinguished due to the cycling nature of Golgi function.
Our data supports a dual role for syntaxin 5, like membrin, in ER/Golgi as well as intraGolgi transport. The 41-kD syntaxin 5, which is presumably more rigorously retrieved by virtue of its N-terminal COPI binding site (Hui et al., 1997), is more abundant in the ER and peripheral structures than the Golgi-centric form (Fig. 8). Our data is consistent with this isoform playing a role in the first fusion event, that between ER-derived rsec22b- and rbet1-containing transport vesicles and the early VTCs (see Fig. 11). This fusion event would presumably be directed by the complex we found between the 41-kD syntaxin 5, rsec22b, rbet1, and membrin (Fig. 9, Table IV). Since the 41-kD isoform exists in the ER as well (Table I, Fig. 8), it is also possible that it functions in the fusion of COPI-mediated retrograde vesicles with the ER, perhaps as a functional analog of the yeast ER SNARE UFE1p (Lewis and Pelham, 1996) that forms a presumed retrograde SNARE complex containing SEC22p and other proteins (Lewis et al., 1997). Our localization and dynamics data are in principle consistent with a recent study documenting a functional role for syntaxin 5 at an early preGolgi stage in transport (Rowe et al., 1998). However, our data do not favor a primary role for syntaxin 5 on ER-derived transport vesicles as this report suggested. Instead we find rsec22b and rbet1 much more prevalent on these structures (Table III), implying that syntaxin 5 may serve as the vesicle receptor on the early VTC membrane (see model, Fig. 11). Later, as the VTC migrates and matures, syntaxin 5 may participate in intra-VTC fusion that may form new Golgi cisternae, an event also perhaps involving the syntaxin 5/rsec22b/rbet1/membrin complex.
The 34-kD syntaxin 5 isoform, on the other hand, is more abundant in the Golgi and Golgi-adjacent VTCs (Fig. 8). In contrast to previous ultrastructural studies localizing syntaxin 5 to Golgi-adjacent VTCs and cis Golgi (Banfield et al., 1994), we find syntaxin 5 immunogold particles nearly equally distributed throughout the Golgi stacks (Table III). In support of a special role for the 34-kD isoform of syntaxin 5 in Golgi trafficking, we have documented a pool of 34-kD syntaxin 5 molecules that associates with GOS-28 in a complex that lacks or excludes the dynamic SNAREs membrin, rsec22b, and rbet1 (Fig. 9, Table IV). We speculate that this complex could serve to anchor a pool of the 34-kD syntaxin 5 in the Golgi, could regulate participation of syntaxin 5 in docking interactions with other SNAREs, or could provide a basis for intraGolgi vesicle/cisternae fusions. In each of these possible capacities, the syntaxin 5/GOS-28 complex would occur within the Golgi, since our studies indicate that GOS-28 and a portion of syntaxin 5 molecules do not travel on VTCs moving between peripheral and Golgi-adjacent locations (Figs. 6 and 8). It is tempting to speculate (see Fig. 11) that the syntaxin 5/GOS-28 complex represents a docking complex between Golgi-derived vesicles containing GOS-28 and Golgi cisternae containing 34-kD syntaxin 5. This possibility would be consistent with a previous report localizing recombinant GOS-28 to Golgi rim vesicles, and demonstrating an inhibitory effect of GOS-28 antisera on intraGolgi transport (Nagahama et al., 1996). It would also be consistent with the finding that overexpression of truncated syntaxin 5 weakly inhibited intraGolgi as well as ER-to-Golgi VSV G protein transport (Dascher et al., 1994). The data seems less consistent with the proposed role for GOS-28 in ER to Golgi transport (Subramaniam et al., 1996; Hay et al., 1997).
We (Hay et al., 1997; this report, Fig. 9) and others (Lowe et al., 1997) have observed coprecipitation of rsec22b with GOS-28 and coprecipitation of GOS-28 with rbet1, but these are on a molar basis minor compared with the major complexes described above. One of the anti-rbet1 antibodies, 4E11, coprecipitated significantly more GOS-28 than the other two rbet1 antisera; however, even in this most efficient precipitation the amount of associated GOS-28 was less than 19% of the associated rsec22b, membrin, or syntaxin 5. These relatively minor GOS-28-rbet1 interactions may arise from lateral associations of distinct SNARE complexes, or may represent intermediates in the transition between the major complexes. For example, although the function of GOS-28 with syntaxin 5 may not depend upon the presence of rbet1 (and vice versa), GOS-28 and rbet1 may not bind to syntaxin 5 in a strictly mutually exclusive manner, allowing some overlap between the major complexes to occur. Such overlap complexes could potentially serve a spatially defined regulatory purpose.
How does intraGolgi traffic work? Unfortunately, defining the distribution of SNAREs through the Golgi does not uniquely favor any one model of intraGolgi transport, including vesicle transport, cisternal maturation, and interstack tubules (Pelham, 1998; Mironov et al., 1998). For example, interstack vesicle transport throughout the Golgi could be mediated by syntaxin 5 (34 kD) and GOS-28 alone. There is no need for distinct t-SNAREs on each stack as long as spatial regulation or polarity is provided by other mechanisms such as microtubules or the Golgi spectrin/ankyrin cytoskeleton (Devarajan et al., 1997; Burkhardt et al., 1997). For completeness, the model shown in Fig. 11 incorporates nonmutually exclusive elements of cisternal maturation as well as interstack vesicle transport. It is possible that rsec22b, rbet1, syntaxin 5, membrin, GOS-28, and perhaps ykt6 (McNew et al., 1997) provide sufficient SNARE machinery to direct cargo from the ER all the way through to the TGN where cargo is sorted into one of several major pathways leading to destinations within the cell or without.
Though a complete picture of ER-to-Golgi and intraGolgi trafficking are not available, we now have enough information about the localization, dynamics, and interactions among ER/Golgi SNAREs to begin formulating useful models and specific testable hypotheses. Syntaxin 5 is likely to emerge from future studies as a common component of several spatially distinct membrane fusion complexes critical for early steps in the secretory pathway. Understanding the composition, regulation, and integration of these complexes with rabs, sec1 proteins, and membrane coat systems will provide the basis for a firm understanding of how cells mobilize and traffic their products.
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Acknowledgments |
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Submitted: 15 April 1998
Revised: 18 May 1998
Jesse C. Hay and Judith Klumpermancontributed equally to this paper. The present address of Jesse C. Hay is Department of Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109-1048.
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