Sequential Involvement of p115, SNAREs, and Rab Proteins in Intra-Golgi Protein Transport*

Michael J. S. GmachlDagger and Christian Wimmer§

From the Dagger  Institute for Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria and the § Basel Institute for Immunology, Grenzacherstraße 487, Ch-4005 Basel, Switzerland

Received for publication, February 16, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Delivery of transport vesicles to their receptor compartment involves tethering, priming, and fusion. Soluble NSF attachment protein-alpha (alpha SNAP) mediates the disruption of SNAREs by N-ethylmaleimide sensitive factor (NSF) and was employed to determine the hierarchy of proteins responsible for intra-Golgi protein transport. The N-terminal 23 amino acids of alpha SNAP are necessary for SNARE binding. The antibody 2F10 recognizes this SNARE interaction domain of alpha SNAP and inhibits intra-Golgi protein transport reversibly. This antibody was applied to modify the transport assay to determine the protein requirements relative to the action of alpha SNAP and NSF. We found that 1) p115 acts independently of alpha SNAP and NSF, 2) SNAREs are required after tethering and interact selectively after activation by alpha SNAP and NSF, and 3) Rab proteins act after SNARE activation and before fusion.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vesicles mediate the transport between membrane-bound compartments of eukaryotic cells (1). To maintain the functional and morphological integrity of the cell, protein and lipid transport has to follow a selective and precise mechanism. After entry of cargo into the budding vesicle, it pinches off and is destined for the target membrane. Short range movement of vesicles is mediated by diffusion, whereas motor proteins move vesicles over a long distance on cytoskeletal tracks. Finally, vesicles dock to and fuse with the target membrane, resulting in the delivery of the cargo. Vesicular transport is a cyclic process accounting for the vectorial flux of cargo while maintaining the structural and functional integrity of the individual compartments (for reviews see Refs. 2-5).

Docking is the morphologically defined event of the association of a vesicle with a target membrane. Tethering is the biochemical equivalent and involves the formation of a stable interaction of a vesicle with a membrane. Priming refers to the activation of the membrane surfaces to obtain fusion competence (6). The SNARE1 hypothesis (7) provides an explanation for specificity in vesicular transport; during targeting, the membrane-bound v-SNARE protein of a vesicle interacts with the corresponding t-SNARE of the target membrane forming a SNARE complex. SNAREs were initially identified in the neuronal system (7), and subsequently homologous proteins involved in other transport steps in the mammalian cell and in yeast were identified. SNAREs of the synapse and of the yeast vacuole are involved in docking and fusion of proteoliposomes and vacuolar membranes, respectively (8-10). Several soluble proteins regulate the v-t-SNARE interaction. Members of the SNAP family of proteins, consisting of the ubiquitously expressed alpha - and gamma SNAP and the neuronal beta SNAP, bind to SNAREs (11, 12). Subsequently, the ATPase NSF binds to SNAREs via the SNAPs (13), and ATP hydrolysis results in the disruption of SNARE complexes (7). Another family of proteins involved in vesicular transport is the Rab family of GTP-binding proteins (14). They cycle between a GTP- and a GDP-bound form, and both forms can be membrane-associated (15). The GDP dissociation inhibitor (GDI) forms a complex with Rabs in their GDP-bound form and removes them from the membrane (16-18). GDP is exchanged for GTP by a guanine nucleotide exchange factor (19, 20).

Several proteins involved in transport through the Golgi apparatus have been identified. The v-SNARE GOS28 was the first SNARE implicated in intra-Golgi protein transport (21, 22). Subsequent studies identified GS27/membrin (23, 24), rbet1 and rsec22b (24, 25), GS32 (26), syntaxin 5 (27), syntaxin 6 (28), syntaxin 10 (29), and syntaxin 16 (30, 31) as additional Golgi SNAREs. Rab family proteins such as Rab6 (32), Rab10 (33), and Rab12 (34) have been localized to the Golgi complex. Rab6 has been shown to be involved in intra-Golgi protein transport (35, 36) and retrograde transport to the endoplasmic reticulum (37). Cytosol fractionation studies revealed p115(TAP) as a protein involved in intra-Golgi transport (38). It forms a homodimer and is composed of a globular head domain and a rodlike tail domain (39). p115 binds to the Golgi-associated proteins GM130 (40) and giantin (41). GM130 is mitotically phosphorylated, and this dissociates the p115-GM130 interaction (40). This is concomitant with the vesiculation of the Golgi complex during mitosis.

Although many proteins involved in the targeting of vesicles have been identified, no consensus exists about the order of involvement of these proteins (42, 43). This study focused on the interaction of Golgi-derived transport vesicles with their target membrane in intra-Golgi transport. GOS28 was chosen as a representative of the SNARE family, and Rab6 was chosen as a representative of the Rab family. Both proteins and p115 are examined for their contribution in intra-Golgi transport relative to alpha SNAP and NSF.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant Material-- Bovine His6-alpha SNAP was purified from Escherichia coli lysate according to Ref. 12, and GST-VAMP was purified as described in Ref. 44. The truncated forms of GSTalpha SNAP designated GSTalpha SNAP-(1-156), GSTalpha SNAP-(24-295), and GSTalpha SNAP-(35-295) were obtained by amplification of the corresponding DNA fragment of alpha SNAP with oligonucleotides introducing a BamHI site at the 5' end and an EcoRI site at the 3' end followed by ligation into the E. coli expression plasmid pGEX4T-1 (Amersham Pharmacia Biotech). GSTalpha SNAP and its deletion mutants were purified as described in Ref. 21, and His6-NSFmyc was purified according to Ref. 7. To bacterially express the head domain of p115 from rat (45), a DNA fragment coding for amino acids 1-651 was amplified by polymerase chain reaction introducing a BamHI site at the 5' end and an EcoRI site at the 3' end. The EcoRI site in the open reading frame was removed without affecting the translated amino acids. This fragment was ligated into pGEX4T-1. The p115 head domain was expressed as a fusion protein with GST and was purified by thrombin cleavage according to the manufacturer's instruction. The cytoplasmic domain of His6-GOS28 was expressed in E. coli (21). Insoluble protein (inclusion bodies) was dissolved in buffer A (8 M urea, 100 mM sodium phosphate, pH 8.0) and cleared by centrifugation. Soluble protein was bound to a nickel-nitrilotriacetic acid column (Qiagen) equilibrated with buffer A. After washing with 50 column volumes of buffer A, a linear gradient against buffer B (100 mM KCl, 10% (w/v) glycerol, 1 mM 2-mercaptoethanol, 20 mM imidazole, 50 mM Tris-HCl, pH 8.0) was applied. Refolded protein was eluted with a linear gradient of buffer B containing 500 mM imidazole. Protein concentration was determined by the method of Bradford (46).

Antibodies-- The following antibodies were used: anti-syntaxin antibody HPC-1 (47), anti-NSF antibody 6E6 (48), anti-alpha SNAP antibody 2F10,2 anti-Rab6 antibody 3A6 (49), and anti-SNAP25 antibody (50). Polyclonal GST antibody was purchased from Santa Cruz Biotechnology, and anti-synaptobrevin/VAMP serum was from Wako. For the preparation of an antibody directed against p115, a rabbit was immunized with the head domain of p115. Antibody was affinity-purified from antigen coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech). Bound antibody was eluted with 0.1 M glycine (pH 2.8), neutralized, concentrated to 1.4 µg/µl in a dialysis tubing placed on dry polyethylene glycol 35000, and dialyzed against 50 mM KCl, 25 mM HEPES (pH 7.4), 5% glycerol, 0.5 mM beta -mercaptoethanol.

Subcellular Fractions and Intra-Golgi Transport Assay-- Extract from bovine brain membranes was prepared as described in Ref. 7, and GDI was purified as described in Ref. 51. M KCl-treated (K-Golgi) membranes were prepared according to Ref. 52. Intra-Golgi protein transport assays were carried out at 37 °C for 60 min as described (53, 54). Reactions containing K-Golgi membranes were carried out in a volume of 100 µl. For the two-stage assay, transport reactions were incubated with 500 ng of 2F10 on ice for 20 min and transferred to 37 °C for 20 min. The reactions were placed on ice, and the cytoplasmic domains of GOS28, GDI, and anti-Rab6 were added, respectively. After the addition of 500 ng of His6-alpha SNAP, the samples were incubated at 37 °C for 1 h. The two-stage assays containing K-Golgi membranes were inhibited with 500 ng of 2F10 as described above, and anti-p115 antibody and 400 ng of His6alpha SNAP were added. Error bars indicate standard deviations of experiments carried out as triplicates (see Fig. 4) or as duplicates (see Figs. 5 and 6).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Domain Analysis of alpha SNAP-- alpha SNAP is required for the disassembly of SNARE complexes by NSF. This dissociation is a key event in vesicular protein transport; therefore, we chose alpha SNAP as a target to examine its role and, in conclusion, that of NSF and SNAREs in protein targeting. To determine the domain structure of alpha SNAP we carried out limited proteolysis with subtilisin, a protease of low sequence specificity. Protein domains of compact and rigid conformation are more resistant to proteolytic degradation by subtilisin, whereas flexible parts of a protein become preferentially hydrolyzed (55). To identify core domains of alpha SNAP we incubated His6-alpha SNAP with increasing amounts of subtilisin. The proteolytic fragments were separated by SDS-PAGE. Fig. 1A shows a typical result of limited proteolysis of alpha SNAP. After transfer to a polyvinylidene fluoride membrane, partial N-terminal amino acid sequencing was performed on the four proteolytic intermediates indicated in Fig. 1A. The largest of the four intermediates starts at position 32 with the smaller fragments beginning at positions 93, 140, and 157 of alpha SNAP, respectively (Fig. 1B). A comparison of the apparent molecular weight of the proteolytic fragments with the calculated molecular weight suggests that the C terminus was not subject to proteolytic degradation (data not shown). The hydrophilic amino acid serine found in positions 24 and 35 flanks the site of the first proteolytic cut at position 32. Truncated GSTalpha SNAP fusion proteins were expressed in E. coli that lacked either the first 23 or 31 amino acids of alpha SNAP. In addition, amino acids 1-156 of alpha SNAP were fused to GST and expressed in E. coli.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 1.   Limited proteolysis of alpha SNAP. A, subtilisin digest of alpha SNAP. In each reaction, 60 µg of alpha SNAP were incubated with 0, 0.025, 0.075, 0.25, 0.75, 2.5, 7.5, and 25 µg of subtilisin (lanes 1-8) in 25 mM HEPES (pH 7.4), 50 mM KCl, 5 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol in a volume of 25 µl at 4 °C for 20 min. The reaction was stopped by the addition of 5 µl of 1 M phenylmethylsulfonyl fluoride, and an aliquot was separated by SDS-PAGE on a 15% gel and stained with Coomassie Brilliant Blue R. Lane 9 contains subtilisin only. B, amino acid sequence of alpha SNAP. The arrowheads indicate the sites of subtilisin hydrolysis. Forward arrows indicate the beginning of N-terminally truncated mutants (24-295 and 35-295), and the reverse arrow indicates the end of the C-terminally truncated form (1) of alpha SNAP expressed in E. coli as a GST fusion protein.

The deletion mutants of alpha SNAP were examined for their ability to bind to neuronal SNARE complexes. SNARE complexes were prepared from bovine brain membranes by lysis with Triton X-100. alpha SNAP and its truncation mutants were incubated with the membrane extract and purified with glutathione-agarose beads. Neuronal SNAREs were detected by SDS-PAGE and transfer to a nitrocellulose membrane followed by Western analysis. Compared with full-length alpha SNAP, removal of 23 amino acids from the N terminus results in a decreased affinity for SNAREs, and removal of 34 amino acids renders SNARE binding undetectable. However, amino acids 1-23 of alpha SNAP are not sufficient for SNARE binding because a mutant alpha SNAP consisting of amino acids 1-156 does not bind SNAREs (Fig. 2A).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   Functional characterization of alpha SNAP mutants. A, binding of neuronal SNAREs to alpha SNAP. 2 µg of the indicated GSTalpha SNAP was incubated with 150 µg of bovine brain membrane proteins and 25 µl of glutathione-agarose in 130 µl of 25 mM HEPES (pH 7.4), 50 mM KCl, 1 mM dithiothreitol, 10% (v/v) glycerol for 2 h. The agarose beads were washed in the same buffer containing 0.2% Triton X-100. An aliquot was applied to SDS-PAGE followed by Western analysis with the antibodies listed. B, epitope mapping of the monoclonal anti-alpha SNAP antibody 2F10. 200 ng of full-length or truncated GSTalpha SNAP were subjected to SDS-PAGE and transfer to nitrocellulose. The membrane was developed with anti-GST serum and the monoclonal antibody 2F10.

The anti-alpha SNAP antibody 2F10 interferes with the binding of SNAREs to alpha SNAP.2 Considering the critical role of the N terminus of alpha SNAP in SNARE binding, we applied the deletion mutants of alpha SNAP in a Western analysis to determine the epitope of 2F10. As depicted in Fig. 2B, the antibody 2F10 recognized full-length alpha SNAP but none of the N-terminal truncation mutants. This coincidence suggests that the N terminus of alpha SNAP is directly involved in binding of neuronal SNAREs. The critical role of the N terminus of alpha SNAP in binding SNAREs is also supported by studies of the interaction of recombinant SNAREs with alpha SNAP (56) and electrophysiological experiments. In this study, a peptide derived from the N terminus of alpha SNAP inhibited calcium-induced exocytosis after microinjection into the giant axon of squid (57).

Development of a Two-stage Intra-Golgi Transport Assay-- An in vitro transport assay that reconstituted vesicular intra-Golgi transport was developed by Rothman and co-workers (54, 58). Subsequent characterization of the transport reaction resulted in a consensus that this assay allows measurement of the fusion of Golgi-derived transport vesicles with early Golgi elements (59, 60), establishing this assay as a tool to identify and characterize components of the transport machinery (61). The antibody 2F10 inhibits the intra-Golgi transport reaction.2 This inhibition is caused by a depletion of alpha SNAP from both cytosol and membranes. The alpha SNAP-2F10 complex is predicted to be soluble and to be minimally invasive during the transport assay. In the first step we determined the time span for which the transport assay is sensitive for 2F10. Standard transport reactions were started, and 2F10 was added after discrete time intervals. As shown in Fig. 3A, the transport signal becomes independent of alpha SNAP within 20 min. The remaining incubation time of 40 min at 37 °C is required for the glycosylation of the vesicular stomatitis virus- encoded glycoprotein. In the second step we tested whether the transport inhibition caused by 2F10 is reversible. Transport was first inhibited by 2F10 for 20 min, and then alpha SNAP was added. As depicted in Fig. 3B, the inhibition by 2F10 is reversible. The reversibility of the inhibition by 2F10 made it possible to divide the intra-Golgi transport reaction into two steps, enabling us to resolve requirements before and after alpha SNAP. In the first step, the reaction is inhibited by 2F10, allowing transport until the first alpha SNAP-dependent step is reached. Cells treated with N-ethylmaleimide accumulate docked vesicles at the Golgi apparatus (62). alpha SNAP and NSF were identified as protein targets of N-ethylmaleimide (63), implying that the transport reaction is inhibited after the accumulation of docked transport intermediates. In the second step, this inhibition is reversed by the addition of recombinant alpha SNAP. At this point, substrates can be tested for their potential to inhibit the two-stage transport reaction, and the quality and quantity of inhibition can be compared with a standard one-stage transport reaction (Fig. 3C).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   alpha SNAP requirement of the intra-Golgi transport reaction. A, susceptibility of the transport reaction for inhibition by the anti-alpha SNAP antibody 2F10. Intra-Golgi transport reactions consisting of 5 µl of donor and 5 µl of acceptor membranes, 12 µl of cytosol, 0.4 µM [3H]UDP-GlcNAc, and an ATP-regenerating system in a total volume of 50 µl (54) were incubated at 37 °C for 1 h. 500 ng of 2F10 were added after the indicated time intervals. Then the membranes were lysed by detergent, and the vesicular stomatitis virus-encoded glycoprotein was immunoprecipitated. B, reversibility of the inhibition by the antibody 2F10. An intra-Golgi transport reaction was carried out for 1 h at 37 °C (lane 1). The same reaction was incubated with 500 ng of 2F10 on ice for 20 min and then for 1 h at 37 °C (lane 2). Another transport reaction was incubated with 500 ng of 2F10 for 20 min on ice and subsequently at 37 °C for 20 min. The reaction was transferred back on ice, 500 ng of alpha SNAP were added, and the incubation at 37 °C was continued for 1 h (lane 3). C, model for the two-stage intra-Golgi transport reaction. The antibody 2F10 depletes the transport assay of alpha SNAP, resulting in a block of the assay at a step preceding the fusion of transport vesicles with the acceptor membrane. The reaction proceeds after the addition of alpha SNAP, and protein requirements relative to alpha SNAP and its interacting proteins NSF and SNAREs can be distinguished.

The Contribution of p115, GOS28, and Rab Proteins to Intra-Golgi Protein Transport-- The proteins p115, GOS28, and Rab6 have been implicated in intra-Golgi protein transport (for references see the Introduction), and we studied their role in the one- and two-stage transport assays. A polyclonal antiserum against the globular head domain of p115 was generated. Affinity-purified antibodies recognized a single band of 115 kDa of Chinese hamster ovary whole cell lysate in Western analysis (data not shown). We added increasing amounts of these antibodies to the transport reactions between 1 M KCl-treated Golgi membranes. As shown in Fig. 4 (top panel), only the one-stage transport reaction but not the two-stage reaction was inhibited by the anti-p115 antibody. The inhibition reached a saturation at 400 ng of antibody/100-µl reaction that could be reversed by the addition of antigen (data not shown). Initially, the antibodies were tested in the standard intra-Golgi transport assay and did not inhibit protein transport (data not shown). Subsequently, K-Golgi membranes were used in the assay. Salt treatment removes p115 quantitatively from Golgi membranes, and cytosol becomes the only source of p115 (38). 100 ng of NSF had to be added to the transport reaction in these experiments because the 1 M KCl wash removes NSF likewise. The degree of GTPgamma S inhibition is an indicator of to what extent vesicular transport as compared with uncoupled fusion contributes to the transport reaction (64). In our assay design, GTPgamma S inhibited the reaction by 60% (Fig. 4, bottom panel), suggesting that most of the transport signal is due to vesicular transport intermediates.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Involvement of p115 in intra-Golgi transport. A, inhibition of intra-Golgi transport by anti-p115 antibodies. A one-stage transport reaction containing 20 µg of cytosol, 10 µl of K-Golgi donor, acceptor membranes, 250 ng of NSF, and an ATP-regenerating system was incubated at 37 °C for 1 h (lane 1). Cytosol was incubated with 1.2 µg of anti-p115 antibody on ice for 10 min, and the same reaction was performed (lane 2). The two-stage reactions were incubated with 500 ng of 2F10 on ice for 20 min and at 37 °C for 20 min. The reactions were transferred on ice, and 400 ng of alpha SNAP (lane 3) and 1.2 µg of anti-p115 (lane 4) were added, kept on ice for 10 min, and incubated for 1 h at 37 °C. B, incubation with a nonhydrolyzable analogue of GTP. One-stage transport reactions were carried out in the absence (lane 1) and the presence (lane 2) of 10 µM GTPgamma S.

We then examined the inhibition pattern of the cytoplasmic domain of GOS28 in the one- and two-stage transport assays. The cytoplasmic domain of GOS28 containing a hexahistidine motif at the N terminus was expressed in E. coli. The protein was purified from inclusion bodies after solubilization in urea. The protein was bound to a nickel-nitrilotriacetic acid matrix, renatured by applying a gradient of a decreasing concentration of urea, and eluted by a gradient of increasing imidazole concentration. In SDS-PAGE a single band of 26 kDa was observed (data not shown). The renatured cytoplasmic domain of GOS28 was added to the one- and two-stage transport assay using standard membranes. In both cases, a similar degree of 70% inhibition was observed. In the two-stage reaction, a 2.5-fold smaller amount of GOS28 was sufficient to achieve inhibition (Fig. 5). Two control reactions for the specificity of GOS28 were carried out. 1) The effect of GOS28 can be neutralized by incubation with antibodies directed against GOS28 (data not shown); 2) the cytoplasmic domain of the plasma membrane SNARE synaptobrevin/VAMP was applied. Soluble recombinant synaptobrevin/VAMP was confirmed to be active by virtue of the fact that it forms a complex with the cytoplasmic domain of syntaxin 1a and SNAP25 that can be disassembled by alpha SNAP and NSF as described in Ref. 65. No inhibition by synaptobrevin/VAMP was observed in the transport reaction (Fig. 5).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Inhibition of intra-Golgi transport by cytoplasmic domains of v-SNAREs. Standard transport reactions (with one stage) as described in the legend for Fig. 3 were carried out with the indicated amounts of the cytoplasmic domain of GOS28 (closed circles) and synaptobrevin/VAMP (open squares). Two-stage transport reactions were incubated with 500 ng of 2F10 for 20 min on ice and then for 20 min at 37 °C. The reactions were transferred on ice to add 500 ng of alpha SNAP and the indicated amounts of the cytoplasmic domain of GOS28 (open circles). The incubation was continued at 37 °C for 1 h.

The contribution of Rab proteins to the intra-Golgi transport was determined by adding GDI or antibodies against Rab6 (3A6) to the one- and two-stage transport assays. GDI depletes the transport reaction of all Rab proteins. Both GDI and anti-Rab6 inhibit the one- and two-stage reactions in a saturable manner by 50% (Fig. 6A and data not shown). Because GDI inactivates Rab proteins, the specific effect of both reagents was determined by simultaneous addition. The combination of GDI and anti-Rab6 exhibits a comparable inhibition that does not differ in a statistically significant manner when applied separately (Fig. 6B), suggesting that both proteins act specifically.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   The involvement of Rab proteins in intra-Golgi transport. A, inhibition of intra-Golgi transport by GDI and anti-Rab6 antibodies. One-stage (lanes 1-3) and two-stage transport reactions (lanes 4-6) were performed as outlined in the legend for Fig. 5. The one-stage reactions were inhibited by 2.6 µg of GDI (lane 2) and 1.6 µg of anti-Rab6 antibodies (lane 3), and the two-stage reactions were inhibited by the same amounts of GDI (lane 5) and anti-Rab6 (lane 6). B, simultaneous inhibition by GDI and anti-Rab6. A one-stage transport reaction (lane 1) was incubated with 2.6 µg of GDI (lane 2), with 1.4 µg of anti-Rab6 antibodies (lane 3), or with the same amounts of both reagents (lane 4).

Our results define the biochemical sequence of proteins involved in the targeting of vesicles during transport through the Golgi apparatus. The addition of recombinant alpha SNAP to an intra-Golgi transport reaction inhibited by the anti-alpha SNAP antibody 2F10 results in a complete reversal of the inhibition. This observation enabled us to modify the intra-Golgi transport assay to determine the contribution and temporal action of p115, GOS28, and Rab6, three proteins known to be involved in intra-Golgi transport, relative to alpha SNAP and its interaction partner NSF. The data presented can be most easily explained by p115-mediated binding of vesicles to the target membrane followed by the activation of SNAREs on the vesicle and the target membrane, respectively. Rab proteins, in particular Rab6, are required for a step between SNARE activation and fusion.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The SNARE hypothesis predicted that SNAP and NSF bind to SNAREs paired in trans as the result of vesicle docking (7). This prediction placed SNAP and NSF at a defined step of the targeting mechanism. Meanwhile, it was shown for the fusion of yeast vacuoles that only t-SNAREs require priming (66) and that priming can take place without tethering (10). In this study, we examined protein transport through the Golgi apparatus, the central organelle of constitutive secretion. In interpreting our data and the data obtained in the yeast system, we propose that the function of alpha SNAP and NSF is to continuously activate the SNAREs on all membranes involved in constitutive transport. This activation would keep the machinery for the recognition and fusion of membranes continuously active for the consumption of newly delivered vesicles to ensure a high transport rate. As a consequence, no sequential order of priming relative to tethering or fusion can be assigned in constitutive secretion in vivo. In regulated secretion, the cycle of generation and consumption of vesicles becomes arrested and allows one to resolve mechanistic intermediates. The model systems studied suggest a role for alpha SNAP and NSF before and after vesicle fusion; in neuroendocrine cells, a readily releasable pool of vesicles is docked to the plasma membrane and fuses after the influx of calcium ions from the extracellular medium (67, 68). SNAP and NSF are required for a priming step that precedes the calcium-triggered fusion (69). This finding was confirmed in the temperature-sensitive Drosophila NSF mutant named comatose (6). Two lines of evidence suggest an additional role for SNAP and NSF after fusion; the temperature-sensitive phenotype of comatose is reversible, and the recovery kinetic corresponds to the kinetic of the Drosophila dynamin mutant shibire known to be required for the recycling of vesicles (70). In comatose mutants, v-t-SNARE complexes accumulate on synaptic vesicles after a temperature shift (71). This suggests that SNAP and NSF are required to separate v- and t-SNAREs after the fusion to ensure that v-SNAREs enrich on recycling vesicles and t-SNAREs remain at the target membrane.

A promiscuous interaction of bacterially expressed SNAREs involved in unrelated transport steps has been observed (72, 73). We compared the effect of the cytoplasmic domain of GOS28 and the neuronal SNARE synaptobrevin/VAMP in the standard intra-Golgi transport reaction. The neuronal SNAREs synaptobrevin/VAMP, syntaxin 1a, and SNAP25 are, to our knowledge, the only SNAREs that, after expression in E. coli, have been shown to form a complex that can be disassembled with alpha SNAP and NSF (65). Therefore, the v-SNARE synaptobrevin/VAMP was the protein of choice to examine the specificity of SNARE interactions in the intra-Golgi transport assay. The cytoplasmic domain of GOS28 inhibited the transport reaction in a dose-dependent manner, confirming previous studies that showed that an antibody against GOS28 inhibits intra-Golgi transport (21, 22). No inhibition by the cytoplasmic domain of synaptobrevin/VAMP was observed. Our data suggest that SNAREs can interact in a specific manner, and we conclude that the structural organization of the cell is sufficiently preserved in the cell-free intra-Golgi transport assay to retain the key mechanisms in specific vesicular targeting. Compared with the one-stage reaction, the same inhibition results from a lower concentration of the cytoplasmic domain of GOS28 in the two-stage reaction. A kinetic and a steric effect can account for the observed difference. alpha SNAP binds to unpaired t-SNAREs (74), and a depletion by the antibody 2F10 gives the cytoplasmic domain of GOS28 a better access to syntaxin 5; alternatively, the simultaneous addition of alpha SNAP and the cytoplasmic domain of GOS28 in the two-stage reaction results in a higher association rate for the cytoplasmic domain of GOS28.

p115 is involved in transcytosis in hepatocytes and is found on vesicles (45). Independently, it was isolated from cytosol in an assay measuring the transport of proteins between high salt-treated Golgi membranes (38, 39). High salt treatment of membranes removes peripheral membrane proteins, including coatomer, resulting in an increased amount of uncoupled homotypic fusion (64). The antibody against p115 inhibited intra-Golgi transport only after removal of p115 by high salt treatment of the membranes. An inhibition was observed in the one-stage assay but not in the two-stage assay. This result suggests that p115 is recruited to the membrane in an alpha SNAP- and NSF-independent manner and that the p115 N-terminal head domain does not become accessible to antibody during the transport reaction. Ultrastructural analysis of Golgi complexes reveals that vesicles are tethered to Golgi stacks by fibrous structures, unable to diffuse freely (75). p115 mediates binding of vesicles to Golgi membranes (41) and is a candidate for such a string protein. The partial inhibition of the intra-Golgi transport reaction caused by the anti-p115 antibody is consistent with the functional redundancy for interaction of vesicles and target membrane observed in a yeast strain harboring mutated p115. This conditional mutant yeast strain expresses a truncated form of Uso1p, the yeast homologue of p115 (39, 45). The mutant Uso1p lacks the vesicle-binding domain (40, 76), and the thermosensitive phenotype can be suppressed by the overexpression of SNAREs (77), suggesting that SNAREs can bypass the function of Uso1p and mediate the docking of vesicles.

GDI depletes membranes of Rab proteins resulting in a partial inhibition of the one- and two-stage transport assays. An involvement of Rab6 in intra-Golgi transport has been demonstrated (35, 36), and the anti-Rab6 antibody showed an inhibition pattern comparable with GDI. This result suggests that Rab proteins, in particular Rab6, act after or parallel to alpha SNAP and NSF. The GTPase Rab5 is involved in the fusion of early endosomes (78), and several proteins interacting with its GTP-bound form have been identified. One of them is the protein EEA1 (79). Early endosome fusion can be inhibited by GDI and can be stimulated by EEA1 (80). An increased concentration of EEA1 can suppress the inhibition by GDI. The partial inhibition of the intra-Golgi transport assay can be due to an EEA1-like activity that bypasses the Rab6 requirement. A dual requirement for Rab proteins has been resolved for the fusion of yeast vacuoles. Antibodies against the alpha SNAP homologue Sec17p and the NSF homologue Sec18p have been compared with GDI and an antibody against the Rab homologue Ypt7p in a kinetic analysis. Under the assay conditions, GDI and Ypt7p act downstream of Sec17p and Sec18p (81). However, when the concentration of Sec18p present in the assay is increased 50-fold over the amount necessary for saturation (82), the sensitivity of the fusion reaction toward GDI and anti-Ypt7p changes, and a contribution of Ypt7p at a step preceding Sec17p and Sec18p could be demonstrated (83, 84). Biochemical and genetic studies of the endoplasmic reticulum-Golgi transport in yeast corroborate these observations. A genetic analysis of proteins involved in endoplasmic reticulum-Golgi transport revealed an interaction between the Rab homologue YPT1 and the p115 homolgue USO1 (77). In addition, high amounts of GDI remove Ypt1p and Uso1p from membranes (85). On the other hand, transport vesicles containing mutated Ypt1p remain fusion-competent, whereas acceptor membranes carrying the mutant of Ypt1p are fusion-incompetent (86). Two roles for Rab proteins and their associated proteins emerge in the yeast system. One precedes the requirement for Sec17p and Sec18p and catalyzes the tethering of vesicles to membranes. Another not yet defined function of Rab proteins is accomplished after the ATP hydrolysis by Sec18p. In the case of the mammalian Golgi apparatus, vesicles are tethered to the cisternae by vesicle-bound giantin linked via p115 to cisternae-bound GM130. Giantin and GM130 are members of the golgin protein family. Golgins share a Rab6-binding domain that may be required for targeting to the cisternae (87).

    ACKNOWLEDGEMENTS

We thank N. Pavletich for advice concerning the proteolytic digestions, H. Erdjument-Bromage for amino acid sequencing, E. Sztul for the plasmid encoding p115, S. Pfeffer for GDI, and R. Scheller for the expression clone encoding GST-VAMP. The technical assistance of W. Eng is acknowledged. We are grateful to C. Hughes, T. Engel, T. Harder, and J. Pieters for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a postdoctoral fellowship from the Max Kade Foundation (to M. J. S. G.) and a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to C. W.). The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 41616051351; Fax: 41616051364; E-mail: wimmer@bii.ch.

Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M101513200

2 C. Wimmer, M. Amherdt, L. Orci, T. H. Söllner, and J. E. Rothman, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: SNARE, soluble NSF attachment protein receptor; v-SNARE, vesicle localized SNARE; t-SNARE, target membrane localized SNARE; SNAP, soluble NSF attachment protein; NSF, N-ethylmaleimide sensitive factor; GDI, GDP dissociation inhibitor; VAMP, vesicle associated membrane protein; GST, glutathione S-transferase; K-Golgi, 1 M KCl-treated Golgi; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Palade, G. (1975) Science 189, 347-358[Medline] [Order article via Infotrieve]
2. Rothman, J. E., and Wieland, F. T. (1996) Science 272, 227-234[Abstract]
3. Lane, J., and Allan, V. (1998) Biochim. Biophys. Acta 1376, 27-55[Medline] [Order article via Infotrieve]
4. Jahn, R., and Südhof, T. C. (1999) Annu. Rev. Biochem. 68, 863-911[CrossRef][Medline] [Order article via Infotrieve]
5. Wickner, W., and Haas, A. (2000) Annu. Rev. Biochem. 69, 247-275[CrossRef][Medline] [Order article via Infotrieve]
6. Robinson, L. J., and Martin, T. F. (1998) Curr. Opin. Cell Biol. 10, 483-492[CrossRef][Medline] [Order article via Infotrieve]
7. Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318-324[CrossRef][Medline] [Order article via Infotrieve]
8. Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Söllner, T. H., and Rothman, J. E. (1998) Cell 92, 759-772[Medline] [Order article via Infotrieve]
9. Sato, K., and Wickner, W. (1998) Science 281, 700-702[Abstract/Free Full Text]
10. Nichols, B. J., Ungermann, C., Pelham, H. R., Wickner, W. T., and Haas, A. (1997) Nature 387, 199-202[CrossRef][Medline] [Order article via Infotrieve]
11. Clary, D. O., Griff, I. C., and Rothman, J. E. (1990) Cell 61, 709-721[Medline] [Order article via Infotrieve]
12. Whiteheart, S. W., Griff, I. C., Brunner, M., Clary, D. O., Mayer, T., Buhrow, S. A., and Rothman, J. E. (1993) Nature 362, 353-355[CrossRef][Medline] [Order article via Infotrieve]
13. Weidman, P. J., Melancon, P., Block, M. R., and Rothman, J. E. (1989) J. Cell Biol. 108, 1589-1596[Abstract]
14. Salminen, A., and Novick, P. J. (1987) Cell 49, 527-538[Medline] [Order article via Infotrieve]
15. Pfeffer, S. R., Soldati, T., Geissler, H., Rancano, C., and Dirac-Svejstrup, B. (1995) Cold Spring Harbor Symp. Quant. Biol. 60, 221-227[Medline] [Order article via Infotrieve]
16. Nishimura, N., Nakamura, H., Takai, Y., and Sano, K. (1994) J. Biol. Chem. 269, 14191-14198[Abstract/Free Full Text]
17. Shisheva, A., Sudhof, T. C., and Czech, M. P. (1994) Mol. Cell. Biol. 14, 3459-3468[Abstract]
18. Janoueix-Lerosey, I., Jollivet, F., Camonis, J., Marche, P. N., and Goud, B. (1995) J. Biol. Chem. 270, 14801-14808[Abstract/Free Full Text]
19. Burton, J., Roberts, D., Montaldi, M., Novick, P., and De Camilli, P. (1993) Nature 361, 464-467[CrossRef][Medline] [Order article via Infotrieve]
20. Horiuchi, H., Lippe, R., McBride, H. M., Rubino, M., Woodman, P., Stenmark, H., Rybin, V., Wilm, M., Ashman, K., Mann, M., and Zerial, M. (1997) Cell 90, 1149-1159[Medline] [Order article via Infotrieve]
21. Nagahama, M., Orci, L., Ravazzola, M., Amherdt, M., Lacomis, L., Tempst, P., Rothman, J. E., and Söllner, T. H. (1996) J. Cell Biol. 133, 507-516[Abstract]
22. Subramaniam, V. N., Peter, F., Philp, R., Wong, S. H., and Hong, W. (1996) Science 272, 1161-1163[Abstract]
23. Lowe, S. L., Peter, F., Subramaniam, V. N., Wong, S. H., and Hong, W. (1997) Nature 389, 881-884[CrossRef][Medline] [Order article via Infotrieve]
24. Hay, J. C., Chao, D. S., Kuo, C. S., and Scheller, R. H. (1997) Cell 89, 149-158[Medline] [Order article via Infotrieve]
25. Xu, Y., Wong, S. H., Zhang, T., Subramaniam, V. N., and Hong, W. (1997) J. Biol. Chem. 272, 20162-20166[Abstract/Free Full Text]
26. Wong, S. H., Xu, Y., Zhang, T., Griffiths, G., Lowe, S. L., Subramaniam, V. N., Seow, K. T., and Hong, W. (1999) Mol. Biol. Cell 10, 119-134[Abstract/Free Full Text]
27. Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, 863-873[Medline] [Order article via Infotrieve]
28. Bock, J. B., Klumperman, J., Davanger, S., and Scheller, R. H. (1997) Mol. Biol. Cell 8, 1261-1271[Abstract]
29. Tang, B. L., Low, D. Y., Tan, A. E., and Hong, W. (1998) Biochem. Biophys. Res. Commun. 242, 345-350[CrossRef][Medline] [Order article via Infotrieve]
30. Simonsen, A., Bremnes, B., Ronning, E., Aasland, R., and Stenmark, H. (1998) Eur. J. Cell Biol. 75, 223-231[Medline] [Order article via Infotrieve]
31. Tang, B. L., Low, D. Y., Lee, S. S., Tan, A. E., and Hong, W. (1998) Biochem. Biophys. Res. Commun. 242, 673-679[CrossRef][Medline] [Order article via Infotrieve]
32. Antony, C., Cibert, C., Geraud, G., Santa Maria, A., Maro, B., Mayau, V., and Goud, B. (1992) J. Cell Sci. 103, 785-796[Abstract/Free Full Text]
33. Chen, Y. T., Holcomb, C., and Moore, H. P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6508-6512[Abstract]
34. Olkkonen, V. M., Dupree, P., Killisch, I., Lutcke, A., Zerial, M., and Simons, K. (1993) J. Cell Sci. 106, 1249-1261[Abstract/Free Full Text]
35. Mayer, T., Touchot, N., and Elazar, Z. (1996) J. Biol. Chem. 271, 16097-16103[Abstract/Free Full Text]
36. Martinez, O., Schmidt, A., Salamero, J., Hoflack, B., Roa, M., and Goud, B. (1994) J. Cell Biol. 127, 1575-1588[Abstract]
37. White, J., Johannes, L., Mallard, F., Girod, A., Grill, S., Reinsch, S., Keller, P., Tzschaschel, B., Echard, A., Goud, B., and Stelzer, E. H. (1999) J. Cell Biol. 147, 743-760[Abstract/Free Full Text]
38. Waters, M. G., Clary, D. O., and Rothman, J. E. (1992) J. Cell Biol. 118, 1015-1026[Abstract]
39. Sapperstein, S. K., Walter, D. M., Grosvenor, A. R., Heuser, J. E., and Waters, M. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 522-526[Abstract]
40. Nakamura, N., Lowe, M., Levine, T. P., Rabouille, C., and Warren, G. (1997) Cell 89, 445-455[Medline] [Order article via Infotrieve]
41. Sönnichsen, B., Lowe, M., Levine, T., Jamsa, E., Dirac-Svejstrup, B., and Warren, G. (1998) J. Cell Biol. 140, 1013-1021[Abstract/Free Full Text]
42. Mellman, I., and Warren, G. (2000) Cell 100, 99-112[Medline] [Order article via Infotrieve]
43. Brennwald, P. (2000) J. Cell Biol. 149, 1-4[Free Full Text]
44. Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263, 1146-1149[Medline] [Order article via Infotrieve]
45. Barroso, M., Nelson, D. S., and Sztul, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 527-531[Abstract]
46. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
47. Barnstable, C. J., Hofstein, R., and Akagawa, K. (1985) Brain Res. 352, 286-290[CrossRef][Medline] [Order article via Infotrieve]
48. Tagaya, M., Wilson, D. W., Brunner, M., Arango, N., and Rothman, J. E. (1993) J. Biol. Chem. 268, 2662-2666[Abstract/Free Full Text]
49. Elazar, Z., Mayer, T., and Rothman, J. E. (1994) J. Biol. Chem. 269, 794-797[Abstract/Free Full Text]
50. Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418[Medline] [Order article via Infotrieve]
51. Sasaki, T., Kikuchi, A., Araki, S., Hata, Y., Isomura, M., Kuroda, S., and Takai, Y. (1990) J. Biol. Chem. 265, 2333-2337[Abstract/Free Full Text]
52. Clary, D. O., and Rothman, J. E. (1990) J. Biol. Chem. 265, 10109-10117[Abstract/Free Full Text]
53. Balch, W. E., Glick, B. S., and Rothman, J. E. (1984) Cell 39, 525-536[Medline] [Order article via Infotrieve]
54. Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984) Cell 39, 405-416[Medline] [Order article via Infotrieve]
55. Pavletich, N. P., Chambers, K. A., and Pabo, C. O. (1993) Genes Dev. 7, 2556-2564[Abstract]
56. Hayashi, T., Yamasaki, S., Nauenburg, S., Binz, T., and Niemann, H. (1995) EMBO J. 14, 2317-2325[Abstract]
57. DeBello, W. M., O'Connor, V., Dresbach, T., Whiteheart, S. W., Wang, S. S., Schweizer, F. E., Betz, H., Rothman, J. E., and Augustine, G. J. (1995) Nature 373, 626-630[CrossRef][Medline] [Order article via Infotrieve]
58. Fries, E., and Rothman, J. E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3870-3874[Abstract]
59. Balch, W. E., and Rothman, J. E. (1985) Arch. Biochem. Biophys. 240, 413-425[Medline] [Order article via Infotrieve]
60. Lin, C. C., Love, H. D., Gushue, J. N., Bergeron, J. J., and Ostermann, J. (1999) J. Cell Biol. 147, 1457-1472[Abstract/Free Full Text]
61. Mellman, I., and Simons, K. (1992) Cell 68, 829-840[Medline] [Order article via Infotrieve]
62. Malhotra, V., Orci, L., Glick, B. S., Block, M. R., and Rothman, J. E. (1988) Cell 54, 221-227[Medline] [Order article via Infotrieve]
63. Wattenberg, B. W., Raub, T. J., Hiebsch, R. R., and Weidman, P. J. (1992) J. Cell Biol. 118, 1321-1332[Abstract]
64. Elazar, Z., Orci, L., Ostermann, J., Amherdt, M., Tanigawa, G., and Rothman, J. E. (1994) J. Cell Biol. 124, 415-424[Abstract]
65. Kee, Y., Lin, R. C., Hsu, S. C., and Scheller, R. H. (1995) Neuron 14, 991-998[Medline] [Order article via Infotrieve]
66. Ungermann, C., Nichols, B. J., Pelham, H. R., and Wickner, W. (1998) J. Cell Biol. 140, 61-69[Abstract/Free Full Text]
67. Plattner, H., Artalejo, A. R., and Neher, E. (1997) J. Cell Biol. 139, 1709-1717[Abstract/Free Full Text]
68. Steyer, J. A., Horstmann, H., and Almers, W. (1997) Nature 388, 474-478[CrossRef][Medline] [Order article via Infotrieve]
69. Banerjee, A., Barry, V. A., DasGupta, B. R., and Martin, T. F. J. (1996) J. Biol. Chem. 271, 20223-20226[Abstract/Free Full Text]
70. Morgan, A. (1996) Nature 382, 680[Medline] [Order article via Infotrieve]
71. Littleton, J. T., Chapman, E. R., Kreber, R., Garment, M. B., Carlson, S. D., and Ganetzky, B. (1998) Neuron 21, 401-413[Medline] [Order article via Infotrieve]
72. Yang, B., Gonzalez, L., Jr., Prekeris, R., Steegmaier, M., Advani, R. J., and Scheller, R. H. (1999) J. Biol. Chem. 274, 5649-5653[Abstract/Free Full Text]
73. Fasshauer, D., Antonin, W., Margittai, M., Pabst, S., and Jahn, R. (1999) J. Biol. Chem. 274, 15440-15446[Abstract/Free Full Text]
74. Hanson, P. I., Otto, H., Barton, N., and Jahn, R. (1995) J. Biol. Chem. 270, 16955-16961[Abstract/Free Full Text]
75. Orci, L., Perrelet, A., and Rothman, J. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2279-2283[Abstract/Free Full Text]
76. Seog, D. H., Kito, M., Yoda, K., and Yamasaki, M. (1994) J. Biochem. (Tokyo) 116, 1341-1345[Abstract]
77. Sapperstein, S. K., Lupashin, V. V., Schmitt, H. D., and Waters, M. G. (1996) J. Cell Biol. 132, 755-767[Abstract]
78. Gorvel, J. P., Chavrier, P., Zerial, M., and Gruenberg, J. (1991) Cell 64, 915-925[Medline] [Order article via Infotrieve]
79. Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J. M., Brech, A., Callaghan, J., Toh, B. H., Murphy, C., Zerial, M., and Stenmark, H. (1998) Nature 394, 494-498[CrossRef][Medline] [Order article via Infotrieve]
80. Christoforidis, S., McBride, H. M., Burgoyne, R. D., and Zerial, M. (1999) Nature 397, 621-625[CrossRef][Medline] [Order article via Infotrieve]
81. Mayer, A., Wickner, W., and Haas, A. (1996) Cell 85, 83-94[Medline] [Order article via Infotrieve]
82. Haas, A., and Wickner, W. (1996) EMBO J. 15, 3296-3305[Abstract]
83. Ungermann, C., Sato, K., and Wickner, W. (1998) Nature 396, 543-548[CrossRef][Medline] [Order article via Infotrieve]
84. Ungermann, C., Price, A., and Wickner, W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8889-8891[Abstract/Free Full Text]
85. Cao, X., Ballew, N., and Barlowe, C. (1998) EMBO J. 17, 2156-2165[Abstract/Free Full Text]
86. Cao, X., and Barlowe, C. (2000) J. Cell Biol. 149, 55-66[Abstract/Free Full Text]
87. Barr, F. A. (1999) Curr. Biol. 9, 381-384[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.