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Vol. 18, Issue 8, 2852-2863, August 2007
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Biozentrum, University of Basel, CH-4056 Basel, Switzerland
Submitted August 28, 2006;
Revised April 16, 2007;
Accepted May 15, 2007
Monitoring Editor: Randy Schekman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the "activated" SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bonifacino and Glick, 2004). Factors involved in these processes show a high degree of conservation between such distantly related species as yeast and mammals. To bud a vesicle, small GTPases of the ADP-ribosylation factor (Arf)-family are first recruited to the donor membrane. Arf1p is activated through interaction with an ARF guanine nucleotide exchange factor (ArfGEF) at the membrane (Donaldson and Jackson, 2000). Subsequently, coatomer is recruited and forms a complex with Arf1p, cargo, or soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and an ARF-GTPase activating protein (ArfGAP), a so-called primer (Springer et al., 1999; Spang, 2002). This interaction does not require the deactivation of Arf1p by a GAP. If more such priming complexes are formed, the coat polymerizes laterally and deforms the membrane until the donor membrane is severed from the nascent vesicle. Thus, ArfGAPs are not only important to stimulate GTP hydrolysis on Arf1p but also are important at a much earlier step in vesicle biogenesis.
Before vesicle fusion the coat has to be shed to expose the vesicle (v)-SNAREs on the vesicle. Four SNARE domains residing in both vesicle and target membrane interact in trans and zipper up into a coiled-coil bundle, pulling the membranes into proximity (Nichols et al., 1997; Ungermann et al., 1998; Weber et al., 1998). Then, a fusion pore is created that opens up, and finally, membrane fusion takes place. Through the fusion event, the trans-SNARE complex becomes a cis-SNARE complex, which needs to be resolved to recycle the SNAREs for the next round of fusion. Therefore, -soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) and the AAA-ATPase N-ethylmaleimide-sensitive factor (NSF) (Sec17p and Sec18p in Saccharomyces cerevisiae, respectively) are recruited to cis-SNARE complexes and catalytically unwind the helix bundle (Sollner et al., 1993; Morgan et al., 1994; Mayer et al., 1996). The driving force to overcome the strong interaction within the cis-SNARE complex is provided through ATP hydrolysis by NSF (Whiteheart et al., 1994). The involvement of -SNAP and NSF in the priming step of vesicle fusion with the target membrane has been shown to be essential for all vesicle fusion events examined so far as well as for homotypic vacuole fusion in yeast (Wickner, 2002; Morgan and Burgoyne, 2004; Sollner, 2004). However, it remains unclear whether unwinding the remains of the previous fusion event satisfies the Sec18p/NSF requirement before fusion.
Other factors that intimately interact with SNAREs are the ArfGAPs Gcs1p and Glo3p, which have been shown to catalytically induce a conformational change in SNAREs involved in endoplasmic reticulum (ER)–Golgi trafficking (Rein et al., 2002). SNAREs in this altered conformation were able to recruit Arf1p. This recruitment step required neither other factors nor the activation of the GTPase by a GEF. The interaction between Arf1p and SNAREs might provide a mechanism by which each vesicle carries at least one SNARE protein and would therefore be capable of fusing with the target membrane (Rein et al., 2002). However, membrane-anchored cargo proteins may also provide a platform for Arf1p binding.
Previously, we reported that the ArfGAPs Glo3p and Gcs1p induce a conformational change in v-SNAREs involved in ER–Golgi and post-Golgi trafficking (Rein et al., 2002; Robinson et al., 2006). In this article, we extended these results to t-SNAREs (target-SNAREs on the target membrane) and show that Gcs1p accelerates v-t-SNARE complex formation in vitro. The ArfGAPs may promote the conversion of a high-energy to a low-energy state in SNAREs and thereby help to form SNARE complexes. These v-t-SNARE complexes were not dead-end complexes, because they could be resolved by the action of Sec17p and Sec18p in the presence of ATP. Furthermore, we revealed an additional function of Sec18p as it could displace Arf1p from single SNAREs in vitro. Surprisingly, this binding to single SNAREs was independent from ATP hydrolysis and Sec17p. Thus, Sec18p may have an additional role during the priming step in vesicle fusion: to ensure that the SNAREs are free to engage in v/t-SNARE complexes and that they are not masked by coat proteins bound to the SNAREs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). GST-Sed5p was expressed from pGEX2T, GST-Snc1p from pGEX4T1, GST-Vam3p and GST-Nyv1p from pGEX4T3, and GST-Tlg2p from pGEX6p-1. His-tagged SNAREs were obtained by expression using vector pET24b (Novagen, Madison, WI). Sec17p and Sec18p were expressed from pQE9 (QIAGEN, Valencia, CA), Sec18E350Qp was expressed from pQE30 (QIAGEN). The vectors for expression of Gcs1p, Glo3p, and Age2p and Arf1
N17p-Q71L have been described previously (Poon et al., 1996, 1999, 2001; Rein et al., 2002).
Protein Purification
The different SNARE-GST fusion proteins were expressed in Escherichia coli BL21* and purified over glutathione-agarose. Cells from 1-liter culture were resuspended in 20 ml of STE (25% [wt/vol] sucrose, 50 mM Tris-Cl, pH 8.0, and 40 mM EDTA). After 1 mg/ml lysozyme treatment, 8 ml of 50 mM Tris-Cl, pH 8.0, 0.2% Triton X-100, and 100 mM MgCl2 was added, and the suspension was subjected to sonication. The cell lysate was cleared by centrifugation (10,000 x g for 20 min). The supernatant was bound to 1 ml glutathione-agarose beads (Sigma-Aldrich, St. Louis, MO) for 2 h at 4°C. Beads were washed several times in phosphate-buffered saline (PBS), 15% glycerol and transferred into a Poly-Prep column (Bio-Rad, Hercules, CA). Protein was eluted with 50 mM glutathione, 150 mM Tris, pH 8.0, 120 mM NaCl, 5 mM dithiothreitol (DTT), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein-containing fractions were pooled and dialyzed against PBS, 5% glycerol. His6-tagged SNARE proteins and His6-Sec17p were expressed in E. coli BL21* and purified using Ni2+-nitrilotriacetic acid (NTA)-agarose (QIAGEN) according to the manufacturer's instructions. Arf1N17p was purified as described previously (Rein et al., 2002). His6-Sec18p and His6-Sec18E350Qp were purified according to Whiteheart et al., (1994) with the following modifications: Ni2+-NTA-agarose with bound proteins was washed in batch twice with 10 column volumes of wash buffer 20 mM HEPES-KOH, pH 6.8, 400 mM KOAc, 0.5 mM ATP, 1 mM MgCl2, 1 mM 2-mercaptoethanol, and 10% glycerol, and 2 x 10 column volumes of wash buffer20, 2 x 10 column volumes of wash buffer50 (wash buffer containing 20 and 50 mM imidazole, respectively). After the last wash, beads were resuspended in wash buffer100 (wash buffer containing 100 mM imidazole), transferred into a Poly-Prep column, and washed with five column volumes of wash buffer100. Protein was eluted with elution buffer (wash buffer containing 250 mM imidazole). Eluted protein was dialyzed against 20 mM HEPES, pH 6.8, 150 mM KOAc, 5 mM Mg(OAc)2, 15% glycerol, 1 mM ATP, 1 mM DTT, and 1 mM PMSF. ArfGAPs were purified and the GAP activity was determined as described previously (Huber et al., 2001; Rein et al., 2002).
SNARE Binding Assay
SNARE binding assays were performed as described previously (Rein et al., 2002). Arf1N17p-Q71L (5 µM) was used in the reaction. When indicated, the reaction contained 20 nM Gcs1p, Glo3p, or Age2p. Sec17p and Sec18p were used at concentrations of 0.6 µM His6-Sec17p, 0.6 µM His6-Sec18p, or 0.6 µM His6-Sec18(E350Q)p. For the competition assays with Arf1N17p-Q71L, 15 nM to 3.5 µM His6-Sec18p was used. ATP (1 mM) and 0.5 mM GTP were added where indicated. Incubation steps with Arf1N17p-Q71L or ArfGAP were performed for 1 h at 4°C, whereas His6-Sec17p, His6-Sec18p, or His6-Sec18(E350Q)p were incubated for 1 h at room temperature (RT). The recruitment of Arf1N17p-Q71L to SNAREs was visualized by Coomassie blue staining. SDS-polyacrylamide gel electrophoresis (PAGE) containing assays performed with His6-Sec17p or His6-Sec18p were stained with SyproRed (Invitrogen, Carlsbad, CA) or according to Fairbanks et al. (1971), which is 2–4 times more sensitive than conventional Coomassie staining. Images were acquired with a Storm PhosphorImager (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Bands were quantified using the ImageQuant software (GE Healthcare).
Assembly and Disassembly of SNARE Complexes
SNARE complex assembly was performed for indicated times at 4°C as described previously (Peng and Gallwitz, 2002) with minor modifications: 5 µg of Sed5p GST-fusion protein was immobilized on glutathione-agarose beads and was incubated with 10 µg of Bet1p-His6, 20 µg of Bos1p-His6, and 20 µg of Sec22p-His6 in a total assay volume of 200 µl at 4°C, in the presence or absence of 10 nM Gcs1p. The reaction was stopped at indicated time points by centrifugation, followed by three washes in buffer C (25 mM Tris-Cl, pH 7.5, 150 mM KCl, 10% glycerol, 1% Triton X-100, and 2 mM 
-mercaptoethanol) and a final wash in 20 mM Tris-Cl, pH 7.5. Bound proteins were dissolved in sample buffer by heating the beads 10 min at 65°C. The proteins were separated by SDS-PAGE and visualized by Fairbanks staining. Band intensities were determined using an Odyssey imaging system (LI-COR, Lincoln, NE). To dissolve SNARE complexes, they were formed as described above for 18–22 h, washed twice in buffer C, and once in disassembly buffer containing 30 mM HEPES-KOH, pH 7.4, 70 mM KCl, 5 mM MgCl2, 2.5 mM EGTA, 0.5 mM DTT, and 2 mM ATP. SNARE complex disassembly was performed by addition of 1.35 µM His6-Sec17p and 1.5 µM His6-Sec18p in an assay volume of 100 µl. The assay was incubated for 1 h at RT, and then it was stopped immediately by the addition of 1 ml of ice-cold disassembly buffer. The beads were settled 30 min on ice. Unbound protein was removed by two washes in disassembly buffer and a final wash in 20 mM HEPES, pH 7.4. Bound protein was analyzed as described above.
Microsome Binding Assay
Preparation of microsomal membranes and the assay for binding to microsomal membranes was carried out as described previously (Rein et al., 2002), with the following modifications: The membranes containing 
8 µg protein were incubated with 8 nM Gcs1p, 10 nM His6-Sec18p in the presence of 0.5 mM ATP in a total volume of 50 µl with gentle agitation for 30 min at 30°C. Unbound protein was removed by centrifugation. After washing, microsomal membranes were dissolved in sample buffer containing 8 M urea, resolved by SDS-PAGE, and analyzed by immunoblot.
Blue Native Gel Electrophoresis
Fifty micrograms of SNARE-GST or GST was immobilized onto 100 µl of glutathione-agarose beads. After removal of unbound proteins, beads were split evenly. One half was incubated overnight (O/N) at 4°C with 13.3 nM Gcs1p, whereas to the other half an equal volume of buffer was added. The proteins were eluted from the beads in the presence of 100 mM glutathione and loaded on a 10% native gel. Blue native gel electrophoresis was performed according to Schaegger and von Jagow (1991) with minor modifications: 10% minigels were used instead of gradient gels. After the run, proteins were blotted onto polyvinylidene difluoride and analyzed using antibodies against GST.
Circular Dichroism (CD) Spectroscopy
Sed5-His6 was mixed with 50 mM potassium phosphate buffer, pH 6.8, in a total volume of 560 µl. The sample was split evenly, and 20 µl of Glo3p or 20 µl of ArfGAP dialysis buffer was added. The final protein concentration of Sed5-His6 was 2.2 µM in the assay and 6.7 nM for Glo3p. For the 2,2,2-trifluorethanol (TFE) treatment, the samples were prepared accordingly, and 150 µl of TFE was added to the reaction mix. The assays were incubated at 4°C for 14–16 h. CD spectra were recorded at 4°C by using a JASCO J720 CD spectrometer. Measurements were performed in Hellma quartz cuvettes with a path length of 0.1 cm. Spectra from 15 consecutive scans (250-190 nm; 50 nm/min scan rate; 1-nm step size; 2-nm bandwidth) were averaged.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N17p to v- and t-SNAREs; Robinson et al., 2006). SNAREs in this primed conformation are able to recruit Arf1p. We aimed to test whether this ArfGAP activity is limited to v-SNAREs or could be extended to t-SNAREs as well. To test this, we used recombinantly expressed ArfGAP and Arf1N17p-Q71L. Arf1N17p lacks the N-terminal 17 amino acids that are strongly hydrophobic and carry the myristoylation site; thus, they would interfere with purification and in vitro assays. The Q71L point mutant reflects the predominantly activated form of Arf1p. We have shown previously that Arf1p does not have to be activated to bind to v-SNAREs (Rein et al., 2002). Despite this finding, we used Arf1N17p-Q71L in most experiments.
We performed GST pull-down assays with SNARE proteins (Table 1) that carried a GST fusion either at the N terminus or which replaced the C-terminal transmembrane domain. Similarly to what we reported previously (Rein et al., 2002), the ArfGAPs Gcs1p and Glo3p were able to induce a conformational change in most SNARE proteins tested, albeit with varying efficiencies (Figure 1). ArfGAPs promoted Arf1N17p-Q71L and Arf1N17p binding to the v-SNAREs Bet1p, Bos1p, Sec22p, Ykt6p, and Snc1p (Rein et al., 2002; Robinson et al., 2006; Figure 1, A and B, and Supplemental Figure S1) without themselves binding in stoichiometric amounts to the SNAREs. Furthermore, ArfGAPs allowed recruitment of the small GTPase on the t-SNARE Sed5p. Arf1N17p-Q71L was also recruited in significant amounts to the GST fusions of the syntaxin t-SNAREs Vam3p, Tlg2p, Sso1p, and Sso2p (Figure 1C; data not shown). In contrast, we did not detect any Arf1N17p-Q71L recruitment to N- or C-terminally tagged Tlg1p or GST-Sso1p. Despite the difference in the level of Arf1pN17p-Q71L recruitment by the two ArfGAPs, both GAPs were able to induce a conformational change on most of the SNAREs tested. In contrast, a third ArfGAP, Age2p, did not lead to the recruitment of stoichiometric amounts of Arf1N17p-Q71L to SNAREs (data not shown). The GTPase Arf1p is involved not only in the generation of COPI-coated vesicles at the ER–Golgi interface but also in the formation of several clathrin- and nonclathrin-coated vesicles. Thus, Arf1p recruitment to SNARE proteins in the "primed" conformation occurs throughout the secretory and endocytic pathway. These interactions seem to provide a mechanism to include SNAREs into nascent vesicles and could provide the means by which not only v-SNAREs but also t-SNAREs reach their final compartment. Furthermore, the conformational change induced by ArfGAPs could be important for vesicle fusion at the target membrane.
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N17Q71L to SNAREs. As expected, Age2p did not accelerate SNARE complex formation (Figure 2, G–I). Thus, the conformational change induced by the ArfGAPs Glo3p and Gcs1p catalyzes the zippering process of SNAREs.
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-Helical ContentN17Q71Lp to bind to the SNAREs was assayed at various times after the preincubation step (Figure 3, A–D). No significant loss of the Arf1N17Q71Lp binding to the SNAREs was observed even after 48 h at 23°C after the preincubation with ArfGAP. This result demonstrates that the conformational change induced by the ArfGAPs Glo3p and Gcs1p is stable and that SNAREs do not revert their conformation spontaneously.
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-helical content, we first determined the maximal signal we could expect by incubating Sed5p-His6 with the -helix-stabilizing agent TFE (Figure 3E). Because significant changes were observed with TFE, we recorded CD spectra for Sed5p-His6 in the presence and absence of Glo3p (Figure 3F). For this experiment, we increased the molar ratio of the SNARE over the GAP about another 10-fold to ensure that we would not detected any signal related to Glo3p. Indeed, under these conditions Glo3p did not give a signal over the buffer only control (data not shown). In the presence of Glo3p, Sed5p-His6 showed a small but reproducible increase in -helical content (Figure 3F), demonstrating that Glo3p induces a conformational change on Sed5p-His6. These data are in agreement with our previous published results showing that SNAREs become more protease-resistant after treatment with Glo3p (Rein et al., 2002).
Sec17p and Sec18p Disassemble SNARE Complexes Formed in the Presence of ArfGAP
We tested next whether the addition of Sec17p (the yeast homologue of -SNAP) and Sec18p (the yeast homologue of NSF) but not of an ATPase-deficient Sec18p [Sec18(E350Q)p] (Steel et al., 1999) could dissolve the SNARE complexes again, and whether this process requires energy. Sec18(E350Q)p can still bind ATP and thus forms the characteristic hexameric complex; yet, it is deficient in ATP hydrolysis. Indeed, Sec17p, together with Sec18p but not with mutant Sec18(E350Q)p, disassembled SNARE complexes (Figure 4, compare lines 2 and 3 and lines 7 and 8), implying that ArfGAPs may render SNAREs fusion competent already upon inclusion into transport vesicles. Furthermore, these results strongly suggest that ArfGAPs can indeed promote SNARE complex formation, perhaps by acting like a chaperone.
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Gcs1p Enhances Sec18p Binding to Microsomal Membranes
We wanted to extend our in vitro findings that Sec18p bound to SNAREs in an ArfGAP-dependent manner, to conditions that would better reflect the in vivo situation. Therefore, we tested whether Sec18p would be recruited to microsomal membranes in an ArfGAP-dependent manner. Microsomal membranes were incubated with Sec18p and ATP in the presence or absence of Gcs1p. After incubation at 30°C, the membrane fraction was isolated and analyzed by immunoblot (Figure 6). Although Sec18p binding to membranes did not necessitate the presence of ArfGAP, Gcs1p strongly enhanced the interaction of Sec18p with SNAREs on microsomal membranes. Sec17p is still present on the microsomal membranes and may recruit Sec18p in the absence of ArfGAP. It is very unlikely that SNARE complex formation was induced by Gcs1p, especially because there are no transport vesicles formed under these conditions. Therefore, it seems more likely that Gcs1p permits binding of Sec18p to single SNAREs. Together, our data demonstrate an ArfGAP-dependent Sec18p recruitment to SNAREs and that this recruitment might not correlate to the Sec18p function to resolve SNARE complexes.
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). We next asked where Sec17p and Sec18p might bind to Bet1p. Fragments of the cytoplasmic domain of Bet1p (Figure 7A) were used to determine the binding sites of Sec17p and Sec18p. Sec17p bound most efficiently to the coiled-coil SNARE domain made up of Bet1p(residues 41-119) and to a smaller fragment of the membrane-proximal region, Bet1p(79-119) (Figure 7B). However, strong binding also occurred to the membrane-distal region of the SNARE domain [Bet1p(41-79)]. Because Sec17p migrates at the same position on the gel as the N-terminal fragment Bet1p(1-65), Sec17p binding to Bet1p(1-65), was determined instead by semiquantitative immunoblot and was found to be close to background levels (data not shown). Thus, Sec17p binds to the SNARE domain of Bet1p. Sec18p also bound to the SNARE domain, although a preference for the membrane-proximal part was detectable (Figure 7, C and D).
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demonstrated that not only Sec17p and Sec18p bind to a SNARE complex containing Vam3p and Nyv1p but also that there is a Sec17p-independent function of Sec18p after unwinding of the complex. The Vam3p(1-144)-GST construct contained the SNARE domain, which was deleted in the Nyv1p(1-136)-GST construct. Although Sec18p and Sec17p bound the full-length Vam3p-GST and Nvy1p-GST as well as to the SNARE domain of Vam3p(1-144)-GST alone, no binding was observed to the Nyv1p(1-136)-GST construct lacking the SNARE domain (data not shown). Thus, both Sec17p and Sec18p bind to the SNARE domains of different SNAREs.
Sec18p Competes with Arf1N17p-Q71L for Binding Sites on SNARE Domains
We have reported previously that Arf1p also binds to the SNARE domain, preferentially to the membrane-proximal region. Because of the overlap of the binding sites of Sec18p, Sec17p, and Arf1p, we wanted to test whether these proteins would compete with each other for binding sites on SNAREs. Therefore, we first formed a complex of Arf1N17p-Q71L and Bet1p-GST with the help of Glo3p. After extensive washing, this complex was then incubated with increasing concentrations of Sec18p. Arf1N17p-Q71L was partially displaced from the SNARE at high Sec18p concentrations (Figure 8A). However, the amount of Sec18p recruited to the Arf1N17p–Q71L–SNARE complex was reduced compared with primed SNAREs alone. When we reversed the order of the addition of Arf1N17p-Q71L and Sec18p, binding of Arf1N17p-Q71L was strongly reduced in the presence of stoichiometric amounts of Sec18p on the SNAREs (Figure 8B). We repeated the assays with an intermediate Sec18p concentration (Figure 8, C–F). Under these conditions, Sec18p displaced 
50% of Arf1N17p-Q71L from Bet1p-GST, whereas in the converse experiment, Arf1N17p-Q71L could not compete with Sec18p for binding sites on Bet1p (Figure 8, compare C with F). Yet, not all binding sites could be exchanged; preincubation of Bet1p with Arf1N17p-Q71L reduced Sec18p binding by 50%, and a similar effect of Sec18p on Arf1N17p-Q71L was observed when the order of addition was inverted (Figure 8, D and E). Furthermore, Arf1N17p-Q71L was unable to chase Sec18p off Bet1p-GST (Figure 8F). The Sec18p-mediated loss of Arf1N17p-Q71L from the SNARE was independent of nucleotide in the reaction mix for all conditions tested. Together, Sec18p could partially displace prebound Arf1N17p-Q71L from Bet1p-GST, and this displacement did not require energy.
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N17p-Q71L or Arf1N17p bound to Bet1p-GST (Figure 9B). Furthermore, the ability of Sec18(E350Q)p to bind to Bet1p-GST prebound to Arf1N17p or Arf1N17p-Q71L was similar to that of Sec18p (Figure 9C). Therefore, the competition of Arf1N17p or Arf1N17p-Q71L and Sec18p for binding sites on the SNARE protein is most likely not due to unwinding of SNARE complexes but rather involves steric hindrance by Sec18p.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We tested three different ArfGAPs for their ability to recruit Arf1N17p-Q71L to SNARE proteins as indication for the conformational change. Two of them, Glo3p and Gcs1p, but not Age2p, promoted Arf1N17p-Q71L binding. We have previously demonstrated that the transient interaction Glo3p with the v-SNAREs Sec22p and Bet1p renders the SNAREs more protease resistant (Rein et al., 2002). This conformational change may promote the inclusion of SNAREs into vesicles, and they might even serve as primers for vesicle formation (Springer et al., 1999; Spang, 2002).
Gcs1p and Glo3p are the orthologues of mammalian ARFGAP1 and ARFGAP3, respectively. At least for ARFGAP1, a similar role in the inclusion of the v-SNARE membrin into vesicles has been reported previously (Honda et al., 2005). ARFGAP1 contains a domain that senses membrane curvature via an amphiphatic helix (Bigay et al., 2003). This domain could be as well responsible for the catalytic change on SNAREs. This exact domain is not conserved in Glo3p. However, Glo3p contains a coiled-coil region, which could fulfill a similar function. Age2p contains neither an amphiphatic helix nor a coiled-coil domain. The difference in the structural organization of the ArfGAPs may explain the variation in the ability to act as chaperone on SNAREs. Whether this chaperone-like function is limited to SNAREs or whether ArfGAPs are generally involved in cargo recruitment remains to be established. The KDEL receptor Erd2 and members of the p24 family of proteins, however, require ARFGAP1 for uptake into COPI-coated vesicles (Aoe et al., 1999; Lanoix et al., 2001).
Both v- and t-SNAREs were positively affected by the interaction with ArfGAPs. One explanation for this observation is that this interaction provides the mechanism by which t-SNAREs reach their final compartment and that the conformational change serves as switch form a high- to a low-energy state, which promotes efficient incorporation of also t-SNAREs into transport vesicles. v-SNAREs seem to use this pathway for efficient inclusion into at least COPI-coated vesicles (Rein et al., 2002; Robinson et al., 2006). Alternatively, the conformational change on t-SNAREs may only take place on the target membrane and may accelerate trans-SNARE complex formation.
We found that ArfGAP accelerated the formation of SNARE complexes and that these complexes were resolved by the yeast homologues of NSF and -SNAP, Sec18p and Sec17p, respectively. Surprisingly, Sec18p and Sec17p also bound independently of each other to SNAREs. This interaction was strictly dependent on the induced conformational switch on SNAREs by ArfGAPs. Moreover, Sec18p competed with Arf1N17p for binding sites on SNAREs. Because this interaction did not require nucleotide hydrolysis, this function of Sec18p is most likely unrelated to the well-established unwinding activity of the AAA-ATPase required to dissolve the four-helix bundle of cis-SNARE complexes. Why would Sec18p bind to SNAREs and displace Arf1p? A transport vesicle arrives at the target membrane, and although it is generally thought that all coat has been shed at this point, experimental proof is lacking. The SNAREs on the vesicles must be exposed to engage in trans-SNARE complexes with the SNAREs on the target membrane. Sec18p could bind to SNAREs and displace coat proteins, especially Arf1p. We have shown previously that binding of Arf1N17p binds to SNAREs in a nucleotide-independent manner. Therefore, it is conceivable that Arf1p-GDP might still stick to SNARE proteins after GTP hydrolysis has occurred and most of the coat proteins have left the transport vesicle. Displacement of Arf1p by Sec18p would provide a mechanism, which would ensure the availability of the SNAREs for complex formation. Because Sec17p is not required at this step, Sec18p is unable to unwind the four-helix bundle and efficient membrane fusion could proceed. This function of Sec18p would also be independent of the action of tethering complexes. Tethering complexes may actually require the presence of residual coat components as determinants for the tethering activity (Malsam et al., 2005).
This is the first report demonstrating binding of Sec18p/NSF in stoichiometric amounts to SNAREs in the absence of Sec17p/-SNAP. Supposedly, Sec18p/NSF only binds to SNAREs after Sec17p/-SNAP recruitment to cis-SNARE complexes (Sollner et al., 1993; Morgan et al., 1994). We used, however, single SNARE molecules; therefore, we likely provide a different molecular environment compared with the four-helix bundle of the cis-SNARE complex. The binding of Sec17p to single SNARE molecules has been shown previously, and this binding was independent of ArfGAP (Rossi et al., 1997). However, in the experimental setup of Rossi et al. (1997), only substiochiometric amounts of Sec17p were recruited, which were detected by immunoblot. ArfGAP-mediated binding of Sec17p to SNAREs was stoichiometric and detectable by Coomassie blue staining, despite an at least fivefold lower concentration of Sec17p in the assay. Therefore, the ArfGAP-induced conformational change greatly enhances the binding of Sec17p to SNAREs. Furthermore, Sec18p binding to single SNAREs was probably undetected because of the low affinity of Sec18p to the single SNARE in the high-energy state.
We mapped the binding sites of Sec18p and Sec17p on the SNAREs. Sec17p and Sec18p bound to the SNARE domain and Sec18p had within this domain a slight preference for the membrane proximal part. These data seem to somewhat disagree with previously published reports, where Sec18p/NSF and Sec17p/-SNAP bind to the N-terminal (membrane distal) part of cis-SNARE complexes (Hanson et al., 1997; Hohl et al., 1998). However, our mapping data were performed on single SNAREs, and the binding of Sec18p was determined in the absence of Sec17p. Sec18p binding to cis-SNARE complexes is strictly dependent on Sec17p. Therefore, the mode of interaction of Sec18p with single SNAREs and with SNAREs complexes could be different. However, one could probably expect a high-affinity of Sec18p to SNARE domains and that is exactly what we observe. These data also support our model of dual function of Sec18p in membrane traffic: first, Sec18p ensures that the single SNAREs can engage in trans-SNARE complexes by displacing residual coat components; and second, Sec18p, together with Sec17p, unwinds cis-SNARE complexes to free the SNAREs for another round of transport.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Anne Spang (anne.spang{at}unibas.ch).
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REFERENCES |
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Bigay, J., Gounon, P., Robineau, S., Antonny, B., Schekman, R., and Orci, L. (2003). Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Self-assembly of minimal COPII cages. Nature 426, 563–566.[CrossRef][Medline]
Bonifacino, J. S., and Glick, B. S. (2004). The mechanisms of vesicle budding and fusion. Cell 116, 153–166.[CrossRef][Medline]
Donaldson, J. G., and Jackson, C. L. (2000). Regulators and effectors of the ARF GTPases. Curr. Opin. Cell Biol 12, 475–482.[CrossRef][Medline]
Fairbanks, G., Steck, T. L., and Wallach, D. F. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606–2617.[CrossRef][Medline]
Hanson, P. I., Roth, R., Morisaki, H., Jahn, R., and Heuser, J. E. (1997). Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. Cell 90, 523–535.[CrossRef][Medline]
Hohl, T. M., Parlati, F., Wimmer, C., Rothman, J. E., Sollner, T. H., and Engelhardt, H. (1998). Arrangement of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes. Mol Cell 2, 539–548.[CrossRef][Medline]
Honda, A., Al-Awar, O. S., Hay, J. C., and Donaldson, J. G. (2005). Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE. J. Cell Biol 168, 1039–1051.
Huber, I., Rotman, M., Pick, E., Makler, V., Rothem, L., Cukierman, E., and Cassel, D. (2001). Expression, purification, and properties of ADP-ribosylation factor (ARF) GTPase activating protein-1. Methods Enzymol 329, 307–316.[CrossRef][Medline]
Kirchhausen, T. (2000). Three ways to make a vesicle. Nat. Rev. Mol. Cell Biol 1, 187–198.[CrossRef][Medline]
Lanoix, J., Ouwendijk, J., Stark, A., Szafer, E., Cassel, D., Dejgaard, K., Weiss, M., and Nilsson, T. (2001). Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1. J. Cell Biol 155, 1199–1212.
Malsam, J., Satoh, A., Pelletier, L., and Warren, G. (2005). Golgin tethers define subpopulations of COPI vesicles. Science 307, 1095–1098.
Mayer, A., Wickner, W., and Haas, A. (1996). Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85, 83–94.[CrossRef][Medline]
Morgan, A., and Burgoyne, R. D. (2004). Membrane traffic: controlling membrane fusion by modifying NSF. Curr. Biol 14, R968–R970.[CrossRef][Medline]
Morgan, A., Dimaline, R., and Burgoyne, R. D. (1994). The ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF) is regulated by soluble NSF attachment proteins. J. Biol. Chem 269, 29347–29350.
Nichols, B. J., Ungermann, C., Pelham, H. R., Wickner, W. T., and Haas, A. (1997). Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature 387, 199–202.[CrossRef][Medline]
Peng, R., and Gallwitz, D. (2002). Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes. J. Cell Biol 157, 645–655.
Poon, P. P., Cassel, D., Spang, A., Rotman, M., Pick, E., Singer, R. A., and Johnston, G. C. (1999). Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function. EMBO J 18, 555–564.[CrossRef][Medline]
Poon, P. P., Nothwehr, S. F., Singer, R. A., and Johnston, G. C. (2001). The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network. J. Cell Biol 155, 1239–1250.
Poon, P. P., Wang, X., Rotman, M., Huber, I., Cukierman, E., Cassel, D., Singer, R. A., and Johnston, G. C. (1996). Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein. Proc. Natl. Acad. Sci. USA 93, 10074–10077.
Rein, U., Andag, U., Duden, R., Schmitt, H. D., and Spang, A. (2002). ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat. J. Cell Biol 157, 395–404.
Robinson, M., Poon, P. P., Schindler, C., Murray, L. E., Kama, R., Gabriely, G., Singer, R. A., Spang, A., Johnston, G. C., and Gerst, J. E. (2006). The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the Golgi in yeast. Mol. Biol. Cell 1, 1.
Rossi, G., Salminen, A., Rice, L. M., Brunger, A. T., and Brennwald, P. (1997). Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9. J. Biol. Chem 272, 16610–16617.
Schaegger, H., and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem 199, 223–231.[CrossRef][Medline]
Sharrocks, A. D. (1994). A T7 expression vector for producing N- and C-terminal fusion proteins with glutathione S-transferase. Gene 138, 105–108.[CrossRef][Medline]
Sollner, T. H. (2004). Intracellular and viral membrane fusion: a uniting mechanism. Curr. Opin. Cell Biol 16, 429–435.[CrossRef][Medline]
Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993). SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324.[CrossRef][Medline]
Spang, A. (2002). ARF1 regulatory factors and COPI vesicle formation. Curr. Opin. Cell Biol 14, 423.[CrossRef][Medline]
Springer, S., Spang, A., and Schekman, R. (1999). A primer on vesicle budding. Cell 97, 145–148.[CrossRef][Medline]
Steel, G. J., Laude, A. J., Boojawan, A., Harvey, D. J., and Morgan, A. (1999). Biochemical analysis of the Saccharomyces cerevisiae SEC18 gene product: implications for the molecular mechanism of membrane fusion. Biochemistry 38, 7764–7772.[CrossRef][Medline]
Ungermann, C., Nichols, B. J., Pelham, H. R., and Wickner, W. (1998). A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion. J. Cell Biol 140, 61–69.
Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T. H., and Rothman, J. E. (1998). SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772.[CrossRef][Medline]
Whiteheart, S. W., Rossnagel, K., Buhrow, S. A., Brunner, M., Jaenicke, R., and Rothman, J. E. (1994). N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion. J. Cell Biol 126, 945–954.
Wickner, W. (2002). Yeast vacuoles and membrane fusion pathways. EMBO J 21, 1241–1247.[CrossRef][Medline]
Xu, Z., Sato, K., and Wickner, W. (1998). LMA1 binds to vacuoles at Sec18p (NSF), transfers upon ATP hydrolysis to a t-SNARE (Vam3p) complex, and is released during fusion. Cell 93, 1125–1134.[CrossRef][Medline]
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