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J. Biol. Chem., Vol. 282, Issue 20, 14861-14867, May 18, 2007

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Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification^*

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

Received for publication, February 1, 2007 , and in revised form, March 8, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
SNARE proteins form bundles of four -helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q·1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7^Q283R, yet Vam7^Q283R is preferred by vacuoles with Nyv1^R191Q. Rotation of the position of the arginine in the 0-layer increases the K_m for Vam7p but does not affect the maximal rate of fusion. Vam7^Q283R forms stable 2Q·2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122 [GenBank] , perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
SNARE⁴ proteins (1) are vital for membrane fusion. Initially discovered in neuronal tissues, SNAREs are found on all organelles of the exocytic and endocytic pathways, from yeast to humans. Their characteristic feature is a heptad-repeat "SNARE domain," flanked by varied N-domains and by C-terminal membrane anchors, either a single membrane-spanning apolar polypeptide or an acyl anchor. SNAREs form 4-helical complexes through their SNARE domains (2). Although the residues in each SNARE that face the others in a 4-helical complex are generally apolar, 3 glutamine (Q) and 1 arginine (R) near the center of the 4-complexed SNARE domains form a conserved and polar "0-layer" (3). Recombinant neuronal SNAREs spontaneously assemble into stable bundles, yet SNARE complex assembly in vivo requires SNARE-binding proteins of the Sec1-Munc18 "SM" family. SNARE complexes are disassembled by two chaperones: Sec17p/-SNAP, which binds directly to SNARE complexes, and Sec18p/NSF, which binds to Sec17p/-SNAP and couples the energy of ATP binding and hydrolysis to SNARE complex disassembly (4). SNARE complexes form in cis, with each SNARE anchored to the same membrane, or in trans, with SNAREs anchored to apposed, "tethered" membranes.

SNARE function has been studied in vivo, on isolated organelles, and through the reconstitution of recombinant SNAREs into proteoliposomes. When several SNAREs, which are found in vivo on a "target" membrane, are co-reconstituted into one population of liposomes, they form a t-SNARE complex. These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7, 8). This reconstituted reaction shows impressive specificity for cognate SNARE pairs (9) and can be directly promoted by other fusion-regulatory factors such as synaptotagmin and calcium (10) or Sec1p (11) under conditions that minimize lysis (12).

SNAREs are required for the homotypic fusion of yeast vacuoles (13). Vacuoles fission and fuse during cell division and organelle inheritance (14) and in response to growth medium osmolarity (15). Purified vacuoles undergo a multistep fusion pathway, which can be monitored by content-mixing assays (16). In the initial "priming" step, Sec18p/Sec17p disassemble cis-SNARE complexes (17). The following tethering stage of "docking" requires the Rab-family GTPase Ypt7p (18) and its effector complex, termed HOPS (homotypic fusion and vacuole protein sorting) (19), which has Vps11p, Vps16p, Vps18p, Vps33p, Vps39p, and Vps41p as subunits (20, 21). Tethered vacuoles are drawn against each other at closely apposed disc-shaped microdomains, termed "boundary membranes" to distinguish them from the "outside membranes," which are not in contact (22). The proteins (Ypt7p, HOPS, and SNAREs) and lipids (phosphoinositides, ergosterol, and diacylglycerol), which are required for fusion undergo striking, interdependent enrichment at a ring-shaped microdomain, termed the "vertex ring," which surrounds the boundary membrane (22–25). Vacuole SNAREs form trans-complexes between the apposed organelles (17). Ypt7p and HOPS are needed for SNARE complex assembly, and HOPS is an integral part of the assembled SNARE complex (26). Fusion occurs around the vertex ring, joining the outside membrane from each vacuole to form the larger, fused organelle and joining the two apposed boundary membrane discs to yield a luminal vesicle (22, 27).

Vacuoles have four SNAREs; their roles have been studied in the organelle-based fusion reaction (26, 28) and in liposome-based studies (29). Vam3p, Vam7p, and Vit1p are Q-SNAREs, which constitute the t-SNARE, and the R-SNARE Nyv1p can serve as the v-SNARE. Three of these have C-terminal trans-membrane anchors, but Vam7p has no apolar membrane anchor. Whereas most purified SNAREs require detergent for their solubility, and thus cannot be directly added to purified organelles without causing lysis, recombinant Vam7p is water-soluble without detergent and supports vacuole fusion (30).

Although SNAREs are central to fusion, it remains unclear how they act. SNAREs may apply torque to membranes (31, 32), destabilize bilayers through poorly fitting TM domains (33, 34), gather fusogenic lipids (e.g. diacylglycerol) into fusion microdomains (25), form the walls of a fusion pore (35), or act by other means. Studies of integral membrane SNAREs in the context of their native membrane and organelle are limited to producing and characterizing mutants, which may have significant effects on organelle composition and cell growth. The vacuolar Vam7p SNARE affords a unique window into SNARE function, because its cloned recombinant form is functional for fusion in vitro, it can readily be expressed and purified in soluble recombinant form, and many subreaction assays of vacuole fusion are available. Vam7p is a Q-SNARE with an N-terminal PX (Phox homology) domain, which directly binds phosphatidylinositol 3-phosphate (36) and HOPS (19), and a C-terminal SNARE domain. We now compare the functions of wild-type Vam7p with Vam7p bearing a Q283R mutation, changing the 0-layer Gln to Arg, in the context of purified vacuoles with the wild-type R-SNARE Nyv1p or with its 0-layer Arg changed to Gln. We find that the 3Q·1R rule (37) is important but not inviolate. Substantial fusion is seen with SNARE complexes having "rotated" positions of the 3Q·1R or with 4Q complexes, each showing dramatically elevated K_m for Vam7p but similar maximal rates of fusion. 2Q·2R SNAREs form SNARE complex without fusion, but substantial fusion can be restored by the addition of chlorpromazine or U73122. [GenBank] Either of these agents modifies the lipid bilayer, suggesting that 0-layer function is directly coupled to the lipid bilayer properties.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Strains—The strains used for fusion assays were BJ3505 (MAT pep4::HIS3 prb1-1.6R his3–200 lys2–801 trp1101 (gal3) ura3–52 gal2 can1) (38) and DKY6281 (MAT leu2–3 leu 2–112 ura3–52 his3-200 trp1-901 lys2–801) (16). BJ3505 nyv1 and DKY6281 nyv1 were described previously (13). Nyv1^R192Q was introduced into BJ3505 nyv1 and DK6281 nyv1 by recombination. The promoter region of the NYV1 gene was amplified by PCR from DKY6281 genomic DNA with the forward primer 5'-CGATATTAAGCTTGCCCTAATTACTAGG-3' containing a HindIII site (bold) and reverse primer 5'-GCTGTTAGAGCATTGGACTTTTATATTTTTACCAAGGATCCGCG-3' containing a BamHI site. The product was subcloned into pRS406 using HindIII and BamHI sites, generating the plasmid pRS406-pNYV1. The NYV1 3'-untranslated region containing the terminator was amplified from DKY6281 genomic DNA with forward primer 5'-TCCCTCTAGAATGAAACGCTTTAATGGTATGTAT-3' containing an XbaI site and reverse primer 5'-GTGCCACTTGTGAGAAGTCTAAGCCCGCGGGGA-3' with a SacII site. The resulting PCR product was subcloned into pRS406-pNYV1 using XbaI and SacII sites, generating pRS406-pNYV1-NYV1. QuikChange mutagenesis (39) was used to generate pRS406-nyv1^R192Q using the forward primer 5'-AACATCGACAAGTTCTTGGAGCAACAAGAAAGAGTTTCTTTATTGTG-3' and reverse primer 5'-CACCAATAAAGAAACTCTTTCTTGTTGCTCCAAGAACTTGTCGATGTT-3'. RFY1 and RFY2 were generated by transforming BJ3505 nyv1 and DKY6281 nyv1, respectively, with pRS406-nyv1^R192Q by standard lithium acetate methods and plated on complete synthetic media lacking uracil.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Equivalent function of wild-type and mutant Vam7p in inhibitory 3sQ complexes. Standard fusion reactions ("Experimental Procedures") were incubated for 90 min in the absence or presence of equimolar mixtures of soluble His6-Vam3p, MBP-Vti1p, and GST·Vam7p. Mixtures contained either wild-type Vam7p, Vam7pY42A (Y42A), Vam7pQ283R (Q283R), or Vam7pY42A/Q283R (YA/QR).

Recombinant Vam7p—The VAM7 open reading frame, excluding the first methionine, was amplified from BY4742 chromosomal DNA using forward primer 5'-GCGGGATCCGCAGCTAATTCTGTAGGGAAA-3' with a BamHI restriction site (bold), and reverse primer 5'-CGCGAATTCTCAAGCACTGTTGTTAAAATG-3' with an EcoRI site. Digested PCR product was ligated to EcoRI/BamHI-linearized pET42a(+) to generate pET42a-GST·Vam7p. Sequenced plasmid was transformed into Escherichia coli Rosetta-2 (DE3) pLysS (Novagen) and grown on Luria broth with kanamycin and chloramphenicol. Using pET42a-GST-VAM7 as a template, QuikChange mutagenesis was used to create VAM7^Y42A, VAM7^Q283R, and VAM7^Y42A/Q283R. Forward primer 5'-AACAAGCGCCTTTACAAAAGGGCATCCGAGTTTTGGAAACTGAAG-3' and reverse primer 5'-CTTCAGTTTCCAAAACTCGGATGCCCTTTTGTAAAGGCGCTTGTT-3' were used to make VAM7^Y42A. To make VAM7^Q283R we used 5'-GAGATGAACGAGGAGCTGCAAACACGGAATGAGCTACTTACAGCACTT-3' and 5'-AAGTGCTGTAAGTAGCTCATTCCGTGTTTGCAGCTCCTCGTTCATCTC-3'.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Regulation of fusion by the 0-layer of Vam7p and Nyv1p. Fusion assays were performed using vacuoles harvested from yeast strains BJ3505 and DKY6281 (A–C), which are wild-type for SNAREs but permit assay of fusion ("Experimental Procedures") or from their isogenic strains RFY1 and RFY2 harboring Nyv1pR192Q (D–F). Vacuoles were incubated in the absence or presence of added recombinant wild-type or mutant Vam7p at the indicated concentrations. Vam7p was present from the start of the fusion incubation. Reactions were under standard conditions, as under "Experimental Procedures" (A and D) or -Sec17 bypass (B and E) or no ATP bypass conditions (C and F) as described in (30). Fusion was measured by alkaline phosphatase activity and expressed in units.

Vacuole Isolation and in Vitro Fusion Assay—Vacuoles were isolated from the yeast strains BJ3505 (38) and DKY6281 (16). Fusion reactions (30 µl) contained 3 µg of BJ3505 vacuoles with inactive pro-Pho8p (pro-alkaline phosphatase) and lacking the protease Pep4p, 3 µg of DKY6281 vacuoles containing Pep4p but lacking Pho8p, standard fusion reaction buffer (125 mM KCl, 5 mM MgCl₂, 20 mM PIPES-KOH, pH 6.8, 200 mM sorbitol), ATP regenerating system (1 mM ATP, 40 mM creatine phosphate, 0.1 mg/ml creatine kinase), 10 µM coenzyme A (18), and 930 nM (inhibitor of protease B). After 90 min at 27 °C, Pho8p activity was assayed in 250 mM Tris-Cl, pH 8.5, 0.4% Triton X-100, 10 mM MgCl₂, 1 mM p-nitrophenyl phosphate. One unit of fusion is 1 µmol of p-nitrophenolate produced per min per µg of BJ3505 vacuoles. p-Nitrophenolate absorbance was measured at 400 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Because Vam7p has no apolar membrane anchor, it can be produced in bacteria, purified in the absence of detergent, and added to in vitro fusion assays. To assess the role of the Vam7p C-terminal SNARE domain, we mutated its zero-layer glutamine to arginine (Q283R) to disrupt the 3Q·1R ratio. As a first test of this mutant Vam7p, we mixed bacterially expressed recombinant Vam7p or Vam7p^Q283R with the recombinant soluble cytoplasmic domains of Vti1p and Vam3p prior to introducing vacuoles and assaying vacuole fusion. The mixed soluble domains of the three Q-SNAREs interact directly with Nyv1p to form nonfunctional SNARE bundles and block vacuole fusion (43). Although this inhibition requires all three of the Q-SNARE-soluble domains, it is simpler than the physiological SNARE complex assembly, because it bypasses regulation by Ypt7p (43). The inhibitory effect of the mixed soluble domains of the t-SNAREs was undiminished by violation of the 3Q·1R rule or by another mutation, which we characterize in detail elsewhere,⁵ that disrupts the affinity of Vam7p for phosphatidylinositol 3-phosphate (Fig. 1). Because Vam7p^Q283R can still interact with other SNAREs to regulate fusion, it warranted further characterization.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Vacuoles discriminate between Vam7p and Vam7pQ283R according to the 0-layer. Vacuoles blocked for priming with -Sec17p antibody or by ATP omission (30) were incubated with indicated amounts of premixed wild-type Vam7p and Vam7pQ283R. Reactions were incubated for 90 min, and fusion was measured by alkaline phosphatase activity. Data represent mean fusion ± S.E. (n = 3).

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Vam7p forms complexes with SNAREs and HOPS. A, reaction scheme. B and C, large scale (180 µl) fusion reactions were incubated with -Sec17 to block priming. After 15 min, inhibitors (353 nM -Vam3p or 32 nM -Vps33p) were added, and the mixture was incubated for 5 min before addition of 400 nM Vam7p or Vam7pQ283R. After 90 min, reactions were placed on ice and 30-µl aliquots were used to measure fusion. The remaining vacuoles were sedimented (11,000 x g, 10 min, 4 °C), and pellets were extracted with solubilization buffer (20 mM HEPES-KOH, pH 7.4, 100 mM NaCl, 2 mM EDTA, 20% glycerol, 0.5% Triton X-100, 1 mM dithiothreitol) with protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 10 µM Pefabloc-SC, 5 µM pepstatin A, and 1 µM leupeptin) on ice for 20 min. Insoluble debris was sedimented (16,000 x g, 10 min, 4 °C), and supernatants were incubated with glutathione-Sepharose 4B (15 h and 4 °C) while nutating. Beads were washed 5x with 1 ml of solubilization buffer, and bound material was eluted by heating with reducing SDS-PAGE sample buffer. GST-Vam7p complexes were examined by Western blotting. Bars represent mean fusion ± S.E. (n = 3).

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Vacuole 2Q·2R fusion, with Vam7Q283R and vacuoles bearing wild-type Nyv1p, can be restored by U73122 or chlorpromazine. Bypass fusion (30) was assayed either without ATP or with ATP present but in the presence of antibody to Sec17p. Fusion reactions were incubated for 10 min, and inhibitors were added and incubated for 5 min before addition of 1.6 µM U73122 (A) or 150 µM chlorpromazine (CPZ) (B). U73122 and chlorpromazine were allowed to act for 5 min before addition of 100 nM Vam7pQ283R. Reactions were incubated for an additional 70 min, and then assayed for fusion. Fusion inhibitors were 120 nM affinity-purified -Nyv1p, 353 nM -Vam3p IgG, 133 nM affinity-purified -Ypt7p, 32 nM affinity-purified -Vps33p, 2.8 µM GDP dissociation inhibitor protein (GDI), and 11.4 µM Gyp1–46. Data represent mean fusion ± S.E. (n = 3).

In the absence of ATP (Fig. 2F), Vam7^Q283R promoted fusion of Nyv1p^R192Q vacuoles whereas wild type Vam7p did not, just as vacuoles bearing wild type Nyv1p could only undergo bypass fusion with wild-type Vam7p (Fig. 2C). However, the concentration of Vam7^Q283R needed for fusion of Nyv1p^R192Q vacuoles was 100 times greater than with wild-type Vam7p and Nyv1p (Fig. 2, F versus C). It may take far more energy to form SNARE bundles containing both Vam7p^Q283R and Nyv1p^R192Q than with wild-type Vam7p and Nyv1p, even though both have 3Q·1R 0-layers. This is consistent with energy being needed to distort the SNARE -helical backbone at the 0-layer to accommodate the altered positions of bulky arginine versus the less bulky glutamine, or with "proofreading" by a SNARE-bound factor such as HOPS. Once formed, these 3Q·1R complexes have the same capacity to promote fusion. A detailed examination of the specific protein and lipid requirements for fusion supported by each form of Vam7p, with wild-type vacuoles or Nyv1^R192Q vacuoles, is presented in the supplemental Fig. S1.

Physical Interactions of Vam7p with SNAREs and HOPS—Because Vam7p mutant proteins bind to vacuoles in a manner indistinguishable from wild-type Vam7p yet differentially support fusion, we examined their capacities to form protein complexes with SNAREs and HOPS. For this study, vacuole fusion was blocked by antibody to Sec17p for 15 min, secondary inhibitors were added, and, after 5 min, the priming block was rescued by the addition of Vam7p (Fig. 4A). As reported (26), wild-type Vam7p relieves the anti-Sec17p block to allow fusion (Fig. 4B, filled bars) and enters into complexes with SNARES and HOPS (Fig. 4C, left panel). These interactions were inhibited by antibodies to Vam3p or Vps33p. Strikingly, Vam7p^Q283R, although it does not support the fusion of wild-type vacuoles (Figs. 2B, 2C, and 4B, open symbols), forms stable and isolable HOPS·SNARE complexes (Fig. 4C) which contained Nyv1p and are thus 2Q·2R. The formation of this complex is also inhibited by antibody to Vam3p or to the HOPS subunit Vps33p, the SM protein of the vacuole.

Bypass of the 2Q·2R Block—Because Vam7p^Q283R binds to vacuoles and enters SNARE complexes as readily as wild-type Vam7p, but with little or no consequent fusion, we sought conditions that might activate fusion in this 2Q·2R system when priming was blocked by either antibody to Sec17p or by the omission of ATP. Bypass fusion with Vam7p^Q283R in the absence of ATP was restored by the addition of the phospholipase C inhibitor U73122 [GenBank] , but restoration was not seen in the presence of ATP (Fig. 5A). Strikingly, bypass fusion with Vam7p^Q283R in the presence of ATP, when priming was blocked by antibody to Sec17p, was restored by the addition of chlorpromazine (Fig. 5B), and this restoration required ATP. In each case, the restored fusion required the normal fusion pathway, because it was blocked by characterized inhibitors that target SNAREs, Ypt7p, and HOPS. Even though bypass fusion with wild-type Vam7p is unaffected by ATP (30), ATP directly regulates the ability of U73122 [GenBank] or chlorpromazine to restore 2Q·2R fusion. To further examine this bypass, we characterized the effects of chlorpromazine on both the physical associations of added Vam7p^Q283R as well as on the functional restoration of fusion for the same samples (Fig. 6). Chlorpromazine had no measurable effect on the binding of Vam7p^Q283R to vacuoles (Fig. 6B, top panel). Chlorpromazine had only a small effect on the association of vacuolebound Vam7p^Q283R with HOPS or with other SNAREs (Fig. 6B, lane 4 versus 5) but was absolutely required for fusion (Fig. 6C). In the presence of chlorpromazine, both the physical association of the Vam7p^Q283R with HOPS and with SNAREs and the function of restored fusion were still dependent on the R-SNARE Nyv1p (Figs. 5, 6B (lane 8), and 6C), showing that fusion occurred using a 2Q·2R SNARE complex, and on Vam3p, Ypt7p, and the SM Vps33p subunit of HOPS (Fig. 6, B (lanes 7, 9–11) and C).

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Chlorpromazine permits Vam7pQ283R to form normal complexes with SNAREs and HOPS. A, reaction scheme. B, fusion reactions were incubated with -Sec17 to block priming. After 10 min, inhibitors were added and the mixture was incubated for 5 min before addition of 150 µM chlorpromazine (CPZ). Chlorpromazine was allowed to act for 5 min before addition of 100 nM Vam7pQ283R. Reactions were incubated for an additional 70 min, and then assayed for Vam7pQ283R-associated proteins as described in Fig. 4. C, fusion was determined by alkaline phosphatase activity. Bars represent mean fusion ± S.E. (n = 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

What is the role of the 0-layer in vacuole fusion? Our studies show that the composition of glutamine and arginine residues in the 0-layer and their precise spatial distribution control the energy needed to assemble functional SNARE complexes, because normal bypass fusion has a K_m for Vam7p of only 3–8 nM, whereas far higher concentrations of mutant or wild-type Vam7p are needed for 4Q fusion (Fig. 2). Once formed, SNARE complexes with 2Q·2R are stable but inactive, yet fusion can be restored when the membrane is perturbed by the intercalating amphiphile chlorpromazine or by the phospholipase C inhibitor U73122. [GenBank] These may modulate bilayer properties such that a "weakened" 2Q·2R complex is still capable of driving fusion.

Chlorpromazine is an amphipathic molecule that partitions into the negatively curved inner leaflets of membrane bilayers. Once intercalated into membranes, chlorpromazine alters the physical properties of bilayers. Upon insertion into inner leaflets, chlorpromazine deforms membranes to induce cupping (48) and can alter lateral diffusion and membrane tension (48). In doing so, chlorpromazine reduces relaxation times of membrane tethers by lowering thresholds for remodeling. This was seen in force measurement experiments using optical tweezers to pull membranes (49). Membrane remodeling by chlorpromazine may also alter phosphoinositide metabolism by activating both phosphoinositide kinases and phospholipase C (50). Each of these actions of chlorpromazine may lower the threshold for the bilayer rearrangements of fusion, permitting 2Q·2R SNARE complexes to function. Similarly, U73122 [GenBank] inhibits vacuolar phospholipase C activities (51) and thereby alters the ratio of two vacuolar lipids that are crucial for fusion, phosphatidylinositol 4,5-bisphosphate and diacylglycerol. Restoration of 2Q·2R fusion by chlorpromazine or U73122 [GenBank] is regulated by the presence or absence of ATP; one of the effects of ATP in this restoration may be to support phosphoinositide synthesis.

SNAREs may promote membrane fusion by several means: 1) They may provide physical stress on the bilayer, which could lower the activation energy for lipid rearrangements for fusion (5). 2) SNAREs are needed to gather lipids such as diacylglycerol, which promote the bilayer rearrangements of fusion, to the fusion site (25). 3) The trans-membrane domain of certain SNAREs will destabilize bilayers (33, 52), and SNARE-driven bilayer stress and destabilization can lead to the lipid rearrangements of lysis as well as fusion (7, 8). 4) SNAREs may also serve as a binding platform to localize and even activate other fusion factors, such as surface-active and calcium-triggered synaptotagmin or lipid-binding HOPS. Assays of each of these four separate SNARE contributions to fusion will be required to determine the importance of each means of SNARE action and the role of the 0-layer in their regulation. Our current findings suggest that SNARE 0-layer function is intimately tied to the lipid bilayer composition and physical properties.

	FOOTNOTES

 
^* This work was supported in part by a grant from NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation. Current address: Dept. of Biochemistry, University of Illinois, Urbana-Champaign, IL 61801-3732.

² Current address: Dept. of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, CT 06520-8024.

³ To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Bldg., Hanover, NH 03755. Tel.: 603-650-1701; Fax: 603-650-1353; E-mail: Bill.Wickner{at}dartmouth.edu.

⁴ The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; HOPS, homotypic fusion and vacuole protein sorting complex; SNAP, soluble NSF attachment protein; NSF, N-ethylmaleimide-sensitive protein.

⁵ Fratti, R. A., and Wickner, W. (2007) J. Biol. Chem. 282, 13133–13138.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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