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Complex Lipid Requirements for SNARE- and SNARE Chaperone-dependent Membrane Fusion

2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês

10.1074/jbc.m109.010223

ISSN

1083-351X

Autores

Joji Mima, William Wickner,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), diacylglycerol (DAG), and phosphatidylinositol 3-phosphate (PI3P). We now report that many of these lipids are required for rapid and efficient fusion of the reconstituted SNARE proteoliposomes in the presence of SNARE chaperones. Omission of either PE, PA, or PI3P from the complete set of lipids strongly reduces fusion, and PC, PE, PA, and PI3P constitute a minimal set of lipids for fusion. PA could neither be replaced by other lipids with small headgroups such as DAG or ERG nor by the acidic lipids PS or PI. PA is needed for full association of HOPS and Sec18p with proteoliposomes having a minimal set of lipids. Strikingly, PA and PE are as essential for SNARE complex assembly as for fusion, suggesting that these lipids facilitate functional interactions among SNAREs and SNARE chaperones. Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), diacylglycerol (DAG), and phosphatidylinositol 3-phosphate (PI3P). We now report that many of these lipids are required for rapid and efficient fusion of the reconstituted SNARE proteoliposomes in the presence of SNARE chaperones. Omission of either PE, PA, or PI3P from the complete set of lipids strongly reduces fusion, and PC, PE, PA, and PI3P constitute a minimal set of lipids for fusion. PA could neither be replaced by other lipids with small headgroups such as DAG or ERG nor by the acidic lipids PS or PI. PA is needed for full association of HOPS and Sec18p with proteoliposomes having a minimal set of lipids. Strikingly, PA and PE are as essential for SNARE complex assembly as for fusion, suggesting that these lipids facilitate functional interactions among SNAREs and SNARE chaperones. Biological membrane fusion is the regulated rearrangement of the lipids in two apposed sealed membranes to form one bilayer while mixing lumenal contents without leakage or lysis. It is fundamental for intracellular vesicular traffic, cell growth and division, regulated secretion of hormones and other blood proteins, and neurotransmission and thus has attracted wide and sustained study (1Jahn R. Lang T. Südhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1200) Google Scholar, 2Wickner W. Schekman R. Nat. Struct. Mol. Biol. 2008; 15: 658-664Crossref PubMed Scopus (343) Google Scholar). Its fundamental mechanisms are conserved and employ a Rab-family GTPase, proteins which bind to the GTP-bound form of a Rab, termed its "effectors" (3Grosshans B.L. Ortiz D. Novick P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11821-11827Crossref PubMed Scopus (794) Google Scholar), and SNARE 3The abbreviations used are: SNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptorsPCphosphatidylcholinePIphosphatidylinositolPEphosphatidylethanolaminePSphosphatidylserinePAphosphatidic acidCLcardiolipinERGergosterolDAGdiacylglycerolPI3Pphosphatidylinositol 3-phosphatePI(4,5)P2phosphatidylinositol (4,5)-bisphosphateRPLreconstituted proteoliposomeHOPShomotypic vacuole fusion and protein sorting complexGSTglutathione S-transferase. 3The abbreviations used are: SNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptorsPCphosphatidylcholinePIphosphatidylinositolPEphosphatidylethanolaminePSphosphatidylserinePAphosphatidic acidCLcardiolipinERGergosterolDAGdiacylglycerolPI3Pphosphatidylinositol 3-phosphatePI(4,5)P2phosphatidylinositol (4,5)-bisphosphateRPLreconstituted proteoliposomeHOPShomotypic vacuole fusion and protein sorting complexGSTglutathione S-transferase. (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins (4Jahn R. Scheller R.H. Nat. Rev. Mol. Cell Biol. 2006; 7: 631-643Crossref PubMed Scopus (1832) Google Scholar) with their attendant chaperones. SNAREs are integral or peripheral membrane proteins with characteristic heptad-repeat domains, which can associate in 4-helical coiled-coils (5Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1881) Google Scholar), termed "cis-SNARE complexes," if they are all anchored to the same membrane bilayer, or "trans-SNARE complexes" if they are anchored to apposed membranes. soluble N-ethylmaleimide-sensitive factor attachment protein receptors phosphatidylcholine phosphatidylinositol phosphatidylethanolamine phosphatidylserine phosphatidic acid cardiolipin ergosterol diacylglycerol phosphatidylinositol 3-phosphate phosphatidylinositol (4,5)-bisphosphate reconstituted proteoliposome homotypic vacuole fusion and protein sorting complex glutathione S-transferase. soluble N-ethylmaleimide-sensitive factor attachment protein receptors phosphatidylcholine phosphatidylinositol phosphatidylethanolamine phosphatidylserine phosphatidic acid cardiolipin ergosterol diacylglycerol phosphatidylinositol 3-phosphate phosphatidylinositol (4,5)-bisphosphate reconstituted proteoliposome homotypic vacuole fusion and protein sorting complex glutathione S-transferase. Stable membrane proximity (docking) does not suffice for fusion. Studies in model systems have shown that fusion can be promoted by any of several agents, which promote bilayer rearrangement, such as diacylglycerol (6Allan D. Thomas P. Michell R.H. Nature. 1978; 276: 289-290Crossref PubMed Scopus (109) Google Scholar), high levels of calcium (7Ingolia T.D. Koshland Jr., D.E. J. Biol. Chem. 1978; 253: 3821-3829Abstract Full Text PDF PubMed Google Scholar), viral-encoded fusion proteins (8Melikyan G.B. Brener S.A. Ok D.C. Cohen F.S. J. Cell Biol. 1997; 136: 995-1005Crossref PubMed Scopus (139) Google Scholar, 9Armstrong R.T. Kushnir A.S. White J.M. J. Cell Biol. 2000; 151: 425-437Crossref PubMed Scopus (174) Google Scholar), or SNAREs (10Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Söllner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (1989) Google Scholar, 11McNew J.A. Weber T. Parlati F. Johnston R.J. Melia T.J. Söllner T.H. Rothman J.E. J. Cell Biol. 2000; 150: 105-117Crossref PubMed Scopus (246) Google Scholar). These studies frequently employed liposomes or proteoliposomes of simple lipid composition, suggesting that fusion may not have stringent requirements of lipid head group species. However, each of these model fusion reactions is accompanied by substantial lysis (12Burgess S.W. McIntosh T.J. Lentz B.R. Biochemistry. 1992; 31: 2653-2661Crossref PubMed Scopus (84) Google Scholar, 13Nickel W. Weber T. McNew J.A. Parlati F. Söllner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12571-12576Crossref PubMed Scopus (157) Google Scholar, 14Dennison S.M. Bowen M.E. Brunger A.T. Lentz B.R. Biophys. J. 2006; 90: 1661-1675Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 15Lau W.L. Ege D.S. Lear J.D. Hammer D.A. DeGrado W.F. Biophys. J. 2004; 86: 272-284Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), whereas the preservation of subcellular compartments is a hallmark of physiological membrane fusion. We have studied membrane fusion with the vacuole (lysosome) of Saccharomyces cerevisiae (reviewed in Ref. 16Ostrowicz C.W. Meiringer C.T. Ungermann C. Autophagy. 2008; 4: 5-19Crossref PubMed Scopus (83) Google Scholar). The fusion of isolated vacuoles requires the Rab Ypt7p, 4 SNAREs (Vam3p, Vti1p, Vam7p, and Nyv1p), the SNARE chaperones Sec17p (α-soluble N-ethylmaleimide-sensitive factor attachment protein)/Sec18p (N-ethylmaleimide-sensitive factor) and the hexameric HOPS complex (17Stroupe C. Collins K.M. Fratti R.A. Wickner W. EMBO J. 2006; 25: 1579-1589Crossref PubMed Scopus (185) Google Scholar), and key "regulatory" lipids including ERG, phosphoinositides, and DAG (18Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar). HOPS interacts physically or functionally with each component of this fusion system. HOPS stably associates with Ypt7p in its GTP-bound state (19Seals D.F. Eitzen G. Margolis N. Wickner W.T. Price A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9402-9407Crossref PubMed Scopus (357) Google Scholar). One HOPS subunit, Vps33p, is a member of the Sec1-Munc18 family of SNARE-binding proteins, and HOPS exhibits direct affinity for SNAREs (17Stroupe C. Collins K.M. Fratti R.A. Wickner W. EMBO J. 2006; 25: 1579-1589Crossref PubMed Scopus (185) Google Scholar, 20Price A. Seals D. Wickner W. Ungermann C. J. Cell Biol. 2000; 148: 1231-1238Crossref PubMed Scopus (171) Google Scholar, 21Sato T.K. Rehling P. Peterson M.R. Emr S.D. Mol. Cell. 2000; 6: 661-671Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 22Collins K.M. Thorngren N.L. Fratti R.A. Wickner W.T. EMBO J. 2005; 24: 1775-1786Crossref PubMed Scopus (87) Google Scholar) and proofreads correct vacuolar SNARE pairing (23Starai V.J. Hickey C.M. Wickner W. Mol. Biol. Cell. 2008; 19: 2500-2508Crossref PubMed Scopus (96) Google Scholar). HOPS also has direct affinity for phosphoinositides (17Stroupe C. Collins K.M. Fratti R.A. Wickner W. EMBO J. 2006; 25: 1579-1589Crossref PubMed Scopus (185) Google Scholar). The SNAREs on isolated vacuoles are in cis-complexes, which are disassembled by Sec17p, Sec18p, and ATP (24Ungermann C. Nichols B.J. Pelham H.R. Wickner W. J. Cell Biol. 1998; 140: 61-69Crossref PubMed Scopus (208) Google Scholar). Docking requires Ypt7p (25Mayer A. Wickner W. J. Cell Biol. 1997; 136: 307-317Crossref PubMed Scopus (202) Google Scholar) and HOPS (17Stroupe C. Collins K.M. Fratti R.A. Wickner W. EMBO J. 2006; 25: 1579-1589Crossref PubMed Scopus (185) Google Scholar). During docking, vacuoles are drawn against each other until each has a substantial membrane domain tightly apposed to the other. Each of the proteins (26Wang L. Seeley E.S. Wickner W. Merz A.J. Cell. 2002; 108: 357-369Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and lipids (18Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar) required for fusion becomes enriched in a ring-shaped microdomain, the "vertex ring," which surrounds the two tightly apposed membrane domains. Not only do the proteins depend on each other, in a cascade fashion, for vertex ring enrichment, and the lipids depend on each other for their vertex ring enrichment as well, but the lipids and proteins are mutually interdependent for their enrichment at this ring-shaped microdomain (18Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar, 27Wang L. Merz A.J. Collins K.M. Wickner W. J. Cell Biol. 2003; 160: 365-374Crossref PubMed Scopus (102) Google Scholar). Fusion occurs around the ring, joining the two organelles. The fusion of vacuoles bearing physiological fusion constituents does not cause measurable organelle lysis, although fusion supported exclusively by higher levels of SNARE proteins is accompanied by massive lysis (28Starai V.J. Jun Y. Wickner W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13551-13558Crossref PubMed Scopus (65) Google Scholar), in accord with model liposome studies (14Dennison S.M. Bowen M.E. Brunger A.T. Lentz B.R. Biophys. J. 2006; 90: 1661-1675Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus fusion microdomain assembly and the coordinate action of SNAREs with other proteins and lipids to promote fusion without lysis are central topics in membrane fusion studies. Reconstitution of fusion with pure components allows chemical definition of essential elements of this biologically important reaction. Although SNAREs can drive a slow fusion of PC/PS proteoliposomes (29Fukuda R. McNew J.A. Weber T. Parlati F. Engel T. Nickel W. Rothman J.E. Söllner T.H. Nature. 2000; 407: 198-202Crossref PubMed Scopus (188) Google Scholar), this was not stimulated by HOPS and Sec17p/Sec18p (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). SNARE proteoliposomes bearing all the vacuolar lipids (18Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar, 31Zinser E. Sperka-Gottlieb C.D. Fasch E.V. Kohlwein S.D. Paltauf F. Daum G. J. Bacteriol. 1991; 173: 2026-2034Crossref PubMed Scopus (498) Google Scholar, 32Schneiter R. Brügger B. Sandhoff R. Zellnig G. Leber A. Lampl M. Athenstaedt K. Hrastnik C. Eder S. Daum G. Paltauf F. Wieland F.T. Kohlwein S.D. J. Cell Biol. 1999; 146: 741-754Crossref PubMed Scopus (380) Google Scholar, 33Jun Y. Fratti R.A. Wickner W. J. Biol. Chem. 2004; 279: 53186-53195Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), PC, PE, PI, PS, CL, PA, ERG, DAG, PI3P, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), showed rapid and efficient fusion that was fully dependent on Sec17p/Sec18p and HOPS (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). The omission of either DAG, ERG, or phosphoinositide from the liposomes caused a marked reduction in fusion (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). We now report that PE and PA are also necessary for rapid and efficient fusion, function in distinct manners, and are required for efficient assembly of newly formed SNARE complexes by the SNARE chaperones Sec17p/Sec18p and HOPS. Three yeast vacuolar SNAREs, Vam3p, Vti1p, and Nyv1p, were produced in Escherichia coli Rosetta2 (Novagen) and purified as described previously (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). For Vam7p (yeast vacuolar Qc-SNARE), the GST and linker sequences upstream of VAM7 were deleted from the expression vector for GST-Vam7p (18Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar) by QuikChange mutagenesis kit (Stratagene) to purify full-length untagged Vam7p from inclusion bodies in E. coli Rosetta2(DE3)pLys (Novagen). After the induction at 37 °C for 3 h by 1 mm isopropyl 1-thio-β-d-galactopyranoside, cells from a 2-liter culture were harvested, suspended in 80 ml of buffer A (20 mm Hepes-NaOH (pH 8.0), 0.5 m NaCl) containing 1 mm phenylmethylsulfonyl fluoride and 1 μg/ml pepstatin, lysed by French press at 4 °C, and centrifuged (60-Ti, Beckman, 50,000 rpm, 30 min, 4 °C). Pellets were washed with 40 ml of buffer A containing 50 mm β-octylglucoside followed by 80 ml of buffer A, re-suspended with 20 ml of buffer A containing 50 mm dithiothreitol, 1 mm EDTA, and 6 m guanidine hydrochloride, and incubated at 37 °C for 1 h with shaking. The suspension was centrifuged (60-Ti, Beckman, 20,000 rpm, 30 min, 4 °C), and the supernatant was dialyzed against 2-liter portions of buffer A at 4 °C for 12 h, then 3 h, and centrifuged again (60-Ti, Beckman, 50,000 rpm, 30 min, 4 °C), yielding re-folded Vam7p in the supernatant. Tobacco etch virus protease (23Starai V.J. Hickey C.M. Wickner W. Mol. Biol. Cell. 2008; 19: 2500-2508Crossref PubMed Scopus (96) Google Scholar), affinity-purified antibodies against Vam3p, Vti1p, Vam7p, Nyv1p, Sec17p, Sec18p, and Vps33p (22Collins K.M. Thorngren N.L. Fratti R.A. Wickner W.T. EMBO J. 2005; 24: 1775-1786Crossref PubMed Scopus (87) Google Scholar), His6-Sec17p (34Haas A. Wickner W. EMBO J. 1996; 15: 3296-3305Crossref PubMed Scopus (149) Google Scholar), His6-Sec18p (34Haas A. Wickner W. EMBO J. 1996; 15: 3296-3305Crossref PubMed Scopus (149) Google Scholar), and HOPS-GST (23Starai V.J. Hickey C.M. Wickner W. Mol. Biol. Cell. 2008; 19: 2500-2508Crossref PubMed Scopus (96) Google Scholar) were purified as described. Reconstituted proteoliposomes (RPLs) were prepared with purified yeast vacuolar SNAREs as described (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). Detergent-mixed micellar solutions of lipids and SNAREs, including the inherently water-soluble Vam7p SNARE, were dialyzed to allow spontaneous proteoliposome formation and any assembly of SNAREs into cis-SNARE complexes (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). The RPLs were reconstituted with a variety of lipid compositions as shown in Table 1, and diC8-PI3P (Echelon) was the only phosphoinositide used in this study.TABLE 1Lipid compositions of the SNARE RPLs used in this studyRPL lipid compositionLipidsPOPCPOPESoyPIERGDAGPOPSPOPACL%, mol/molPC97/99PC/PE79/8118PC/PE/PI61/631818PC/PE/PI/ERG/DAG52/54181881Complete44/461818814.421.6PC/PE/PS61/631818PC/PE/PA61/631818PC/PE/ERG61/631818PC/PE/DAG61/631818PC/PA79/8118Complete minus PA46/481818814.41.6Complete minus PE62/6418814.421.6 Open table in a new tab Lipid mixing between the SNARE-RPLs was assayed as described (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar), with modifications. RPL reaction mixtures (RB150 (20 mm HEPES-NaOH (pH 7.4), 0.15 m NaCl, 10% glycerol) with 1 mm ATP, 2 mm MgCl2, 50 μm donor RPLs, 400 μm acceptor RPLs, and 90 μm diC8-PI3P) were prepared in black 384-well plates (#3676, Corning) on ice and incubated at 27 °C for 10 min in a SpectraMAX Gemini XPS plate reader (Molecular Devices) pre-equilibrated at 27 °C. After 10 min, His6-Sec17p (1.2 μm), His6-Sec18p (1.0 μm), HOPS-GST (55 nm), and Vam7p (600 nm) were added to the RPL reactions where indicated, followed by further 60-min incubation at 27 °C in the plate reader. During the lipid mixing reactions, NBD fluorescence was monitored. For calculating the ratios of NBD fluorescence, F/F0, the fluorescence just before the Sec17p/Sec18p/HOPS addition (at 0 min) was used for F0, except in Fig. 6, where the lowest values during the initial 10-min incubation were used for F0. Topology of lipid mixing (Fig. 2D) was analyzed by addition of sodium dithionite (Sigma) to lipid mixing reactions while monitoring NBD fluorescence, as described previously (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). 4-SNARE RPL reactions (100 μl each, 450 μm donor RPLs) in RB150, with 1 mm ATP, 2 mm MgCl2, 90 μm diC8-PI3P, 1.2 μm His6-Sec17p, 1.0 μm His6-Sec18p, and 55 nm HOPS-GST were incubated at 27 °C for 30 min, placed on ice for 10 min, and mixed with 400 μl of 50% Histodenz (Sigma) in RB150 to a final Histodenz concentration of 40%, then transferred to 11 × 60 mm tubes, overlaid with 1.5 ml of 35% Histodenz in RB150, 2 ml of 30% Histodenz in RB150, and 200 μl of RB150, and centrifuged (SW60Ti, 55,000 rpm, 3 h, 4 °C). RPLs, which floated, were harvested in 400 μl from the top of the gradients and analyzed by SDS-PAGE and immunoblotting with affinity-purified antibodies against Vti1p, Vam7p, Sec17p, Sec18p, and Vps33p. RPL lipid mixing reactions (20 μl each), which had been prepared with Qabc-SNARE RPLs, R-SNARE RPLs, diC8-PI3P, and SNARE chaperones, as described above, were incubated at 27 °C for 40 min while monitoring NBD fluorescence, then transferred to 1.7-ml microcentrifuge tubes on ice and left for 10 min. The RPL reactions were mixed with αVam3p (5 μl each, 1.7 μm final), incubated on ice for 10 min, further mixed with 230 μl each of Protein A-Sepharose (Amersham Biosciences) in RB150 with 1% Triton X-100, and nutated at 4 °C for 30 min. The Protein A-Sepharose beads were washed with 500 μl each of RB150 with 1% Triton X-100 three times. Vam3p and Nyv1p bound to the Protein A-Sepharose beads were eluted from the beads with 5× SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting. Membrane fusion can be assayed by the resultant lipid mixing. Two sets of liposomes are prepared, one bearing NBD-PE and sufficient rhodamine-PE to quench the NBD fluorescence and another set of liposomes that have neither of these fluorophores. Upon fusion, dilution of the fluorophores by lipid mixing relieves the quenching, and the resulting NBD fluorescence is a measure of fusion (35Struck D.K. Hoekstra D. Pagano R.E. Biochemistry. 1981; 20: 4093-4099Crossref PubMed Scopus (1128) Google Scholar). Proteoliposomes, which bear the four vacuolar SNAREs, will fuse when given purified HOPS, Sec17p, Sec18p, and ATP if they are composed of vacuolar lipids (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). We express the fusion as F/Fo, the fluorescence F at each time normalized for the fluorescence immediately before the addition of soluble SNARE and SNARE chaperones, Fo. At the end of each incubation, detergent is added and the maximally dequenched NBD fluorescence, Fm, is measured (calculated values of [F − Fo/Fm − Fo] × 100% are presented in the figure legends). The vacuolar lipid composition is PC, PE, PI, PS, CL, PA, ERG, DAG, PI3P, and PI(4,5)P2 (18Fratti R.A. Jun Y. Merz A.J. Margolis N. Wickner W. J. Cell Biol. 2004; 167: 1087-1098Crossref PubMed Scopus (167) Google Scholar, 31Zinser E. Sperka-Gottlieb C.D. Fasch E.V. Kohlwein S.D. Paltauf F. Daum G. J. Bacteriol. 1991; 173: 2026-2034Crossref PubMed Scopus (498) Google Scholar, 32Schneiter R. Brügger B. Sandhoff R. Zellnig G. Leber A. Lampl M. Athenstaedt K. Hrastnik C. Eder S. Daum G. Paltauf F. Wieland F.T. Kohlwein S.D. J. Cell Biol. 1999; 146: 741-754Crossref PubMed Scopus (380) Google Scholar, 33Jun Y. Fratti R.A. Wickner W. J. Biol. Chem. 2004; 279: 53186-53195Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Fusion was shown to be depressed ∼50% by the omission of DAG and to be even more depressed by omission of ERG, PI(4,5)P2, or PI3P (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). We now report that PA and PE have important and distinct roles in fusion as well. SNARE proteoliposomes were prepared by the dialysis method of detergent removal from mixed micellar solutions, which bore all four SNAREs, the R-SNARE Nyv1p alone, the two integral Qab-SNAREs Vti1p and Vam3p, or all three Qabc-SNAREs (supplemental Fig. S1, A–D) and lipid mixtures ranging in complexity from PC alone to the complete vacuolar lipid mixture except for phosphoinositides (Table 1); diC8-PI3P can be added independently (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar) and will fulfill all the roles of phosphoinositides for fusion. 4Mima, J., and Wickner, W. (2009) Proc. Natl. Acad. Sci. U.S.A., in press. In all cases, the level of reconstitution of SNAREs was unaffected by the lipid composition (supplemental Fig. S1). Nevertheless, as shown below, lipid composition has a profound effect on fusion. Proteoliposomes with all four vacuolar SNAREs and the complete vacuole lipid mixture were incubated with diC8-PI3P, then mixed with Sec17p, Sec18p, and HOPS to initiate rapid and complete fusion (Fig. 1A, open circles), as reported.4 Omission of three of the acidic lipids, PS, PA, and CL, caused a 14-fold reduction in the initial rate of fusion (filled squares), which was further reduced 2.3-fold by the omission of small headgroup lipids ERG and DAG (filled diamonds). Removal of PI from this mixture abolished fusion entirely; we therefore determined whether PI, or another lipid, would optimally complement PC and PE for fusion. PA was optimal for this function, and rapid fusion was seen with RPLs bearing PC, PE, and PA (filled circles). Other acidic (PS) or small headgroup (DAG and ERG) lipids could not substitute for PA at all. Thus PC, PE, PA, and PI3P are a minimal set of lipids for reconstitution of fusion; the single omission of any of the latter three of this set causes drastic loss of fusion (Fig. 1A). Were PA and PE only important for fusion with a minimal lipid composition, or did they also have an important role in the complex, complete vacuolar lipid mixture? The rapid initial rate of fusion characteristic of the complete lipid mixture (Fig. 1A, open circles) was diminished 2.9-fold by the single omission of PA and 9.8-fold by the single omission of PE. Although there is some redundancy to lipid function, optimal fusion requires both of these lipids. To determine whether the lipid mixing between proteoliposomes bearing four vacuolar SNAREs and PC, PE, PA, and PI3P in the presence of the SNARE chaperones Sec17p/Sec18p and HOPS represents authentic fusion between sealed membrane compartments or lysis and re-annealing, we exploited a published assay (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar, 36McIntyre J.C. Sleight R.G. Biochemistry. 1991; 30: 11819-11827Crossref PubMed Scopus (427) Google Scholar, 37Meers P. Ali S. Erukulla R. Janoff A.S. Biochim. Biophys. Acta. 2000; 1467: 227-243Crossref PubMed Scopus (53) Google Scholar) that uses the membrane-impermeable reductant dithionite (S2O42−). The fluorescence donor proteoliposomes in our fusion reactions have NBD-PE and rhodamine-PE on both outer and inner monolayers. Exposure of these proteoliposomes to dithionite reduced the NBD-PE to N-7-amino-2,1,3-benzoxadiazol-4-yl-PE, destroying its fluorescence (Fig. 1B, filled circles, −32 to −26 min). The NBD-PE on the inner monolayer of these sealed proteoliposomes, though inaccessible to S2O42−, remained strongly quenched by rhodamine-PE. After 32 min, the S2O42− in this solution is fully oxidized and can no longer destroy the NBD fluorescence (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar). Addition of the HOPS, Sec17p, and Sec18p SNARE-chaperones triggered lipid mixing. An identical mixture of proteoliposomes and diC8-PI3P was incubated in parallel but without S2O42−, then mixed with S2O42− only 6 min before the addition of SNARE chaperones (open circles). Although the S2O42− in this second sample was largely active at the time of chaperone-triggered lipid mixing (30Mima J. Hickey C.M. Xu H. Jun Y. Wickner W. EMBO J. 2008; 27: 2031-2042Crossref PubMed Scopus (126) Google Scholar), mixing-induced dequenching occurred at the same rate in each sample, showing that this lipid mixing was accompanied by maintenance of a sealed membrane, which excluded the S2O42− reductant. We next determined whether PE or PA were required for the steady-state association of peripherally bound fusion proteins to the proteoliposomal membrane. 4-SNARE proteoliposomes were mixed with diC8-PI3P or control buffer, and then fusion was initiated with a mixture of the three SNARE chaperones (Sec17p, Sec18p, and HOPS). After 30 min, proteoliposomes were re-isolated by flotation and assayed by immunoblot for bound Vti1p (an integral membrane SNARE, as control) or peripheral membrane proteins Vam7p, Sec17p, Sec18p, and HOPS (Fig. 2). HOPS (detected via its Vps33p subunit), Sec18p, Sec17p, and Vam7p associated with proteoliposomes of PC, PE, and PA (lane 8, no PI3P; lane 9, with PI3P) to a similar extent as with proteoliposomes bearing the complete vacuolar mixture (lanes 12 and 13). Omission of PE from the minimal mixture had little effect on the association of peripheral membrane proteins (lanes 6 and 7), but omission of PA (lanes 4 and 5) or substitution of PS for PA (lanes 10 and 11) reduced the level of bound Sec18p and HOPS (Fig. 2, C and D). Fusion between 4-SNARE-RPLs was diminished by reduced Sec18p or HOPS (Fig. S2). Purified HOPS has direct affinity for protein-free liposomes bearing PA in addition to its affinity for protein-free liposomes bearing phosphoinositides (17Stroupe C. Collins K.M. Fratti R.A. Wickner W. EMBO J. 2006; 25: 1579-1589Crossref PubMed Scopus (185) Google Scholar). The requirements for PE and PA for fusion were even more strict when the proteoliposomes, which were assayed for fusion, bore separate SNARE populations (Fig. 3). With 3Q- and 1R-SNARE proteoliposomes, chaperone-dependent fusion was seen with PC/PE/PA proteoliposomes in the presence of diC8-PI3P (Fig. 3A), but omission of either PE (Fig. 3B) or PA (Fig. 3C), or substitution of PS for PA (Fig. 3D), abolished fusion. However, when the two integral membrane Qab-SNAREs were on the acceptor proteoliposomes and the soluble Qc-SNARE Vam7p was added along with SNARE chaperones (Fig. 4), even PC/PE/PA proteoliposomes, which were supplemented with PI3P, 600 nm Vam7p, and the SNARE chaperones, could not support fusion (Fig. 4A), although the full vacuolar lipid mix supported fusion well (Fig. 4B). Excess Vam7p (6 μm) could support mixing of lipids between PC/PE/PA SNARE proteoliposomes with PI3P, but this was independent of SNARE chaperones and showed little response to their addition (data not shown).FIGURE 4The minimal lipids do not support fusion with Qab-SNARE RPLs, R-SNARE RPLs, and exogenous Qc-SNARE Vam7p.

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