title: Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis.
abstract: The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-D-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis.
authors: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.
authors: Paola C
authors: PC
authors: Zucchi
authors: Michael
authors: M
authors: Zick
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001710:Biotin / Q000493:pharmacokinetics; D002451:Cell Compartmentation; D004227:Dithionite / Q000493:pharmacokinetics; D006923:Hymecromone / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D007425:Intracellular Membranes; D008055:Lipids / Q000737:chemistry; D008561:Membrane Fusion; D018832:Molecular Chaperones / Q000378:metabolism*; D010539:Permeability; D021381:Protein Transport; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D008055:Lipids; D018832:Molecular Chaperones; D011510:Proteolipids; C075137:SEC17 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; C020485:proteoliposomes; D004227:Dithionite; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D001710:Biotin; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins