Membrane fusion is a conserved process which is essential for cell growth, hormone secretion, and neurotransmission, and is vital to human health and pathophysiology. We have established the yeast vacuole as a technically advanced system for studying fusion mechanisms. Vacuole fusion occurs in stages: Priming is the ATP-dependent disassembly of SNARE complexes and the synthesis of phosphoinositides, tethering is the initial stage of vacuole adhesion, docking is the enrichment of fusion proteins and lipids into a fusion-dedicated microdomain which culminates in the association of proteins in trans, between the vacuoles, and hemifusion and fusion are the lipid rearrangements which permit compartment mixing. These are catalyzed by SNAREs which bind to each other, in cis or in trans, SNARE chaperones (Sec17p/18p), the Rab GTPase Ypt7p, regulatory lipids (sterol, diacylglycerol, and phosphoinositides), and the HOPS complex which interacts with each of these key elements and links their functions. We have studied fusion with the intact organelle, and have purified each key element and reconstituted their functions with liposomes. We now propose to: 1. Exploit our liposomal reconstitution of fusion to understand its mechanism, 2. Analyze the proteins and dynamics of trans-SNARE complexes, 3. Determine the factors which cause SNARE-mediated bilayer disruption to lead to fusion rather than lysis, and 4. Measure the affinities between fusion catalysts and rate constants for subreactions to lay the foundation for quantitative understanding o fusion.

Public Health Relevance

Membrane fusion is fundamental to cell growth, hormone secretion, and neurotransmission, yet its molecular mechanisms remain unclear. We study membrane fusion with yeast vacuoles, and have defined the lipids and proteins which catalyze vacuole fusion, purified each, reconstituted authentic fusion with these pure proteins, and established fundamental outlines of fusion mechanisms. Our proposed studies will exploit this system to determine how the fusion catalysts and lipids work together to give orderly fusion while maintaining organelle integrity, a fundamental process for human health and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Ainsztein, Alexandra M
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Dartmouth College
Schools of Medicine
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Zick, Michael; Stroupe, Christopher; Orr, Amy et al. (2014) Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion. Elife 3:e01879
Zick, Michael; Wickner, William T (2014) A distinct tethering step is vital for vacuole membrane fusion. Elife 3:e03251
Karunakaran, Vidya; Wickner, William (2013) Fusion proteins and select lipids cooperate as membrane receptors for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vam7p. J Biol Chem 288:28557-66
Stroupe, Christopher (2012) The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex. Biochem J 443:205-11
Xu, Hao; Zick, Michael; Wickner, William T et al. (2011) A lipid-anchored SNARE supports membrane fusion. Proc Natl Acad Sci U S A 108:17325-30
Zucchi, Paola C; Zick, Michael (2011) Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis. Mol Biol Cell 22:4635-46
Xu, Hao; Wickner, William (2010) Phosphoinositides function asymmetrically for membrane fusion, promoting tethering and 3Q-SNARE subcomplex assembly. J Biol Chem 285:39359-65
Xu, Hao; Jun, Youngsoo; Thompson, James et al. (2010) HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion. EMBO J 29:1948-60
Hickey, Christopher M; Wickner, William (2010) HOPS initiates vacuole docking by tethering membranes before trans-SNARE complex assembly. Mol Biol Cell 21:2297-305
Stroupe, Christopher; Hickey, Christopher M; Mima, Joji et al. (2009) Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components. Proc Natl Acad Sci U S A 106:17626-33

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