Membrane fusion is central to cell growth, protein secretion, organelle biogenesis, and neurotransmission. We have established yeast vacuole homotypic fusion for studying membrane fusion mechanisms- with excellent genetics, organelle isolation, organelle cytology, and a rapid quantitative in vitro assay. Docked vacuoles are drawn together, each with a disc-like regions of closely apposed membrane. The proteins and lipids needed for docking and fusion are highly enriched at the periphery of these apposed discs, the """"""""vertex ring domain"""""""". Fusion occurs around these vertex rings. Vacuole fusion requires SNAREs, SM and NSF/SNAP chaperones of SNAREs, Rab/Ypt proteins, Rho GTPases, actin and its regulatory proteins, regulatory lipids (sterol, diacylglycerol, and 3- and 4- phosphoinositides), and calcium, each implicated in other fusion reactions as well. To investigate their interrelationships, We have purified the soluble :and peripheral membrane proteins which support vacuole fusion, assembled antibody, lipid ligand, and drug inhibitors, and characterized the stages of the reaction: priming (Sec18/17/ATP-driven SNARE disassembly, phosphoinositide synthesis), docking (consisting of tethering, vertex ring enrichment of selective proteins and lipids, and trans-pairing of SNAREs), Ca2+ efflux, and fusion (compartment mixing). We now propose to: 1. Discover the rest of the proteins and lipids of the vertex ring and map their assembly relationships. 2. Develop assays of the bound-nucleotide and functional relationships of the 4 GTPases of vacuole fusion. 3. Examine the receptors, regulation, and functions of remodeling of vacuole-bound actin. 4. Uncover the channel of Ca2+ efflux from the vacuole and its downstream effectors. 5. Establish biochemical assays of the function of the heterohexameric HOPS complex, which integrates Ypt/Rab activation, phosphoinositide binding, and SM-mediated SNARE complex assembly. 6. Exploit additional functional assays, including lipid mixing and patch-clamping docked vacuoles, and seek reconstitution of vacuole fusion subreactions.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cell Development and Function Integrated Review Group (CDF)
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Shapiro, Bert I
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Dartmouth College
Schools of Medicine
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Orr, Amy; Song, Hongki; Rusin, Scott F et al. (2017) HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex. Mol Biol Cell 28:975-983
Song, Hongki; Wickner, William (2017) A short region upstream of the yeast vacuolar Qa-SNARE heptad-repeats promotes membrane fusion through enhanced SNARE complex assembly. Mol Biol Cell 28:2282-2289
Song, Hongki; Orr, Amy; Duan, Mengtong et al. (2017) Sec17/Sec18 act twice, enhancing membrane fusion and then disassembling cis-SNARE complexes. Elife 6:
Schwartz, Matthew L; Nickerson, Daniel P; Lobingier, Braden T et al. (2017) Sec17 (?-SNAP) and an SM-tethering complex regulate the outcome of SNARE zippering in vitro and in vivo. Elife 6:
Zick, Michael; Wickner, William (2016) Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement. Mol Biol Cell 27:2590-7
Zick, Michael; Orr, Amy; Schwartz, Matthew L et al. (2015) Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18. Proc Natl Acad Sci U S A 112:E2290-7
Orr, Amy; Wickner, William; Rusin, Scott F et al. (2015) Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion. Mol Biol Cell 26:305-15
Baker, Richard W; Jeffrey, Philip D; Zick, Michael et al. (2015) A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly. Science 349:1111-4
Zick, Michael; Wickner, William T (2014) A distinct tethering step is vital for vacuole membrane fusion. Elife 3:e03251
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

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