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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023377-37
Application #
8286295
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
1976-05-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
37
Fiscal Year
2012
Total Cost
$1,212,675
Indirect Cost
$445,159
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
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; 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
Zick, Michael; Wickner, William (2013) The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes. Mol Biol Cell 24:3746-53
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
Xu, Hao; Wickner, William T (2012) N-terminal domain of vacuolar SNARE Vam7p promotes trans-SNARE complex assembly. Proc Natl Acad Sci U S A 109:17936-41
Zick, Michael; Wickner, William (2012) Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion. Mol Biol Cell 23:3429-37

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