Docking and fusion events in the endomembrane system are regulated and executed by Rab GTPases, SNARE proteins, and their cofactors. A great deal has been learned about these proteins and their interactions. However, the molecular events leading to fusion are not understood in detail for any intracellular transport step. The ability to resolve individual events rather than the aggregate behavior of event populations has potentiated major advances in many areas of biology. We believe that the mechanistic dissection of membrane docking and fusion would be greatly facilitated if we could trace and perturb the subreactions of tethering, docking, and fusion at the level of individual docking and fusion events. In this proposal we combine direct attacks on the problem of monitoring single events with biochemical and genetic studies that will simultaneously characterize critical molecules and yield new reagents and probes for the single-event work. Our experimental system, the yeast vacuole, offers numerous experimental advantages for these studies including its size, which is large enough to facilitate light microscopy, the genetic and genomic toolkit of budding yeast, and a cell-free fusion system that is unsurpassed in its experimental flexibility and existing knowledge base. These experiments will build on- andsubstantially extend - approaches developed during the Pi's postdoctoral work in Dr. William Wickner's group. Microfabrication techniques and low-light, quantitative fluorescence microscopy will be; combined with the cell-free vacuole fusion system to monitor and perturb individual yeast vacuoles as they tether, dock, and fuse. We will focus our efforts on Ypt7p, the vacuole Rab, its Vps-C/HOPS effector complex, and Vam7p, a soluble SNARE. These proteins have critical functions over the entire span of the tethering-to fusion sequence, and many functional and physical interactions link them. The biological questions that we address are straightforward. Does GTP hydrolysis and exchange on the Rab Ypt7p influence the force or reversibility of tethering, or the dynamics of docking junction assembly (Aim 1)? What does the SNARE Vam7p touch as it executes its various functions in docking and fusion (Aim 2)? How is the Vps-C complex organized, and do subunits of this complex undergo structural rearrangements in response to Ypt7p-binding or other events of docking or fusion (Aim 3)? Does Ypt7p """"""""know"""""""" when tethering, docking, or fusion have occurred (Aim 4)?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077349-03
Application #
7368053
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Shapiro, Bert I
Project Start
2006-03-01
Project End
2011-02-28
Budget Start
2008-03-01
Budget End
2009-02-28
Support Year
3
Fiscal Year
2008
Total Cost
$281,226
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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