Membrane trafficking is a fundamental process crucial for normal functioning of eukaryotic cells and organisms. Rab proteins are small G proteins that are required for membrane tethering, docking and fusion in eukaryotic cells. The molecular mechanisms underlying the action of Rab proteins and their downstream effectors are poorly understood. Here I propose experiments that will provide insight into the biochemical events immediately downstream of Ypt7p, a yeast Rab protein involved in vacuole fusion. These experiments will use a yeast in vitro vacuole fusion assay that is rapid, quantitative, and amenable to genetic and biochemical manipulation. I will purify the Ypt7p effector, a six-protein complex known as the HOPS/VpsC (hereafter referred to as HOPS) complex, and demonstrate that the HOPS complex mediates membrane tethering via its interaction with GTP-bound Ypt7p I will examine the role of the HOPS complex in controlling activation of Rho1p and Cdc42p, two small G proteins that are also required for vacuole membrane docking. Finally, I will examine whether the HOPS complex binds, prior to bilayer fusion, to the SNARE complex, a complex of integral membrane proteins required for membrane fusion. These experiments will, for the first time, establish the biochemical events directly under the control of a Rab protein and its effector.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM065710-01
Application #
6487071
Study Section
Special Emphasis Panel (ZRG1-F05 (20))
Program Officer
Flicker, Paula F
Project Start
2002-07-01
Project End
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
1
Fiscal Year
2002
Total Cost
$38,320
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Stroupe, Christopher; Collins, Kevin M; Fratti, Rutilio A et al. (2006) Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p. EMBO J 25:1579-89