Mechanism of action of Ypt7p and the HOPS/VpsC complex

Dartmouth College, Hanover NH

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Abstract

DESCRIPTION (provided by applicant) 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.