Characterization of Rab Interacting Proteins in S. cerevisiae

Cornell Univ - State: Awds Made Prior May 2010, Ithaca NY

Investigators

Abstract

Eukaryotic cells are distinguished by the presence of a number of intracellular membrane-enclosed organelles. All eukaryotic life depends on the transport of material into, out of, and between compartments of the cell. The movement of proteins and lipids between these organelles is mediated by small membrane containers or vesicles which form from one donor compartment and then travel to and fuse with a second target compartment. This traffic must be selective in order to maintain the identities of organelles as well as allow traffic between them. Thus, each stage of transport; the budding, movement, docking and fusion of transport vesicles is subject to regulatory controls. A variety of experimental strategies, using different cell types, has begun to converge onto a common set of elements which appear to be conserved in all eukaryotic cells. These conserved elements include members of the Rab GTPase family, SNARE proteins, NSF/Sec18p, SNAP/Sec17p, Sec1 proteins and docking complexes that represent Rab effectors. The Rab family represents a distinct sub-group of the Ras superfamily of small GTPases. Each stage of membrane traffic through both the constitutive and regulated secretory pathways of all eukaryotic cells is associated with a distinct Rab protein which regulates the cascade of events that lead to SNARE-mediated membrane fusion. This project is designed to yield insights into the mechanisms by which Rab proteins are recruited onto membranes. Recruitment onto the surface of a particular subcellular membrane is a crucial step prior to activation of the Rab GTPase. It is clear that a protein machinery exists to recruit the Rabs onto their donor organelles although no component of this process has been identified to date. The recruitment machinery is an essential part of membrane transport because it determines the site on the membrane where the Rab protein exerts its activity. The prenylated Rabs are found in the cytosol bound to the protein GDP Dissociation Inhibitor (GDI) and it is the soluble GDI-Rab heterodimer that is delivered to the recruitment machinery. Recently, Dr. Collins has identified a protein in yeast, Yip1p which may play a role in regulating Rab protein action. Yip1p is a membrane protein that binds Rabs in a non-specific manner. YIP1 is an essential gene which is highly conserved in evolution. The goal of this proposal is to exploit molecular genetics in yeast together with biochemical approaches to gain information that can be used to test the physiological roles of this factor. Interestingly, it was found that the requirements of Yip1p interaction with Rabs are the same as the features of Rab protein required for GDI interaction. This suggests that the role of Yip1p could be the mirror image of the role played by GDI,which is also a pleiotropic factor. While GDI retrieves Rabs from membranes into the cytosol, Yip1p acts to retrieve Rabs from the cytosol onto membranes. Yip1p may represent a pivotal intervention point that higher eukaryotic cells use to control membrane transport. Regulated membrane transport is essential for many cellular processes in plants and animals, including the secretion of extracellular materials such as enzymes, hormones, extracellular matrix components and neurotransmitters. In addition, the pleiotropic nature of Yip1p implies the existence of other factors that impart organelle specificity into the recruitment process. Dr. Collins has used Yip1p to identify a novel membrane protein termed Yop1p that physically interacts with Yip1p. The possibility will be explored that Yop1p also plays an important role in mediating the action of Rab proteins. The result of these studies are expected to provide important new insights into the fundamental mechanisms underlying the regulation of Rab proteins and their role in intracellular trafficking and secretion.