Rab7 and Accessory Protein Function in Late Endocytosis

University Of New Mexico Health Sciences Center, Albuquerque NM

Investigators

Abstract

This project is a continuation of studies initiated with support from NSF CAREER Award MCB9507206. The overall objective is to investigate the molecular mechanisms underlying late endocytic membrane transport. Endocytosis is the process whereby eukaryotic cells internalize materials from their environment and transfer them to appropriate intracellular compartments. Endocytosis allows cells to sample their environment, to receive specific signals from other cells, and to remove materials from the environment. The process involves the involution of a portion of the plasma membrane of the cell surrounding the material to be internalized, followed by a pinching-off of this membrane to form the endosome. The endosome then moves from the periphery of the cell to the interior, where it may fuse with other membrane-enclosed compartments, such as lysosomes, to deliver its cargo. Detailed analyses of endosome morphology and numerous studies tracing the movement of molecules along both the exo- and endocytic pathways have engendered a great appreciation for the complexity of both the organelles and the pathways, which includes the function of effector proteins such as G-proteins. In particular, the small G-protein, rab7, has formed the focal point for ongoing studies in this laboratory because of its unique localization to the late endosome and its demonstrated importance in regulating late endocytic membrane transport. During the previous CAREER award several key observations were made that are relevant to the current project. First, rab7 was shown to be uniquely required for transport from early to late endosomes and to have no function in subsequent transport from late endosomes to lysosomes. Therefore, rab7 is an ideal 'molecular handle' for further dissection of this important transport step. As proof of principle, several rab7 interacting partners, likely to be further components of the transport machinery, have already been identified by genetic and biochemical means. Most notably, rab7 was shown to interact with a phosphatidyl inositol 3-kinase whose activity was critical for transport from early to late endosomes. An additional requirement for a phosphatidyl inositol 3-kinase activity in transport from late endosomes to lysosomes was also observed, consistent with the idea that rab proteins serve as specific activators of more general downstream effectors controlling membrane fusion. Second, a clarification of the pathways interconnecting the Golgi and endosomes was achieved. The information gained about the routes whereby newly synthesized lysosomal proteins and internalized molecules flux to and through the endocytic pathway is relevant to the proposed studies on endosome dynamics. Third, rab7-positive endosomes (visualized with green fluorescent protein-rab7 chimeras) were found to translocate along microtubules implicating microtubule motors as components of the transport machinery. The reagents, technologies and aggregate experience gained through CAREER award funding place the laboratory in a unique position to conduct the current project. Three specific aims are proposed that will further clarify the protein components regulating late endocytic membrane transport. These are: 1, examination of the role of the vps34 phosphatidyl inositol 3-kinase, which was observed to interact with rab7 in a rab7-conformation-dependent manner, in endocytic transport; 2, analysis of the functions of two novel rab7-interacting proteins, previously identified in a yeast two-hybrid assay, using an established in vitro transport assay based on sequential cleavage of VSV G protein; and 3, monitoring of endosome dynamics in real time using green fluorescent protein-rab7 chimeras. Completion of this project will advance knowledge of how molecules flux through endosomes and what regulates these processes. Such information serves as a paradigm for the molecular mechanisms underlying all membrane transport processes, including those involved in virus entry, antigen processing and presentation, and lysosomal function.