Receptor-mediated endocytosis is a fundamental process carried out by all cells and is characterized by the internalization of plasma membrane-derived coated vesicles and a series of ordered and specific fusion events among coated vesicles, endosomes, and lysosomes. During the last grant period, in vitro assays were developed to reconstituted early fusion events. Heterotrimeric G proteins and a variety of other factors emerged as important regulators of endocytic vesicle fusion. These assays will now be used to identify key GTP binding proteins and other novel regulators such as phospholipase A2 and characterize their mechanisms of action. Dr. Stahl plans four major specific aims. (1) To use density shift and/or immunoisolation techniques to prepare enriched preparations of endosomes for biochemical and morphological analysis. These fractions will be used to identify cytosolic and membrane proteins associated with endosomes that have been """"""""primed"""""""" for recognition and fusion. Ultrastructural characterization of putative fusion pores will be continued. Finally, monoclonal antibodies will be prepared that inhibit fusion. (2) To elucidate the role of heterotrimeric G proteins and ADPribosylation factor (ARF) in fusion, G proteins present in the enriched vesicle fraction will be identified by western blot and mutant or wild type G protein subunits and different ARF family members overexpressed in intact cells using the Sindbis virus expression system. Effects on in vitro fusion or endocytosis in intact cells will be sought. (3) Since G proteins are likely to be activated by upstream effectors, such effectors will be sought. Given that clathrin adaptor proteins have an effect on the in vitro fusion assay, the possibility that adaptins themselves serve this effector function will be evaluated. The role of downstream effectors, such as rab5, NSF and phospholipase A2 will also be evaluated. (4) Endosome-lysosome fusion will be reconstituted in permeabilized cell systems or by using Xenopus oocytes injected with mRNA encoding molecules of interest.
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