Identification and Characterization of Effectors/Binding Molecules for the Small GTPase Rab15
Endocytic trafficking is a key mechanism for regulating receptor availability on\r\nthe plasma membrane as well as receptor degradation. Clathrin-dependent endocytosis\r\ninvolves receptor internalization into early endosomes. Here internalized receptors are\r\nsorted for degradation in lysosomes, direct recycling back to the cell surface or indirect\r\nrecycling via a second recycling compartment called the pericentriolar recycling\r\nendosome. Rab GTPases regulate specific membrane trafficking steps including vesicle\r\nbudding, vesicle transport and fusion with downstream target compartments. Rab\r\nfunction is mediated by the cyclical binding and hydrolysis of GTP, which in turn\r\nregulates the recruitment of downstream effector molecules directly involved in\r\nmembrane transport steps. This dissertation focuses on the endocytic GTPase Rab15.\r\nRab15 localizes to early and pericentriolar recycling endosomes, and differentially\r\nregulates receptor transport at these distinct organelles. For example, over expression of\r\nGTP-bound Rab15 inhibits internalization of the Transferrin Receptor and inhibits\r\nhomotypic endosome fusion in vitro. Conversely, over expression of Rab15-GDP\r\ndifferentially stimulates Transferrin receptor recycling from the early endosome and\r\npericentriolar recycling endosome respectively. Rab15 may differentially regulate\r\nreceptor trafficking through these distinct endocytic compartments by binding\r\ncompartment specific effectors. To test this hypothesis, I performed yeast two-hybrid\r\nscreens to identify and characterize Rab15 binding partners. This dissertation is the\r\nfunctional characterization of three Rab15 binding proteins; Mammalian Suppressor of\r\nSec4, Rab15 Effector Protein and Rab15 Binding Protein. Using molecular, biochemical\r\nand imaging approaches, I demonstrated that interactions between Rab15 and Mss4\r\nmodulate the inhibitory effect of Rab15-GTP on receptor entry into early endosomes.\r\nThe second binding partner, Rab15 Effector Protein, localized specifically to the\r\npericentriolar recycling endosome where it regulated Transferrin receptor recycling back\r\nto the cell surface. Finally, Rab15 Binding Protein is a neural specific protein of\r\nunknown function, suggesting an important regulatory function for Rab15 in neural\r\nreceptor trafficking. These results confirm that Rab15 is a bi-functional GTPase, which\r\ndifferentially regulates receptor trafficking through early and pericentriolar recycling\r\nendosomes, by binding specific effector proteins. Moreover, identification of putative\r\nRab15 effector molecules further defines the endocytic pathway, thus providing valuable\r\ninformation for the characterization of trafficking-related diseases and potential drug\r\ntargets in the future.