Identification and Characterization of Effectors/Binding Molecules for the Small GTPase Rab15

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Title: Identification and Characterization of Effectors/Binding Molecules for the Small GTPase Rab15
Author: David Jay Strick
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .3 /63
Date: 2005-03-18

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Identification and Characterization of Effectors/Binding Molecules for the Small GTPase Rab15. Doctoral dissertation, The University of Texas Medical Branch. Available electronically from http : / /hdl .handle .net /2152 .3 /63 .

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