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Description:
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Endocytic trafficking is a key mechanism for regulating receptor availability on
the plasma membrane as well as receptor degradation . Clathrin -dependent endocytosis
involves receptor internalization into early endosomes . Here internalized receptors are
sorted for degradation in lysosomes , direct recycling back to the cell surface or indirect
recycling via a second recycling compartment called the pericentriolar recycling
endosome . Rab GTPases regulate specific membrane trafficking steps including vesicle
budding , vesicle transport and fusion with downstream target compartments . Rab
function is mediated by the cyclical binding and hydrolysis of GTP , which in turn
regulates the recruitment of downstream effector molecules directly involved in
membrane transport steps . This dissertation focuses on the endocytic GTPase Rab15 .
Rab15 localizes to early and pericentriolar recycling endosomes , and differentially
regulates receptor transport at these distinct organelles . For example , over expression of
GTP -bound Rab15 inhibits internalization of the Transferrin Receptor and inhibits
homotypic endosome fusion in vitro . Conversely , over expression of Rab15 -GDP
differentially stimulates Transferrin receptor recycling from the early endosome and
pericentriolar recycling endosome respectively . Rab15 may differentially regulate
receptor trafficking through these distinct endocytic compartments by binding
compartment specific effectors . To test this hypothesis , I performed yeast two -hybrid
screens to identify and characterize Rab15 binding partners . This dissertation is the
functional characterization of three Rab15 binding proteins ; Mammalian Suppressor of
Sec4 , Rab15 Effector Protein and Rab15 Binding Protein . Using molecular , biochemical
and imaging approaches , I demonstrated that interactions between Rab15 and Mss4
modulate the inhibitory effect of Rab15 -GTP on receptor entry into early endosomes .
The second binding partner , Rab15 Effector Protein , localized specifically to the
pericentriolar recycling endosome where it regulated Transferrin receptor recycling back
to the cell surface . Finally , Rab15 Binding Protein is a neural specific protein of
unknown function , suggesting an important regulatory function for Rab15 in neural
receptor trafficking . These results confirm that Rab15 is a bi -functional GTPase , which
differentially regulates receptor trafficking through early and pericentriolar recycling
endosomes , by binding specific effector proteins . Moreover , identification of putative
Rab15 effector molecules further defines the endocytic pathway , thus providing valuable
information for the characterization of trafficking -related diseases and potential drug
targets in the future . |