UBC Theses and Dissertations
Molecular and genetic analyses of complexes and pathways for retrograde transport to the Golgi Quenneville, Nicole R.
Proteins and lipids are selectively transported between the Golgi, plasma membrane and endosomes by a network of vesicle-mediated endosomal transport pathways. Trafficking specificity requires the coordination of multiple protein assemblies and signals of compartment identity. Genetic screens, and molecular and biochemical techniques, have revealed many components for endosomal transport, but questions regarding the mechanisms of specificity and the coordination of trafficking pathways remain. The Golgi Associated Retrograde Protein (GARP) complex is required to tether vesicles derived from multiple types of endosomes with the Golgi. In the absence of GARP, retrograde transport from endosomes to the Golgi is abolished, and numerous cargoes are missorted. Mutation of the GARP subunit Vps54 causes motor neuron disease in the mouse, emphasizing the physiological importance of GARP. Tethering requires recognition of multiple membranes, but how GARP recognizes vesicles derived from multiple upstream compartments is not known. In my first body of work, the function of the GARP subunit Vps54 was addressed. The N-terminal portion of Vps54 was found to be important for GARP complex assembly and stability, while the C-terminal portion localized to a compartment with features of an early endosome. In the absence of the C-terminal domain, retrieval of early endosome cargo became dependent on late-endosome retrograde transport. This body of work supports the model that tethers recognize, and possibly distinguish between, upstream compartments. The machinery involved in retrograde transport from endosomes is not fully understood. In my second body of work, genes involved endosomal transport were systematically identified by screening mutant collections with a reporter of early endosome dysfunction. To evaluate the relationships between genes and pathways discovered in this screen, genetic interaction analyses with two phenotypes, growth and endosomal dysfunction, were performed. An analysis of genetic interactions based on trafficking dysfunction revealed interesting genetic relationships between endosomal coat proteins and their regulators. This body of work provides insight into the relationships between endosomal transport pathways and presents a framework to discover relationships between genes and pathways discovered in a genomic screen. Together, this thesis presents a molecular and pathway perspective of endosomal transport that provides insight into pathway specificity and the relationships between pathway components.
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