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|Title:||Initial Characterization of a Vam7 Domain Necessary for Interaction with the HOPS Subunit, Vps18|
|Abstract:||Vesicular trafficking is an essential process within the eukaryotic cell by which mobile vesicles transport cargo to designated target membranes. The final step in this process, membrane fusion, requires the actions of SNARE proteins, which assemble to form membrane-bridging trans-SNARE complexes that catalyze the fusion of apposed membranes. Membrane- (or multisubunit-) tethering complexes (MTCs) regulate membrane fusion by interacting with SNAREs, but little is known of the structural details of these interactions. The homotypic fusion and vacuole protein sorting (HOPS) complex is an MTC that has many functions including interaction with the soluble SNARE, Vam7. Specifically, one member of the HOPS complex, Vps18, has been shown to be responsible for Vam7 targeting in vitro and in vivo. A high-resolution structure of the Vps18-Vam7 interaction will provide understanding of how the HOPS complex tethers membranes, allow further insight into the Vam7 recruitment mechanism, and will serve as a guide for future biochemical assays allowing us to further understand the Vps18- Vam7 binding interface. In this study, we identify a minimal binding region between Chaetomium thermophilum Vps18 and Vam7. Domain mapping studies reveal a previously uncharacterized domain within Vam7 necessary for Vps18 binding. Lastly, we describe two techniques used to purify the Vps18-Vam7 complex for crystallization trials. Future work includes further crystallization trials towards a structural characterization of the Vps18-Vam7 interaction, followed by mutagenic studies both in vitro and in vivo.|
|Type of Material:||Princeton University Senior Theses|
|Appears in Collections:||Molecular Biology, 1954-2020|
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