Princeton University Library Catalog

Structure-Function Studies of Uso1, an ER-to-Golgi Vesicle Tethering Factor

Wu, Zhexiang [Browse]
Senior thesis
Hughson, Fred [Browse]
Princeton University. Department of Molecular Biology [Browse]
Class year:
57 pages
Restrictions note:
Walk-in Access. This thesis can only be viewed on computer terminals at the Mudd Manuscript Library.
Summary note:
Tethering factors play an essential role in intracellular vesicle transport between membrane-bound organelles by forming a link between vesicles and their target membranes. Uso1 is an essential protein that has been shown to dock ER-derived vesicles to the Golgi membrane in Saccharomyces cerevisiae. Uso1 exists as a homodimer and is composed of a 724-residue N-terminal globular “head” domain and a 1066-residue C-terminal coiled-coil “tail” domain. Yet, its mechanism is not well-understood. A crystal structure of the Uso1 head domain was uncovered by previous work in the Hughson lab. In this study, plasmid shuffling was used to show that a Uso1head deletion yields a lethal phenotype, demonstrating that the head domain is essential. Using the crystal structure as a basis, conserved surface clusters on the Uso1 head domain were identified and disruptively mutated. Plasmid shuffling performed on the missense mutants showed that a triple mutation near the N-terminus is lethal and that a double mutation yields a slow-growth phenotype. Immunoaffinity precipitation and tandem mass spectrometry were used to test whether the residues disrupted in these mutants act as binding sites for essential interactions. Two COPI coat subunits, clathrin heavy chain, and a guanine nucleotide exchange factor involved in vesicle budding were identified as potential binding partners of Uso1head. However, the phenotypes of the lethal triple mutant and slow-growth double mutant remain mostly unexplained. Together, this study builds a foundation for exploring the mechanism of the Uso1head and shows that vesicle transport mechanisms may be conserved across species.