The molecular mechanism of biological membrane fusion in the yeast Saccharomyces cerevisiae
McNew, James A.
Doctor of Philosophy
This work is focused on elucidating the molecular mechanism of biological membrane fusion using S.cerevisiae as a model system. Membrane fusion is vital for the survival of all eukaryotes, yet many aspects still remain unclear. The work is composed of two projects: (i) Identifying cell-cell membrane fusion proteins (fusogens) in yeast mating; (ii) Illuminating regulatory mechanisms of intracellular membrane fusion in exocytosis. First, I conducted a pilot exploration to identify potential fusogens driving direct cell-cell plasma membrane fusion during yeast mating. The strategy was to screen for pheromone-inducible fusogen candidates from late secretory vesicles. In this study, post-Golgi late secretory vesicles were successfully purified from pheromone-treated cells and vesicle proteins have been separated by two-dimensional electrophoresis. However, few pheromone-induced proteins have been detected due to a low signal to background ratio. I thus developed an alternative method, which involves identifying fusogen candidates on the plasma membrane. Preliminary data yielded a greatly increased ratio of signal to background, suggesting that the new approach is more promising. Further mass spectrometry analysis is underway to identify the potential fusogen candidates. Second, I dissected domain functions of the yeast syntaxin Sso1p in SNARE-mediated membrane fusion during exocytosis. In vivo functional analyses suggest that both the Sso1p polybasic juxtamembrane region and the N-terminal regulatory domain (NRD) are essential for regulating membrane fusion. The polybasic juxtamembrane region, which is required for survival, likely interacts with the negatively charged lipid groups in the plasma membrane, whereas the NRD is required for assembly of the Sso1p-Sec9p t-SNARE complex possibly by preventing formation of inappropriate SNARE complexes. Furthermore, I found that the yeast t-SNAREs and their mammalian homologues are functionally interchangeable in vitro, but not in vivo unless regulation is circumvented. That indicates the core machinery for intracellular membrane fusion has been well conserved, while regulation is diversified in evolution. My studies of yeast cell-cell membrane fusion and intracellular membrane fusion shed lights on the mechanism of different aspects of membrane fusion and will facilitate clinical application of the fusion mechanism to in vitro drug delivery and regenerative medicine.
Molecular biology; Genetics; Cell biology