Intra- and inter-cellular membrane fusion mechanisms in Saccharomyces cerevisiae
McNew, James A.
Doctor of Philosophy
Intra- and inter-cellular membrane fusion processes are all mediated by specialized membrane fusion proteins and baker's yeast Saccharomyces cerevisiae has been an invaluable model system to study these fusion events. Intracellular fusion in yeast is mediated by a large family of proteins named SNAREs, however, the molecular machinery mediating intercellular membrane fusion between yeast haploid cells remains unknown. Despite the identification of SNAREs as the minimal membrane fusion machinery for yeast intracellular fusion, additional regulatory mechanisms remain to be elucidated. Sec9p and Spo20p are two SNAP25 family SNAREs specialized for different developmental stages in yeast. Sec9p interacts with Sso1/2p and Snc1/2p to mediate membrane fusion between post-Golgi vesicles and the plasma membrane during vegetative growth. Spo20p replaces Sec9p in the generation of prospore membranes during sporulation. The function of Spo20p requires phosphatidic acid (PA) to be generated in the prospore membrane, the reason of which has not been fully understood. I compared the in vitro membrane fusion function of Sec9p and Spo20p and found that Spo20p forms a less fusion efficient SNARE complex than Sec9p. Incorporation of PA in the lipid bilayer stimulates Sec9p or Spo20p mediated membrane fusion, likely by decreasing the energetic barrier during membrane merger. I also found that PA could directly interact with the positively charged juxtamembrane region of Sso1p, which contributed to the stimulatory effects of PA as well. These results suggested that the fusion strength of SNAREs, the composition of organelle lipids and lipid-SNARE interactions could be coordinately regulated to control the rate and specificity of membrane fusion. I also tried to identify the fusogens mediating yeast intercellular fusion using proteomic methods. We hypothesized that such fusogens were induced by mating pheromone and transported to the cell-cell fusion sites. I was able to isolate post-Golgi vesicles from yeast cells stimulated with mating pheromone and find yeast intercellular fusion related proteins associated with these vesicles. However, our proteomic analysis of the proteins on these vesicles failed to identify any fusogen candidates.
Cell biology; Biochemistry