The roles of lipids and intracellular trafficking in specialized SNARE-mediated fusion during Saccharomyces cerevisiae sporulation

Gametogenesis is an essential, conserved process in all sexually-reproducing eukaryotes. In Saccharomyces cerevisiae, gametogenesis, or sporulation, generates haploid gametes from diploid cells by coupling meiosis to a complex cellular differentiation program. This process involves multiple intracellular trafficking pathways for the generation of spores competent for survival in a nutrient-deficient environment. The prospore membrane is a novel compartment which forms concomitantly with the second meiotic division and is required for spore morphogenesis. In this work, I have examined the interplay of phospholipids with the soluble NSF attachment receptors (SNAREs) in mediating prospore membrane formation. The current study finds that phosphatidylinositol-4,5-bisphosphate interacts with the autoregulatory domain of the sporulation-specific SNARE Sso1p in vitro. Additionally, elevated levels of this lipid in vivo stimulate prospore membrane formation in the absence of Sso1p; these results suggest a novel lipid-mediated regulatory mechanism is required for synthesis of the prospore membrane. The product of phospholipase D enzymatic activity, phosphatidic acid, is also required for prospore membrane formation. I utilized a high-throughput proteomic approach to screen for potential PA-binding proteins that may be required during sporulation. Of the hundreds of candidates identified, Gga1p, a clathrin adaptor involved in trafficking from the trans-Golgi network, was further examined for its role in sporulation. Together with the functionally redundant Gga2p, it appears to play a role in spore wall production. I also participated in a study on the role of retrograde trafficking protein complexes in sporulation; we found that the SNARE Snc1p is retrieved from the plasma membrane through the endosome to participate in prospore membrane precursor vesicle fusions. This body of work suggests that the process of prospore membrane formation requires the direct interaction of membrane phospholipids and SNARE machinery for fusion and also indicates the requirement of specific trafficking components for the movement of molecules to the site of prospore membrane synthesis.