Browsing by Author "Van Komen, Jeffrey S."
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Item Regulatory regions of Sso1p and their roles in SNARE mediated membrane fusion(2005) Van Komen, Jeffrey S.; McNew, James A.Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p) and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion in the secretory pathway, many aspects of SNARE assembly and regulation are ill defined. I examined the yeast regulatory protein, Sec1p, and its function in exocytosis. I show that the majority of Sec1p localizes to the plasma membrane, even though it is predicted to be a soluble protein. Furthermore, a significant portion of Sec1p colocalizes with Sso1, but is enriched in the bud neck. Sec1p binds to the t-SNARE complex and directly stimulates membrane fusion in vitro. I have also examined several defined structural regions of the yeast plasma membrane t-SNARE component Sso1p for their effect on membrane fusion in vivo and in vitro. To analyze the role of the N-terminal regulatory domain in Sso1p, I generated a chimeric protein that physically links the two separate proteins of the yeast plasma membrane t-SNARE complex, namely a truncated Sec9p and Sso1p. With this chimera, I have shown that the required function of the N-terminal regulatory domain Sso1p can be circumvented when t-SNARE complex formation is made intramolecular, suggesting the N-terminal regulatory domain is required for efficient t-SNARE complex formation and does not recruit necessary scaffolding factors. Next, I used targeted sequence modification, including insertions and replacements, in a conserved, highly charged juxtamembrane region between the transmembrane helix and the core coiled-coil domain of Sso1p. The effects of the modifications were examined both in vitro and in vivo. I found that mutant Sso1 proteins with insertions or duplications show limited function in vivo, whereas replacement of as few as three amino acids preceding the transmembrane domain results in a non-functional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting helical continuity between the transmembrane domain and the core coiled-coil domain is not essential. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrate that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.