Regulatory regions of Sso1p and their roles in SNARE mediated membrane fusion

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dc.contributor.advisorMcNew, James A.en_US
dc.creatorVan Komen, Jeffrey S.en_US
dc.date.accessioned2009-06-04T06:45:03Zen_US
dc.date.available2009-06-04T06:45:03Zen_US
dc.date.issued2005en_US
dc.description.abstractExocytosis 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.en_US
dc.format.extent162 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoTHESIS BIOCHEM. 2006 VAN KOMENen_US
dc.identifier.citationVan Komen, Jeffrey S.. "Regulatory regions of Sso1p and their roles in SNARE mediated membrane fusion." (2005) Diss., Rice University. <a href="https://hdl.handle.net/1911/18988">https://hdl.handle.net/1911/18988</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/18988en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectCell biologyen_US
dc.subjectMicrobiologyen_US
dc.subjectGeochemistryen_US
dc.subjectBiologyen_US
dc.subjectBiochemistryen_US
dc.titleRegulatory regions of Sso1p and their roles in SNARE mediated membrane fusionen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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