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dc.contributor.advisorMary Munson PhD
dc.contributor.authorBrewer, Daniel Niron
dc.date2022-08-11T08:08:42.000
dc.date.accessioned2022-08-23T16:04:55Z
dc.date.available2022-08-23T16:04:55Z
dc.date.issued2009-11-23
dc.date.submitted2010-01-12
dc.identifier.doi10.13028/1dg3-hh20
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31775
dc.description.abstractTrafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicles are targeted to sites of exocytosis on the plasma membrane in part by a conserved multi-subunit protein complex termed the exocyst. In addition to tethering vesicles to the plasma membrane, the exocyst complex and components therein may also add a layer of regulation by directly controlling assembly of the SNARE complex, which is required for membrane fusion, as well as other regulatory factors such as Sec1p. In the past, we have shown that Sec6p interacts with Sec9p in vivo and that that interaction retards binary SNARE complex formation in a SNARE assembly assay. Though many interactions have been mapped using in vitro methods, confirming them in vivoand placing them into the context of a complete model that accounts for all observed interactions (and lack of interactions) has proven difficult. In order to address these problems, I have studied the interactions between Sec6p and other factors involved in exocytosis at the plasma membrane via in vivo methods. My hypothesis was that Sec6p interaction with Sec9p and subsequent inhibition of SNARE complex assembly in vitro was an intermediate state and Sec6p was part of a set of cofactors that accelerated SNARE complex assembly in vivo. To test this hypothesis I showed that the interaction between the plasma membrane t-SNARE Sec9p and the yeast exocyst subunit Sec6p can be observed in vivoand designed point mutations to disrupt that interaction. Interestingly, I also showed that Sec6p:Sec9p interaction involves the free pool of Sec6p rather than the exocyst bound fraction of Sec6p. Point mutations in the N-terminal domain of Sec6p result in temperature sensitive growth and secretion defects, without loss of Sec6p-Sec9p interaction. However, at the non-permissive temperature, the exocyst subunits Sec5p, Sec10p and Sec15p are mislocalized and are absent from the exocyst complex. The resulting subcomplex, containing Sec3p, Sec8p, Exo70p and Exo84p, remains stably assembled and localized at sites of polarized secretion. This subcomplex is likely due to disruption of interaction between Sec6p and Sec5p, and may be similar to that observed at restrictive temperatures in the sec6-54temperature sensitive mutant. Additionally, one of the sec6 temperature sensitive mutants displays a loss of binding to the yeast regulatory protein Sec1p. In vitro binding studies indicate a direct interaction between Sec1p and the free pool of the wild-type Sec6p protein, suggesting close interplay between Sec6p and Sec1p in the regulation of SNARE complexes. A coherent model which incorporates all these interactions has continued to be elusive. However, the results I have found do suggest several hypotheses which should prove testable in the future.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectSNARE Proteins
dc.subjectVesicular Transport Proteins
dc.subjectExocytosis
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectCells
dc.subjectGenetic Phenomena
dc.titleElucidation of the Role of the Exocyst Subunit Sec6p in Exocytosis: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1449&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/446
dc.legacy.embargo2010-12-10T00:00:00-08:00
dc.identifier.contextkey1108905
refterms.dateFOA2022-08-24T03:26:33Z
html.description.abstract<p>Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicles are targeted to sites of exocytosis on the plasma membrane in part by a conserved multi-subunit protein complex termed the exocyst. In addition to tethering vesicles to the plasma membrane, the exocyst complex and components therein may also add a layer of regulation by directly controlling assembly of the SNARE complex, which is required for membrane fusion, as well as other regulatory factors such as Sec1p. In the past, we have shown that Sec6p interacts with Sec9p <em>in vivo</em> and that that interaction retards binary SNARE complex formation in a SNARE assembly assay. Though many interactions have been mapped using <em>in vitro</em> methods, confirming them <em>in vivo</em>and placing them into the context of a complete model that accounts for all observed interactions (and lack of interactions) has proven difficult.</p> <p>In order to address these problems, I have studied the interactions between Sec6p and other factors involved in exocytosis at the plasma membrane via <em>in vivo</em> methods. My hypothesis was that Sec6p interaction with Sec9p and subsequent inhibition of SNARE complex assembly <em>in vitro</em> was an intermediate state and Sec6p was part of a set of cofactors that accelerated SNARE complex assembly <em>in vivo</em>. To test this hypothesis I showed that the interaction between the plasma membrane t-SNARE Sec9p and the yeast exocyst subunit Sec6p can be observed <em>in vivo</em>and designed point mutations to disrupt that interaction. Interestingly, I also showed that Sec6p:Sec9p interaction involves the free pool of Sec6p rather than the exocyst bound fraction of Sec6p.</p> <p>Point mutations in the N-terminal domain of Sec6p result in temperature sensitive growth and secretion defects, without loss of Sec6p-Sec9p interaction. However, at the non-permissive temperature, the exocyst subunits Sec5p, Sec10p and Sec15p are mislocalized and are absent from the exocyst complex. The resulting subcomplex, containing Sec3p, Sec8p, Exo70p and Exo84p, remains stably assembled and localized at sites of polarized secretion. This subcomplex is likely due to disruption of interaction between Sec6p and Sec5p, and may be similar to that observed at restrictive temperatures in the <em>sec6-54</em>temperature sensitive mutant.</p> <p>Additionally, one of the <em>sec6</em> temperature sensitive mutants displays a loss of binding to the yeast regulatory protein Sec1p. <em>In vitro</em> binding studies indicate a direct interaction between Sec1p and the free pool of the wild-type Sec6p protein, suggesting close interplay between Sec6p and Sec1p in the regulation of SNARE complexes. A coherent model which incorporates all these interactions has continued to be elusive. However, the results I have found do suggest several hypotheses which should prove testable in the future.</p>
dc.identifier.submissionpathgsbs_diss/446
dc.contributor.departmentBiochemistry and Molecular Pharmacology
dc.description.thesisprogramBiochemistry and Molecular Pharmacology


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