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dc.contributor.authorDubuke, Michelle L.
dc.contributor.authorManiatis, Stephanie
dc.contributor.authorShaffer, Scott A.
dc.contributor.authorMunson, Mary
dc.date2022-08-11T08:08:56.000
dc.date.accessioned2022-08-23T16:12:55Z
dc.date.available2022-08-23T16:12:55Z
dc.date.issued2015-11-20
dc.date.submitted2017-09-13
dc.identifier.citation<p>J Biol Chem. 2015 Nov 20;290(47):28245-56. doi: 10.1074/jbc.M115.673806. Epub 2015 Oct 7. <a href="https://doi.org/10.1074/jbc.M115.673806">Link to article on publisher's site</a></p>
dc.identifier.issn0021-9258 (Linking)
dc.identifier.doi10.1074/jbc.M115.673806
dc.identifier.pmid26446795
dc.identifier.urihttp://hdl.handle.net/20.500.14038/33499
dc.description.abstractIn eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=26446795&dopt=Abstract">Link to Article in PubMed</a></p>
dc.rights© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Publisher PDF posted after 12 months as allowed by the publisher's author rights policy at http://www.jbc.org/site/misc/Copyright_Permission.xhtml.
dc.subjectMS
dc.subjectSNARE proteins
dc.subjectexocyst
dc.subjectexocytosis
dc.subjectintracellular trafficking
dc.subjectintrinsically disordered protein
dc.subjectprotein cross-linking
dc.subjectprotein-protein interaction
dc.subjecttethering complex
dc.subjectBiochemistry
dc.subjectMolecular Biology
dc.subjectStructural Biology
dc.titleThe Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume290
dc.source.issue47
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=3047&amp;context=gsbs_sp&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_sp/2024
dc.identifier.contextkey10740180
refterms.dateFOA2022-08-23T16:12:55Z
html.description.abstract<p>In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions.</p>
dc.identifier.submissionpathgsbs_sp/2024
dc.contributor.departmentProteomics and Mass Spectrometry Facility
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.source.pages28245-56
dc.contributor.studentMichelle L. Dubuke


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