Biochemistry & Molecular Pharmacology
Department of Biochemistry and Molecular Pharmacology; Proteomics and Mass Spectrometry Facility
Biochemistry | Molecular Biology | Structural Biology
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.
MS, SNARE proteins, exocyst, exocytosis, intracellular trafficking, intrinsically disordered protein, protein cross-linking, protein-protein interaction, tethering complex
Rights and Permissions
© 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.
DOI of Published Version
J Biol Chem. 2015 Nov 20;290(47):28245-56. doi: 10.1074/jbc.M115.673806. Epub 2015 Oct 7. Link to article on publisher's site
The Journal of biological chemistry
Dubuke, Michelle L.; Maniatis, Stephanie; Shaffer, Scott A.; and Munson, Mary, "The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes" (2015). GSBS Student Publications. 2024.