Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology
Medical Subject Headings
Carrier Proteins; Circular Dichroism; Dimerization; Membrane Proteins; Multiprotein Complexes; Protein Folding; Protein Structure, Quaternary; Protein Structure, Tertiary; Qc-SNARE Proteins; Recombinant Fusion Proteins; SNARE Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
Life Sciences | Medicine and Health Sciences
Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation.
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Citation: Biochemistry. 2005 Apr 26;44(16):6302-11. Link to article on publisher's site
Sivaram, Mylavarapu V. S.; Saporita, Jennifer A.; Furgason, Melonnie Lynn Marie; Boettcher, Angela J.; and Munson, Mary, "Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p" (2005). GSBS Student Publications. 1123.