Dimerization of the Exocyst Protein Sec6p and Its Interaction with the t-SNARE Sec9p

Abstract

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.

The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes

“…Consistent with other MTC-SNARE interactions, we have shown previously that the yeast exocyst subunit Sec6 interacts with the C-terminal SNAP-25 domain of the plasma membrane t-SNARE Sec9 (Sec9CT, residues 414 -651) (46). However, Sec6 appeared to inhibit, rather than promote or stabilize, in vitro formation of the Sso1-Sec9 binary SNARE complex (46).…”

“…However, Sec6 appeared to inhibit, rather than promote or stabilize, in vitro formation of the Sso1-Sec9 binary SNARE complex (46). Therefore, we proposed that the Sec6-Sec9 interaction prevented premature assembly of the Sso1-Sec9 SNARE complex and that assembly of the exocyst complex could release Sec9 to form fusogenic SNARE complexes.…”

“…Previous attempts to identify a minimal region of Sec9 necessary for the interaction with Sec6, including limited proteolysis and truncation experiments, were inconclusive (46). Identification of the minimal binding region of Sec6 was hindered by the insolubility of the N-terminal domain of Sec6.…”

“…Identification of the minimal binding region of Sec6 was hindered by the insolubility of the N-terminal domain of Sec6. However, the C-terminal half of the protein has been shown to be insufficient for binding (46). We were also limited by the flexible nature of Sec9.…”

RalA-exocyst-dependent Recycling Endosome Trafficking Is Required for the Completion of Cytokinesis

“…Recent insights into structure of the exocyst have shed light on the architecture and function of this complex, suggesting that the exocyst assembles into an overall rodlike structure, in the process bridging the vesicles to their target membrane (5,6). Consistent with this notion, the exocyst has been found to concentrate on "hotspots" on the plasma membrane where exocytosis actively takes place and has been implicated in different types of membrane trafficking including polarized growth in yeast, neurite growth in the nervous system, glucose transport in fat cells, and basal-lateral trafficking in epithelial cells (4).…”