The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif

Abstract

The exocyst is a large complex that is required for tethering vesicles at the final stages of the exocytic pathway in all eukaryotes. Here we present the structures of the Exo70p subunit of this complex and of the C-terminal domains of Exo84p, at 2.0-A and 2.85-A resolution, respectively. Exo70p forms a 160-A-long rod with a novel fold composed of contiguous alpha-helical bundles. The Exo84p C terminus also forms a long rod (80 A), which unexpectedly has the same fold as the Exo70p N terminus. Our structural results and our experimental observations concerning the interaction between Exo70p and other exocyst subunits or Rho3p GTPase are consistent with an architecture wherein exocyst subunits are composed of mostly helical modules strung together into long rods.

Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells

“…We found that mutations on exo70-1 did not affect its interaction with Sec8 in the cell (Supplementary Figure 1B). This result is consistent with the previous reports in mammalian and yeast cells that the interaction of Exo70 with the other exocyst components for complex assembly is mediated by the N-terminal domains of Exo70 (Inoue et al, 2003;Dong et al, 2005). …”

“…Moreover, the structural analysis of Exo70 provided important insights into the potential mechanism of membrane association of Exo70 (Dong et al, 2005;Hamburger et al, 2006). On the basis of the crystal structure information of yeast Exo70, we have made mutations on the rat Exo70 residues K632 and K635 (exo70-1), which are positively charged amino acids well conserved in the yeast Exo70 sequence.…”

“…As shown in Figure 1B, Exo70 with its C-terminal domain deleted (amino acids 1-408; Exo70-NT) was cytosolic, whereas the C-terminus of Exo70 (amino acids 403-653; Exo70-CT) was enriched at the PM ( Figure 1B), suggesting a critical role of Exo70 C-terminus in membrane association. The crystal structure of yeast Exo70 revealed that this protein is a long rod composed mainly of ␣-helices that fold into four domains (named domains A, B, C, and D; Dong et al, 2005;Hamburger et al, 2006). Domain D (the C-terminal 114 amino acids) is the most evolutionarily conserved domain in Exo70 that contains a number of basic residues that cluster into an electro-positive patch on the surface of the C-terminus.…”

“…In Madin-Darby canine kidney (MDCK) cells, extragenically expressed GFP-tagged Exo70 is localized to the PM near cell-cell contacts, suggesting that Exo70 may mediate PM association independent of the rest exocyst components in these cells (Matern et al, 2001). Recent structural studies have revealed that Exo70 contains a number of conserved basic residues that cluster on a surface patch at the Cterminal end of the tertiary structure that may directly bind to the PM (Dong et al, 2005;Hamburger et al, 2006;Moore et al, 2007). In fact, the C-terminal sequence of Exo70 is the most evolutionarily conserved region of this protein.…”

Structural basis for a human glycosylation disorder caused by mutation of the COG4 gene

“…Subunits of the COG, exocyst, Dsl1p, and Golgiassociated retrograde protein (GARP) complexes share limited sequence homology (7,13) that has been suggested to reflect convergent evolution (13). Structures, most of them partial, have been reported for one COG complex subunit, two Dsl1p complex subunits, and four exocyst subunits (14)(15)(16)(17)(18)(19)(20); these structures contain one (Cog2p), two (Dsl1p, Sec15, Exo84p), three (Sec6p), four (Exo70p), or five (Tip20p) helix bundle domains. In each of the exocyst and Dsl1p complex structures, the tandem helical domains form an extended, rod-like array (20,21).…”