Exposing the Elusive Exocyst Structure

Abstract

A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high-resolution structures of large, multisubunit tethering complexes that spatially and temporally control intracellular membrane fusion. Exocyst is a large hetero-octameric protein complex proposed to tether secretory vesicles at the plasma membrane to provide quality control of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence microscopy, and single-particle cryoelectron microscopy, are providing critical insights into the structure and function of the exocyst. These studies now pose more questions than answers for understanding fundamental functional mechanisms, and they open wide the door for future studies to elucidate interactions with protein and membrane partners, potential conformational changes, and molecular insights into tethering reactions.

Keywords: Rab GTPase; Rho GTPase; SNARE; exocyst; exocytosis; membrane fusion; membrane trafficking; tethering complex.

Figure 1.

Timeline of the major exocyst structural studies. (A) Unfixed and glutaraldehyde-fixed mammalian exocyst complexes, visualized by platinum rotary shadowing (adapted from [44]). (B) High resolution crystal structures of various domains of exocyst subunits (residues as indicated): yeast Exo70 (PDB ID: 2B1E; [45]); yeast Exo84 C-terminal domain (PDB ID: 2D2S; [45]); mammalian Exo84 RalA-binding domain (PDB ID: 1ZC3; [55]); Drosophila Sec15 C-terminal domain (PDB ID 2A2F; [47]); yeast Sec3 N-terminal domain (PDB ID 3HIE; [58]); yeast Sec6 C-terminal domain (PDB ID 2FJI; [46]); and zebrafish Sec10 (PDB ID 5H11; [48]). Overall, high resolution structural information is available for <30% of yeast exocyst. (C) Network of protein-protein interactions between mammalian exocyst subunits, as mapped by a visible immunoprecipitation assay (from [61]). Identified subcomplexes are boxed and colored, subunits that showed homodimerization are outlined in red, and the thickness of the blue interaction lines indicates the strength of the interactions. (D) Network of protein-protein interactions between subunits, as mapped by auxin-induced degradation experiments plus in vitro binding experiments (adapted from [16]). (E) Negative stain EM of intact yeast exocyst (adapted from [16]). (F) Representative model for the yeast exocyst structure, built using distance restraints from in vivo fluorescence measurements (adapted from [38]). The flat ends of the beads represent the N-terminal ends of each subunit, while pointy beads are the C-terminal ends.(G) Moderate resolution model of the intact yeast exocyst structure determined using cryoEM, modeling and crosslinking/mass spectrometry (PDB ID 5YFP; [17]). Note that the subunit colors are not the same as in panel F. (H) In contrast to the compact exocyst, negative stain EM of the COG complex shows an open, highly dynamic structure (adapted from [80]).

Figure 2.

The architecture of the yeast exocyst is assembled from two modules containing four subunits each. The subunits within the Sec3-5-6-8 module and the Sec10-15-70-84 module each contributes a helix to one of two CorEx helical bundles (insets). The modules are stacked one on top of the other in this view, to form the octameric complex observed in the negative stain and cryoEM structures [16, 17]. The biochemical studies, PICT experiments and EM images of the yeast complex demonstrate that no detectable subcomplexes or stable modules are observed in the absence of destabilizing conditions [16, 17, 38]. The subunits are colored as indicated on the right. The most dynamic and/or flexible—and therefore lowest resolution—regions are the C-terminal end of Sec15 at the bottom (red), the C-terminal half of Sec6 across the top (purple) and the C-terminal end of Sec10 (cyan).

Figure 3.

Diagram indicating many of the known interactions between the yeast exocyst and various partners. This structure is rotated ~180° relative to that shown in Figure 2. Each of the partners is color-coded to match the subunit with which they interact. The dissociated yellow domain is the Sec3 N-terminal domain (residues 71-241; PDB ID 3HIE; [58]). The first 610 amino acids of Sec3 do not appear in the cryoEM electron density, likely due to flexibility/dynamics; if extended, the region between 241 and 611 could span a distance up to 15 nm. The exact binding site(s) on Exo70 for Rho3 and Cdc42 have not been determined; binding studies with the prenylated Rho-GTPases indicates that their GTP-dependent interaction requires both the N- and C-terminal regions of Exo70 [74]. Although the diagram suggests that these interactions may all take place simultaneously, the spatial constraints and overlapping binding sites suggest that stepwise interactions and/or conformational changes are likely to take place.