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. 2016 Jan;23(1):59-66.
doi: 10.1038/nsmb.3146. Epub 2015 Dec 14.

Subunit Connectivity, Assembly Determinants and Architecture of the Yeast Exocyst Complex

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Free PMC article

Subunit Connectivity, Assembly Determinants and Architecture of the Yeast Exocyst Complex

Margaret R Heider et al. Nat Struct Mol Biol. .
Free PMC article

Abstract

The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst complexes from Saccharomyces cerevisiae and showed that they are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin induced-degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer and demonstrated that several known exocyst-binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex by using negative-stain electron microscopy; our results indicate that the exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests that the subunits are contiguous helical bundles packed together into a bundle of long rods.

Figures

Figure 1
Figure 1
Purification of intact yeast exocyst complexes. Purified complexes were separated by SDS-PAGE and visualized by Krypton staining (Thermo Scientific). The asterisk corresponds to the PrA-tagged exocyst subunit used as purification handle (shifts the protein molecular weight by 25 kDa). Both the Sec3 and Exo84 protein bands often migrate as multiple species due to phosphorylation, which appear as slightly smeared bands on SDS-PAGE. The resuspension buffer used was 50 mM Hepes pH 7.4, 300 mM NaCl, plus protease inhibitors. Full-size images for this and most gels in Figures 2–5 are shown in Supplementary Data Set 1.
Figure 2
Figure 2
Purified exocyst complexes are stable over a wide range of conditions and are comprised of discrete pairwise interactions. (a) Sec15-PrA exocyst complexes were purified using buffers of different pH and KCl concentration as indicated and visualized using Coomassie-stained SDS-PAGE (b) Sec15-PrA exocyst complexes were purified using 50 mM Hepes pH 7.4, 300 mM NaCl buffer and various commonly used detergents at the following concentrations: 0.1% NP-40, 0.1% Tween-20, 1% Triton X-100, 20 mM Sodium cholate. (c) Destabilizing buffer conditions were used with each exocyst subunit as PrA purification handle in order to isolate subcomplexes and stable subunit pairs. A=20 mM PIPES pH 6.8, 300 mM KCl. B=20 mM Tris pH 8.0, 500 mM KCl. C=20 mM Tris pH 8.0, 300 mM KCl, 500 mM Urea. Asterisks correspond to the PrA-tagged subunit.
Figure 3
Figure 3
Use of the auxin-inducible degron (AID) system to selectively degrade essential exocyst proteins from yeast. (a) Schematic of the AID system. The auxin-inducible degron (AID) tag from Arabidopsis thaliana was fused to the C-terminus of exocyst subunits at their genomic locus in yeast strains constitutively expressing OsTIR1 (F-box transport inhibitor response 1) protein. Upon treatment with the natural plant hormone auxin (IAA=Indole 3-acetic acid), the SCF-OsTIR1 E3 Ubiquitin ligase complex is activated, which then recruits E2 Ubiquitin ligases for polyubiquitination of the AID-tagged protein. The AID-tagged protein is then rapidly degraded by the proteasome,. (b) AID-tagged exocyst strains were tested for growth by serial dilution growth assay on YPD plates containing the indicated amount of IAA. Suppressor colonies can be seen in some dilutions. (c) Degradation of exocyst subunits in these strains was confirmed by western blotting lysates from NaOH/SDS lysis. (−) denotes untreated strains and (+) treated with IAA. All subunits were degraded to <10–12% of starting protein level. Asterisks indicate the AID-tagged exocyst subunit in blots where antibodies also bind non-exocyst subunits.
Figure 4
Figure 4
Most exocyst subunits are critical for maintaining the assembly of two 4-subunit modules within the full octameric complex. (a) Exocyst complexes were purified using the indicated PrA purification handle (blue) from yeast strains where one AID-tagged subunit (magenta) is degraded. The resuspension buffer used was 50 mM Hepes pH 7.4, 150 mM NaCl. Purified complexes were run on SDS-PAGE and visualized with Coomassie staining. (−) denotes untreated and (+) treated with IAA. Exocyst subunits are denoted by their number (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, Exo84 as 3,5,6,8,10,15,70,84). Degradation of 6 of the subunits tested led to the complete separation of exocyst into two 4-subunit modules: 3–5–6–8 and 10–15–70–84 with the connections depicted in the central table. Sec10-AID, Sec15-PrA was not determined (N.D.). Faded symbols represent subunits that showed partial loss from the complex. (b) Model depicting the subunit connectivity within and between each exocyst module (green and purple). Thick lines indicate the strong pairwise connections identified in Fig. 2, Fig. 4a, and Supplementary Figure 4b which are required for stability of the assembled exocyst. The thin line depicts a putative connection between Sec8 and Sec10–Sec15 identified in the AID studies, but Sec8’s direct binding partner within this pair is not known. Dashed lines represent interactions identified in previous in vitro studies using Y2H and recombinant proteins (summarized in ); these are consistent with several additional, weaker pairwise interactions identified here.
Figure 5
Figure 5
Depletion of known exocyst binding partners does not affect the assembly of exocyst complex. (a) Exocyst binding partners Cdc42, Myo2, Sec1, Snc2 (in snc1Δ strain background), and Sec4 were AID-tagged in strains with Sec8-PrA and constitutively expressing OsTIR1. (−) denotes untreated and (+) treated with IAA for 60 minutes. Western blots demonstrate degradation of these proteins from yeast lysate using antibodies specific to the AID-tagged protein of interest. In the Sec1 blot, the Sec1 antibody also reacts with the PrA tag on Sec8-PrA. (b) Exocyst complexes were purified using Sec8-PrA as the purification handle from untreated (−) versus IAA-treated (+) yeast lysates.
Figure 6
Figure 6
Negative stain electron microscopy of purified exocyst complexes. (a) A representative transmission electron micrograph of Sec15-GFP exocyst complexes after negative staining in uranyl acetate. Scale bar is 50 nm. (b) Representative 2D class average (Sec15-GFP) is shown, overlaid with a ribbon diagram of the structure of yeast Exo70 (residues 67–623), PDB ID 2B1E . The orientation and position of Exo70 were arbitrarily chosen to illustrate the similarities in the length and width of the “legs” of the complex and Exo70. (c) Highly populated 2D class averages generated by unsupervised classification for both wild type and Sec15-GFP image datasets, the number of particles per class is indicated next to each 2D average. Four apparent “faces” of the complex are labeled as I-IV. The red arrow points to the more “compact” end of the complex in class I, while the white arrowhead points to the more “open” flexible end in class III. Scale bar is 20 nm.

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