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Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex

Nature Structural & Molecular Biology volume 23, pages 59–66 (2016)Cite this article

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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.

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Figure 1: Purification of intact yeast exocyst complexes.
Figure 2: Purified exocyst complexes are stable over a wide range of conditions and are composed of discrete pairwise interactions.
Figure 3: Use of the auxin-inducible degron (AID) system to selectively degrade essential exocyst proteins from yeast.
Figure 4: Most exocyst subunits are critical for maintaining the assembly of two four-subunit modules within the full octameric complex.
Figure 5: Depletion of known exocyst-binding partners does not affect the assembly of the exocyst complex.
Figure 6: Negative-stain EM of purified exocyst complexes.

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References

  1. Heider, M.R. & Munson, M. Exorcising the exocyst complex. Traffic 13, 898–907 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wickner, W. & Schekman, R. Membrane fusion. Nat. Struct. Mol. Biol. 15, 658–664 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yu, I.M. & Hughson, F.M. Tethering factors as organizers of intracellular vesicular traffic. Annu. Rev. Cell Dev. Biol. 26, 137–156 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Chia, P.Z. & Gleeson, P.A. Membrane tethering. F1000Prime Rep. 6, 74 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Brunet, S. & Sacher, M. Are MTCs tethering complexes or trafficking complexes that may act as tethers? Traffic 15, 1282–1287 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Sivaram, M.V., Saporita, J.A., Furgason, M.L.M., Boettcher, A.J. & Munson, M. Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p. Biochemistry 44, 6302–6311 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Morgera, F. et al. Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1. Mol. Biol. Cell 23, 337–346 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Laufman, O., Hong, W. & Lev, S. The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes. J. Cell Sci. 126, 1506–1516 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. TerBush, D.R., Maurice, T., Roth, D. & Novick, P. The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 6483–6494 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. TerBush, D.R. & Novick, P. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130, 299–312 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Guo, W., Grant, A. & Novick, P. Exo84p is an exocyst protein essential for secretion. J. Biol. Chem. 274, 23558–23564 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Hsu, S.C. et al. The mammalian brain rsec6/8 complex. Neuron 17, 1209–1219 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Koumandou, V.L., Dacks, J.B., Coulson, R.M. & Field, M.C. Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins. BMC Evol. Biol. 7, 29 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Riquelme, M. et al. The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth. Mol. Biol. Cell 25, 1312–1326 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Novick, P., Field, C. & Schekman, R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21, 205–215 (1980).

    Article  CAS  PubMed  Google Scholar 

  16. Guo, W., Roth, D., Walch-Solimena, C. & Novick, P. The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18, 1071–1080 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Friedrich, G.A., Hildebrand, J.D. & Soriano, P. The secretory protein Sec8 is required for paraxial mesoderm formation in the mouse. Dev. Biol. 192, 364–374 (1997).

    Article  CAS  PubMed  Google Scholar 

  18. Murthy, M., Garza, D., Scheller, R.H. & Schwarz, T.L. Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists. Neuron 37, 433–447 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Jin, Y. et al. Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex. Dev. Cell 21, 1156–1170 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Munson, M. & Novick, P. The exocyst defrocked, a framework of rods revealed. Nat. Struct. Mol. Biol. 13, 577–581 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Shen, D. et al. The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p. J. Cell Biol. 202, 509–526 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wu, H., Rossi, G. & Brennwald, P. The ghost in the machine: small GTPases as spatial regulators of exocytosis. Trends Cell Biol. 18, 397–404 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wu, H., Turner, C., Gardner, J., Temple, B. & Brennwald, P. The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis. Mol. Biol. Cell 21, 430–442 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. He, B., Xi, F., Zhang, X., Zhang, J. & Guo, W. Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane. EMBO J. 26, 4053–4065 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhang, X. et al. Membrane association and functional regulation of Sec3 by phospholipids and Cdc42. J. Cell Biol. 180, 145–158 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Baek, K. et al. Structure-function study of the N-terminal domain of exocyst subunit Sec3. J. Biol. Chem. 285, 10424–10433 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Songer, J.A. & Munson, M. Sec6p anchors the assembled exocyst complex at sites of secretion. Mol. Biol. Cell 20, 973–982 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Boyd, C., Hughes, T., Pypaert, M. & Novick, P. Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J. Cell Biol. 167, 889–901 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Katoh, Y., Nozaki, S., Hartanto, D., Miyano, R. & Nakayama, K. Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J. Cell Sci. 128, 2351–2362 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Terbush, D.R., Guo, W., Dunkelbarger, S. & Novick, P. Purification and characterization of yeast exocyst complex. Methods Enzymol. 329, 100–110 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. De Craene, J.O. et al. Rtn1p is involved in structuring the cortical endoplasmic reticulum. Mol. Biol. Cell 17, 3009–3020 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hsu, S.C. et al. Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 20, 1111–1122 (1998).

    Article  CAS  PubMed  Google Scholar 

  33. Oeffinger, M. et al. Rrp17p is a eukaryotic exonuclease required for 5′ end processing of Pre-60S ribosomal RNA. Mol. Cell 36, 768–781 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hakhverdyan, Z. et al. Rapid, optimized interactomic screening. Nat. Methods 12, 553–560 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Richman, D.D., Cleveland, P.H., Oxman, M.N. & Johnson, K.M. The binding of staphylococcal protein A by the sera of different animal species. J. Immunol. 128, 2300–2305 (1982).

    CAS  PubMed  Google Scholar 

  36. Oeffinger, M. et al. Comprehensive analysis of diverse ribonucleoprotein complexes. Nat. Methods 4, 951–956 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Bowser, R., Müller, H., Govindan, B. & Novick, P. Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis. J. Cell Biol. 118, 1041–1056 (1992).

    Article  CAS  PubMed  Google Scholar 

  38. Roth, D., Guo, W. & Novick, P. Dominant negative alleles of SEC10 reveal distinct domains involved in secretion and morphogenesis in yeast. Mol. Biol. Cell 9, 1725–1739 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wiederkehr, A., De Craene, J.O., Ferro-Novick, S. & Novick, P. Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p. J. Cell Biol. 167, 875–887 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Haarer, B.K. et al. SEC3 mutations are synthetically lethal with profilin mutations and cause defects in diploid-specific bud-site selection. Genetics 144, 495–510 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Nishimura, K. & Kanemaki, M.T. Rapid depletion of budding yeast proteins via the fusion of an auxin-inducible degron (AID). Curr. Protoc. Cell Biol. 64, 20.9 (2014).

    Google Scholar 

  43. Adamo, J.E. et al. Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud. J. Cell Biol. 155, 581–592 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hashizume, K., Cheng, Y.S., Hutton, J.L., Chiu, C.H. & Carr, C.M. Yeast Sec1p functions before and after vesicle docking. Mol. Biol. Cell 20, 4673–4685 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang, X. et al. Cdc42 interacts with the exocyst and regulates polarized secretion. J. Biol. Chem. 276, 46745–46750 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Donovan, K.W. & Bretscher, A. Myosin-V is activated by binding secretory cargo and released in coordination with Rab/exocyst function. Dev. Cell 23, 769–781 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Novick, P. & Schekman, R. Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 76, 1858–1862 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Govindan, B., Bowser, R. & Novick, P. The role of Myo2, a yeast class V myosin, in vesicular transport. J. Cell Biol. 128, 1055–1068 (1995).

    Article  CAS  PubMed  Google Scholar 

  49. Croteau, N.J., Furgason, M.L., Devos, D. & Munson, M. Conservation of helical bundle structure between the exocyst subunits. PLoS One 4, e4443 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wu, S., Mehta, S.Q., Pichaud, F., Bellen, H.J. & Quiocho, F.A. Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo. Nat. Struct. Mol. Biol. 12, 879–885 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Dong, G., Hutagalung, A.H., Fu, C., Novick, P. & Reinisch, K.M. The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif. Nat. Struct. Mol. Biol. 12, 1094–1100 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Yamashita, M. et al. Structural basis for the Rho- and phosphoinositide-dependent localization of the exocyst subunit Sec3. Nat. Struct. Mol. Biol. 17, 180–186 (2010).

    Article  CAS  PubMed  Google Scholar 

  53. Hamburger, Z.A., Hamburger, A.E., West, A.P. Jr. & Weis, W.I. Crystal structure of the S.cerevisiae exocyst component Exo70p. J. Mol. Biol. 356, 9–21 (2006).

    Article  CAS  PubMed  Google Scholar 

  54. Sivaram, M.V., Furgason, M.L.M., Brewer, D.N. & Munson, M. The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles. Nat. Struct. Mol. Biol. 13, 555–556 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Moskalenko, S. et al. Ral GTPases regulate exocyst assembly through dual subunit interactions. J. Biol. Chem. 278, 51743–51748 (2003).

    Article  CAS  PubMed  Google Scholar 

  56. Bodemann, B.O. et al. RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 144, 253–267 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Fendrych, M. et al. Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol. Biol. Cell 24, 510–520 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Murthy, M. et al. Sec6 mutations and the Drosophila exocyst complex. J. Cell Sci. 118, 1139–1150 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Lees, J.A., Yip, C.K., Walz, T. & Hughson, F.M. Molecular organization of the COG vesicle tethering complex. Nat. Struct. Mol. Biol. 17, 1292–1297 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ren, Y. et al. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1. Cell 139, 1119–1129 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Sikorski, R.S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Adamo, J.E., Rossi, G. & Brennwald, P. The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Mol. Biol. Cell 10, 4121–4133 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Perkins, E.M. & McCaffery, J.M. in Mitochondria 467–483 (Springer, 2007).

  64. Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

    Article  CAS  PubMed  Google Scholar 

  66. Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank P. Brennwald (University of North Carolina, Chapel Hill), C. Carr (Texas A&M University) and L. Weisman (University of Michigan) for antibodies, P. Novick (University of California, San Diego) for gifts of yeast strains, the Yeast Genome Resource Center in Japan for the AID-system reagents and the Wendland laboratory (Johns Hopkins University) for technical advice. Thanks to W. Holmes, M. Jacques, R. Kalia, L. Hassinger and members of the University of Massachusetts Medical School Core EM Facility for technical assistance. Thanks to R. Gilmore, S. Ryder, P. Pryciak, C. Carr and members of M.M.'s laboratory for critical reading of this manuscript and advice. Work in our laboratories is supported by US National Institutes of Health grants GM068803 (M.M. and A.F.), 1DP2GM110772 (A.F.), U54 GM103511 and P41 GM109824 (M.P.R.), and a Searle Scholars Award (A.F.).

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  1. Laura L Marcotte

    Present address: Present address: Department of Natural Sciences, Assumption College, Worcester, Massachusetts, USA.,

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Margaret R Heider, Caroline M Duffy, Anne M Mirza, Laura L Marcotte, Alexandra C Walls & Mary Munson

  2. Department of Biochemistry, University of Utah, Salt Lake City, Utah, USA

    Mingyu Gu, Nicholas Farrall & Adam Frost

  3. Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA

    Zhanna Hakhverdyan & Michael P Rout

  4. Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, UK

    Mark C Field

  5. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA

    Adam Frost

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Contributions

M.R.H. and M.M. conceived the study, designed the biochemical and cell biology experiments and wrote the manuscript; M.R.H. made yeast strains and performed most of the biochemistry and cell biology experiments with assistance from A.M.M., C.M.D., L.L.M. and A.C.W.; M.G. and A.F. designed, performed and analyzed the EM experiments; early EM optimization work was done by N.F.; development of the purification method was done by Z.H., C.M.D., M.P.R. and M.C.F.; all authors contributed to discussion and approved the final manuscript.

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Integrated supplementary information

Supplementary Figure 1 Characterization of purified yeast exocyst complexes.

(a) Yeast strains with C-terminally PrA-tagged exocyst subunits show normal growth compared to wild-type (WT) by serial dilution at all temperatures tested. PrA-only corresponds to yeast expressing PrA tag alone. (b) Lysate mixing reveals exocyst complexes are not disassembling and reassembling during purification. Sec3-PrA or Exo70-PrA lysate powders were each mixed individually with lysate powder from Sec10-GFP. Exocyst complexes were subsequently purified from the mixed lysates (after 60 min binding at 4°C in 20 mM PIPES pH 6.8, 300 mM KCl) and run on SDS-PAGE for Coomassie staining (left) and Western blot (right). GFP antibody also recognizes exocyst PrA tag. Asterisks indicate PrA-subunit on Coomassie gel. (c) Cryogenic ball mill grinding improves yield and complex integrity. Protein concentrations (mg/ml) were measured using BioRad protein assay and all beads were incubated with the same total protein in the same volume. Purified complexes were separated by SDS-PAGE and visualized by Coomassie staining. (d) Native elution from IgG-beads using a PreScission Protease site engineered between the C-terminus of each exocyst subunit and the PrA tag. Sec15-PrA tagged exocyst complexes bound to IgG-beads were incubated with PPX for 60 minutes at 4°C to elute native, intact complexes into buffer 20 mM PIPES pH 6.8, 300 mM NaCl (sup). IgG-beads were boiled in SDS/DTT loading buffer to release any undigested complexes (bead boil). Heavy chain of Rabbit IgG is indicated.

Supplementary Figure 2 Exocyst complexes purified under physiological conditions interact with known binding partners.

Exocyst complexes were purified from yeast lysate (using 50 mM Hepes pH 7.4, 150 mM NaCl as resuspension buffer), run on SDS-PAGE, and western blotted to look for co-purification of known exocyst interacting partners when compared to a negative control (PrA-expressing strain in (a) and GFP-PrA expressing strain in (b) and (c)). 0.5% lysate input samples were run for the Sec1 and Myo2 binding experiments and 0.4% input for the Snc binding blot. 100% of bound samples were used in all cases. (a) Sec8-PrA and Sec15-PrA were each used as purification handles to co- purify Snc. We blotted our pull-downs for Exo70 to show that we are pulling down assembled exocyst complexes with Sec8-PrA and Sec15-PrA. The rabbit antibody reacts with both Exo70 and the PrA tag. (b) Sec6-PrA was purified and Myo2 binding was detected. (c) Sec8-PrA was purified and Sec1 binding was detected. There was some bleed over of the GFP-PrA bound lane into the input lane of Sec8-PrA.

Supplementary Figure 3 Degradation of one exocyst subunit does not affect the protein levels of the remaining exocyst subunits.

Exocyst-AID strains were grown in YPD 30°C and treated with IAA for 60 minutes. Degradation of the AID-tagged exocyst subunit was confirmed by western blot of yeast lysates from NaOH/SDS lysis. The protein levels for the remaining subunits were blotted in the same strain (same column in the western blot). (–) denotes untreated and (+) treated with IAA. The positions of the untagged exocyst subunits are indicated to the left of the blots and the AID-tagged subunit is marked with (*). All lysates were blotted for ADH as a loading control.

Supplementary Figure 4 Investigating connectivity and assembly determinants by combining PrA purification with IAA-induced subunit depletion.

(a) We constructed yeast strains expressing double-tagged exocyst complexes: one subunit with a C-terminal AID-tag and another with a C-terminal PrA tag. All tags were integrated at the genomic loci under the endogenous promoter. Strains were serially diluted on standard YPD plates or YPD plates containing IAA and grown at 30°C. Exocyst complexes with both AID-tagged and PrA-tagged subunits are functional and yeast strains are inviable on IAA-containing YPD plates. (b) Western blot confirms composition of exocyst subcomplexes following depletion of individual subunits. Exocyst complexes were purified using the indicated PrA purification handle (blue) from yeast strains where one AID-tagged subunit (magenta) is degraded. Purified complexes were run on SDS-PAGE and visualized by Western blotting with antibodies specific to exocyst subunits (antibody indicated in inset box for each blot). (–) denotes untreated and (+) treated with IAA for 40 minutes. Exocyst subunits are denoted by their number. Magenta asterisks indicate the AID-tagged subunit, and green asterisks indicate the subunit whose co-purification is being monitored in that particular blot. Polyclonal antibodies also recognize PrA-tagged subunits, which in all cases is the band running higher than the subunit monitored (green asterisk), with the exception of Sec3, which runs above the PrA- tagged subunit.

Supplementary Figure 5 AID-tagged exocyst-binding partners are functional and have varying levels of growth and secretion defects in IAA-containing medium.

(a) N-terminally AID-tagged Cdc42, Snc2, and Sec4 combined with Sec8-PrA and OsTIR1 were tested for growth on YPD and YPD-IAA plates at 30°C relative to the wild-type (WT) parent strain (BY4742). AID- Sec4/Sec8-PrA demonstrated a mild growth defect on YPD plates and in liquid culture (data not shown); this growth defect was exacerbated slightly in the presence of IAA. AID-Snc2/ snc1Δ/Sec8-PrA showed a slight growth defect in the presence of IAA, and AID-Cdc42/Sec8-PrA was inviable on IAA plates. Sec1-AID/Sec8-PrA and Myo2-AID/Sec8-PrA showed no growth defects when compared to their parent strain (W303-1A) but were inviable on IAA plates. (b) Graphs depict the fold increase of internal Bgl2 levels in AID-tagged partner strains over internal Bgl2 levels in the appropriate WT untreated control strain. Sec1-AID and Sec6-AID showed severe secretion defects, while Myo2-AID and AID-Cdc42 showed minor defects consistent with previous reports43,44,48. AID-Snc and AID-Sec4 showed severe Bgl2 accumulation even before treatment, suggesting a partial loss of function due to the AID tag. Error bars indicate SEM for n=3-4 different treated or untreated yeast cultures. (c) Thin section EM confirms the vesicle accumulation defects observed in the Bgl2 assay. AID-Cdc42 cells also showed a loss of polarity and fewer budding cells. Scale bar=1 µm.

Supplementary Figure 6 The complete class gallery of the Sec15-GFP exocyst complexes.

Class averages with similar orientations are shown in the same row. Each row starts with the most populated class and ends with the least populated class. The number of particles per class is shown near the lower left corners. Although the last row is labeled V as a different class from the others, we cannot rule out that these 2D averages belong to class III and the flexible ends were averaged out.

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Supplementary Figures 1–6 and Supplementary Table 1 (PDF 1561 kb)

Supplementary Data Set 1

Full-size gels and western blots (PDF 18031 kb)

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Heider, M., Gu, M., Duffy, C. et al. Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex. Nat Struct Mol Biol 23, 59–66 (2016). https://doi.org/10.1038/nsmb.3146

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