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-Å and 2.85-Å resolution, respectively. Exo70p forms a 160-Å-long rod with a novel fold composed of contiguous α-helical bundles. The Exo84p C terminus also forms a long rod (80 Å), 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.
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References
Rothman, J.E. Lasker basic medical research award. The machinery and principles of vesicle transport in the cell. Nat. Med. 8, 1059–1062 (2002).
Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S. & Novick, P. Protein complexes in transport vesicle targeting. Trends Cell Biol. 10, 251–255 (2000).
Whyte, J.R. & Munro, S. Vesicle tethering complexes in membrane traffic. J. Cell Sci. 115, 2627–2637 (2002).
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).
Guo, W., Grant, A. & Novick, P. Exo84p is an exocyst protein essential for secretion. J. Biol. Chem. 274, 23558–23564 (1999).
Walch-Solimena, C., Collins, R.N. & Novick, P.J. Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles. J. Cell Biol. 137, 1495–1509 (1997).
Grote, E., Carr, C.M. & Novick, P.J. Ordering the final events in yeast exocytosis. J. Cell Biol. 151, 439–452 (2000).
Kee, Y. et al. Subunit structure of the mammalian exocyst complex. Proc. Natl. Acad. Sci. USA 94, 14438–14443 (1997).
Matern, H.T., Yeaman, C., Nelson, W.J. & Scheller, R.H. The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells. Proc. Natl. Acad. Sci. USA 98, 9648–9653 (2001).
Yeaman, C., Grindstaff, K.K., Wright, J.R. & Nelson, W.J. Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells. J. Cell Biol. 155, 593–604 (2001).
Bowser, R., Muller, 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).
Finger, F.P., Hughes, T.E. & Novick, P. Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92, 559–571 (1998).
Guo, W., Tamanoi, F. & Novick, P. Spatial regulation of the exocyst complex by Rho1 GTPase. Nat. Cell Biol. 3, 353–360 (2001).
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).
Finger, F.P. & Novick, P. Spatial regulation of exocytosis: lessons from yeast. J. Cell Biol. 142, 609–612 (1998).
Zhang, X. et al. Cdc42 interacts with the exocyst and regulates polarized secretion. J. Biol. Chem. 276, 46745–46750 (2001).
Robinson, N.G. et al. Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol. Cell. Biol. 19, 3580–3587 (1999).
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).
Inoue, M., Chang, L., Hwang, J., Chiang, S.H. & Saltiel, A.R. The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422, 629–633 (2003).
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).
Whyte, J.R. & Munro, S. The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic. Dev. Cell 1, 527–537 (2001).
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).
Vega, I.E. & Hsu, S.C. The exocyst complex associates with microtubules to mediate vesicle targeting and neurite outgrowth. J. Neurosci. 21, 3839–3848 (2001).
Jin, R. et al. Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase. EMBO J. 24, 2064–2074 (2005).
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).
Mott, H.R. et al. Structure of the GTPase-binding domain of Sec5 and elucidation of its Ral binding site. J. Biol. Chem. 278, 17053–17059 (2003).
Fukai, S., Matern, H.T., Jagath, J.R., Scheller, R.H. & Brunger, A.T. Structural basis of the interaction between RalA and Sec5, a subunit of the sec6/8 complex. EMBO J. 22, 3267–3278 (2003).
Doublie, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).
Otwinowski, Z. & Minor, W. Processing X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Brunger, A.T. et al. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).
Kleywegt, G.J. & Jones, T.A. Model building and refinement practice. Methods Enzymol. 277, 208–230 (1997).
Kraulis, P.J. MolScript: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).
Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).
Kleywegt, G.J. & Jones, T.A. Detecting folding motifs and similarities in protein structures. Methods Enzymol. 277, 525–545 (1997).
Acknowledgements
We are grateful to the staffs at beamlines X25 and X29 at Brookhaven National Laboratory and at the NE-CAT beamline 8BM at the APS for their help in collecting data. P. Patel was helpful in the initial characterization of Exo70p. We are very grateful to D.W. Rodgers and G. Warren for discussion and their comments regarding this manuscript, to M. Munson for allowing us to cite her structure of Sec6p and to F. Quiocho for sharing with us the manuscript describing the Sec15 structure. K.M.R. is supported by funds from the G. Harold and Leila Y. Mathers Foundation and the Pew Charitable Trust, P.N. is funded by grant GM35370 from the US National Institutes of Health and G.D. and A.H.H. have been supported by fellowships from the American Heart Foundation and the Anna Fuller Fund, respectively.
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Supplementary information
Supplementary Fig. 1
The carboxy-terminal fragment of Sec15 is composed of helical modules strung into a rod, like Exo70p. (PDF 948 kb)
Supplementary Fig. 2
Sequence alignment for the Exo70p C-terminus. (PDF 250 kb)
Supplementary Fig. 3
Sequence alignment of the S. cerevisiae (Sc) Exo84p carboxy-terminal domains. (PDF 446 kb)
Supplementary Table 1
Exo70p truncation constructs (PDF 34 kb)
Supplementary Table 2
Secondary structure and coiled-coil prediction for S. cerevisiae exocyst subunits (PDF 94 kb)
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Dong, G., Hutagalung, A., Fu, C. et al. The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif. Nat Struct Mol Biol 12, 1094–1100 (2005). https://doi.org/10.1038/nsmb1017
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DOI: https://doi.org/10.1038/nsmb1017
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