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Structural basis for the Rho- and phosphoinositide-dependent localization of the exocyst subunit Sec3

Nature Structural & Molecular Biology volume 17, pages 180–186 (2010)Cite this article

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Abstract

The exocyst complex is a hetero-octameric protein complex that functions during cell polarization by tethering the secretory vesicle to the target membrane. The yeast exocyst subunit Sec3 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and the small GTPases Rho1 and Cdc42 via its N-terminal domain (Sec3-N), and these interactions target Sec3 to the plasma membrane. Here we report the crystal structure of the Sec3-N in complex with Rho1 at 2.6-Å resolution. Sec3-N adopts a pleckstrin homology (PH) fold, despite having no detectable sequence homology with other PH domains of known structure. Clusters of conserved basic residues constitute a positively charged cleft, which was identified as a binding site for PtdIns(4,5)P2. Residues Phe77, Ile115 and Leu131 of Sec3 bind to an extended hydrophobic surface formed around switch regions I and II of Rho1. To our knowledge, these are the first structural insights into how an exocyst subunit might interact with both protein and phospholipid factors on the target membrane.

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Figure 1: Overall structure of yeast Sec3-N in complex with the GppNHp-bound Rho1.
Figure 2: Interactions between Sec3-N and Rho1.
Figure 3: Interactions between Sec3-N and PtdIns(4,5)P2.
Figure 4: Conformational switch of Rho1.
Figure 5: In vivo localization of yeast Sec3 mutants.
Figure 6: The membrane attachment model between Sec3-N and the plasma membrane.

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Acknowledgements

We thank C. Toyoshima for support of this research, S. Kaiser for critical reading and improvement of this manuscript, and the beamline staffs at PFAR-NW12A and BL-5A of PF (Tsukuba, Japan) and BL41XU of SPring8 (Hyogo, Japan) for technical help during data collection. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology to S.F., A.Yamagata and H.M. Y.S. and M.Y. are supported by Japan Society for the Promotion of Science research fellowships for young scientists.

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Authors and Affiliations

  1. Life Science Division, Structural Biology Laboratory, Synchrotron Radiation Research Organization and Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan

    Masami Yamashita, Yusuke Sato, Atsushi Yamagata, Hisatoshi Mimura, Azusa Yoshikawa & Shuya Fukai

  2. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan

    Masami Yamashita, Yusuke Sato, Atsushi Yamagata & Shuya Fukai

  3. Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan

    Kazuo Kurokawa & Akihiko Nakano

  4. Department of Biological Information, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Yokohama, Japan

    Azusa Yoshikawa

  5. Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan.,

    Ken Sato

  6. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.,

    Ken Sato & Akihiko Nakano

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Contributions

M.Y. carried out sample preparation, crystallization, structure determination and in vitro binding assays; Y.S., A.Yoshikawa, H.M., A.Yamagata and S.F. assisted with these experiments; K.K. carried out in vivo localization experiments using yeast transformants prepared by M.Y.; M.Y. and S.F. wrote the paper; S.F. designed the research with advice from K.S. and A.N.; all authors discussed the results and commented on the manuscript; S.F. supervised the work.

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Correspondence to Shuya Fukai.

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Yamashita, M., Kurokawa, K., Sato, Y. et al. Structural basis for the Rho- and phosphoinositide-dependent localization of the exocyst subunit Sec3. Nat Struct Mol Biol 17, 180–186 (2010). https://doi.org/10.1038/nsmb.1722

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