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Exocyst function regulated by effector phosphorylation

Nature Cell Biology volume 13, pages 580–588 (2011)Cite this article

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Abstract

The exocyst complex tethers vesicles at sites of fusion through interactions with small GTPases. The G protein RalA resides on Glut4 vesicles, and binds to the exocyst after activation by insulin, but must then disengage to ensure continuous exocytosis. Here we report that, after recognition of the exocyst by activated RalA, disengagement occurs through phosphorylation of its effector Sec5, rather than RalA inactivation. Sec5 undergoes phosphorylation in the G-protein binding domain, allosterically reducing RalA interaction. The phosphorylation event is catalysed by protein kinase C and is reversed by an exocyst-associated phosphatase. Introduction of Sec5 bearing mutations of the phosphorylation site to either alanine or aspartate disrupts insulin-stimulated Glut4 exocytosis, as well as other trafficking processes in polarized epithelial cells and during development of zebrafish embryos. The exocyst thus serves as a ‘gatekeeper’ for exocytic vesicles through a circuit of engagement, disengagement and re-engagement with G proteins.

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Figure 1: RalA promotes exocyst function independently of GTP hydrolysis.
Figure 2: The RalA-interacting exocyst subunit Sec5 undergoes hormone-stimulated phosphorylation in its effector domain.
Figure 3: Sec5 effector-domain phosphorylation disengages the G protein RalA during vesicle targeting.
Figure 4: PKC catalyses Sec5 effector-domain phosphorylation, which negatively regulates RalA interaction.
Figure 5: Sec5 phosphorylation contributes to a cyclic regulatory loop on recognition by RalA.
Figure 6: Sec5 phosphorylation contributes to cellular organization and organism development.
Figure 7: Proposed model describing the role of the cyclic phosphorylation of Sec5 in an engagement–disengagement cycle between the exocyst and its upstream G protein.

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Acknowledgements

The work is supported by NIH RO1 DK061618 and DK076906 to A.R.S., and P60 DK020572 to the Diabetes Research and Training Center at the University of Michigan. D.G. is an investigator of Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Life Sciences Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA

    Xiao-Wei Chen, Dara Leto, Junyu Xiao, Jordan A. Shavit, Tingting Xiong, Genggeng Yu, David Ginsburg, Zhaohui Xu & Alan R. Saltiel

  2. Department of Molecular and Integrative Physiology, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA

    Xiao-Wei Chen, Tingting Xiong & Alan R. Saltiel

  3. Program of Cellular and Molecular Biology, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA

    Dara Leto & Alan R. Saltiel

  4. Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA

    Junyu Xiao, Qian Wang & Zhaohui Xu

  5. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA

    John Goss & Derek Toomre

  6. Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA

    Jordan A. Shavit, David Ginsburg & Alan R. Saltiel

  7. Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA

    Jordan A. Shavit & David Ginsburg

  8. Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA

    David Ginsburg

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Contributions

X.W.C., D.L., J.X., J.G., Q.W., T.X. and G.Y. carried out experiments; X.W.C., J.A.S., D.G., D.T., Z.X. and A.R.S. analysed the data; X.W.C., Q.W., D.G., D.T., Z.X. and A.R.S. designed experiments and wrote the manuscript.

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Correspondence to Alan R. Saltiel.

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Chen, XW., Leto, D., Xiao, J. et al. Exocyst function regulated by effector phosphorylation. Nat Cell Biol 13, 580–588 (2011). https://doi.org/10.1038/ncb2226

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