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Spatial regulation of the exocyst complex by Rho1 GTPase

Abstract

Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis. The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane. One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis. We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae. Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.

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Figure 1: A screen for mutants that affect the polarized localization of the exocyst protein in the budding yeast S. cerevisiae.
Figure 2: Comparison of the localization of Sec3, Sec4, actin and Myo2 in wild-type and various alleles of rho1 mutants.
Figure 3: Sec3–GFP is mislocalized in rho1-2 and rho1-5 mutant cells recovering from G0 arrest at 37 °C.
Figure 4: The interaction of Sec3 and Rho GTPase by the yeast two-hybrid assay.
Figure 5: In vitro binding between Sec3 and Rho1.
Figure 6: Different domains of Sec3 mediate its interaction with Rho1 and the exocyst proteins.
Figure 7: Localization of Sec3 with an N-terminal deletion (Sec3ΔN–GFP) in wild-type cells.
Figure 8: Localization and protein binding of Sec3ΔN–GFP as the sole copy in the cell.
Figure 9: Rho1 coordinates various cellular activities through different effectors for polarized cell growth in the budding yeast S. cerevisiae.

References

  1. 1

    Drubin, D. G. & Nelson, W. J. Origins of cell polarity. Cell 84, 335–344 (1996).

    CAS  Article  Google Scholar 

  2. 2

    Lew, D. J. & Reed, S. I. Cell cycle control of morphogenesis in budding yeast. Curr. Opin. Genet. Dev. 5, 17–23 (1995).

    CAS  Article  Google Scholar 

  3. 3

    Madden, K. & Snyder, M. Cell polarity and morphogenesis in budding yeast. Annu. Rev. Microbiol. 52, 687–744 (1998).

    CAS  Article  Google Scholar 

  4. 4

    Chant, J. Cell polarity in yeast. Annu. Rev. Cell. Dev. Biol. 15, 365–391 (1999).

    CAS  Article  Google Scholar 

  5. 5

    Finger, F. P. & Novick, P. J. Spatial regulation of exocytosis: lessons from yeast. J. Cell Biol. 142, 609–612 (1998).

    CAS  Article  Google Scholar 

  6. 6

    Pruyne, D. W., Schott, D. H. & Bretscher, A. Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast. J. Cell Biol. 143, 1931–1945 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Schott, D., Ho, J., Pruyne, D. & Bretscher, A. The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting. J. Cell Biol. 147, 791–808 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Govindin, 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  Google Scholar 

  9. 9

    Karpova, T. S. et al. Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae. Mol. Biol. Cell 11, 1727–1737 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Pfeffer, S. R. Transport-vesicle targeting: tethers before SNAREs. Nature Cell Biol. 1, E17–E22 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S. & Novick, P. Protein complexes in transport vesicle targeting. Trends Cell Biol. 10, 251–255 (2000).

    CAS  Article  Google Scholar 

  12. 12

    Rothman, J. E. Mechanisms of intracellular protein transport. Nature 372, 55–63 (1994).

    CAS  Article  Google Scholar 

  13. 13

    Novick, P. & Zerial, M. The diversity of Rab proteins in vesicle transport. Curr. Opin. Cell Biol. 9, 496–504 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Lazar, T., Gotte, M. & Gallwitz, D. Vesicular transport: how many Ypt/Rab-GTPases make a eukaryotic cell? Trends Biochem. Sci. 22, 468–472 (1997).

    CAS  Article  Google Scholar 

  15. 15

    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).

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

    Finger, F. P., Hughes, T. E. & Novick, P. Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92, 559–571 (1998).

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

    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).

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

    Brennwald, P. et al. Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell 79, 245–258 (1994).

    CAS  Article  Google Scholar 

  22. 22

    Hazuka, C. D. et al. The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains. J. Neurosci. 19, 1324–1334 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Grindstaff, K. K. et al. Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93, 731–740 (1998).

    CAS  Article  Google Scholar 

  24. 24

    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).

    CAS  Article  Google Scholar 

  25. 25

    Carr, C. M., Grote, E., Munson, M., Hughson, F. M. & Novick, P. J. Sec1p binds to SNARE complexes and concentrates at sites of secretion. J. Cell Biol. 146, 333–344 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Hall, A. Rho GTPases and the actin cytoskeleton. Science 279, 509–514 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Cabib, E., Drgonova, J. & Drgon, T. Role of small G proteins in yeast cell polarization and wall biosynthesis. Annu. Rev. Biochem. 67, 307–333 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Schmidt, A. & Hall, M. N. Signaling to the actin cytoskeleton. Annu. Rev. Cell Dev. Biol. 14, 305–338 (1998).

    CAS  Article  Google Scholar 

  29. 29

    Helliwell, S. B., Schmidt, A., Ohya, Y. & Hall, M. N. The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton. Curr. Biol. 8, 1211–1214 (1998).

    CAS  Article  Google Scholar 

  30. 30

    Ayscough, K. R. et al. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J. Cell Biol. 137, 399–416 (1997); erratum ibid. 146, 1201 (1999).

    CAS  Article  Google Scholar 

  31. 31

    Nonaka, H. et al. A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J. 14, 5931–5938 (1995).

    CAS  Article  Google Scholar 

  32. 32

    Drgonova, J., Drgon, T., Roh, D. H. & Cabib, E. The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell. J. Cell Biol. 146, 373–387 (1999).

    CAS  Article  Google Scholar 

  33. 33

    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  Google Scholar 

  34. 34

    Yamochi, W. et al. Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae. J. Cell Biol. 125, 1077–1093 (1994).

    CAS  Article  Google Scholar 

  35. 35

    Novick, P. & Botstein, D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell 40, 405–416 (1985).

    CAS  Article  Google Scholar 

  36. 36

    Robinson, N. G. 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).

    CAS  Article  Google Scholar 

  37. 37

    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).

    CAS  Article  Google Scholar 

  38. 38

    Kim, S. K. Cell polarity: new PARtners for Cdc42 and Rac. Nature Cell Biol. 2, E143–E145 (2000).

    CAS  Article  Google Scholar 

  39. 39

    Kroschewski, R., Hall, A. & Mellman, I. Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells. Nature Cell Biol. 1, 8–13 (1999).

    CAS  Article  Google Scholar 

  40. 40

    Lin, D. et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nature Cell Biol. 2, 540–547 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Joberty, G., Petersen, C., Gao, L. & Macara, I. G. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nature Cell Biol. 2, 531–539 (2000).

    CAS  Article  Google Scholar 

  42. 42

    Braga, V. M., Machesky, L. M., Hall, A. & Hotchin, N. A. The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J. Cell Biol. 137, 1421–1431 (1997).

    CAS  Article  Google Scholar 

  43. 43

    Reck-Peterson, S. L., Novick, P. J. & Mooseker, M. S. The tail of a yeast class V myosin, myo2p, functions as a localization domain. Mol. Biol. Cell 10, 1001–1017 (1999).

    CAS  Article  Google Scholar 

  44. 44

    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).

    CAS  Article  Google Scholar 

  45. 45

    Kohno, H. et al. Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO J. 15, 6060–6068 (1996).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank N. Robinson for sharing preliminary results; Y. Takai, Y. Ohya, M. Hall, M. Snyder and E. Cabib for providing us with reagents; and P. De Camilli, I. Mellman, L. Du, E. Grote and C. Carr for constructive discussions. W.G. was supported by Brown–Cox and NIH postdoctoral fellowships. This study was supported by a grant to P.N. from the NIH and in part to a grant to F. T. from the NIH.

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Correspondence to Peter Novick.

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Guo, W., Tamanoi, F. & Novick, P. Spatial regulation of the exocyst complex by Rho1 GTPase. Nat Cell Biol 3, 353–360 (2001). https://doi.org/10.1038/35070029

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