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Yeast Cytokinesis pp 205–218Cite as

Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1369))

Abstract

Rho-type small GTPases are involved in cytokinesis in various organisms, but their precise roles and regulation remain unclear. Rho proteins function as molecular switches by cycling between the active GTP-bound and inactive GDP-bound states; the GTP-bound proteins in turn interact with their downstream effectors to transmit the signal. Biochemical assays using Rho-binding domains of effector proteins have been used to specifically pull down GTP-bound Rho proteins from cell extracts. Here, we describe the application of such a method in combination with cell-cycle synchronization in the budding yeast Saccharomyces cerevisiae; this approach allows dissection of the activity of Rho1 at different stages of cytokinesis. We also present data showing the importance of caution in interpreting such biochemical data and of comparing to the results obtained with other approaches where possible. The principle of this protocol is also applicable to analyses of other Rho-type GTPases and cell-cycle events.

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Acknowledgments

We thank Keiko Kono and Satoshi Yoshida for their helpful advice in developing this protocol. This work was supported in part by grants to JRP from the National Institutes of Health (GM31006) and RJEG Foundation and a postdoctoral fellowship to MO from the Uehara Memorial Foundation.

Author information

Authors and Affiliations

  1. Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94025, USA

    Masayuki Onishi

  2. Department of Genetics, Stanford University School of Medicine, MC 5120, 300 Pasteur Drive, M-322 Alway Building, Stanford, CA, 94305-5120, USA

    John R. Pringle

Authors
  1. Masayuki Onishi
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  2. John R. Pringle
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Corresponding author

Correspondence to Masayuki Onishi .

Editor information

Editors and Affiliations

  1. Departamento de Biología Molecular, Universidad de Cantabria, Facultad de Medicina, Cardenal Herrera Oria s/n, Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria, CSIC, Santander, Spain

    Alberto Sanchez-Diaz

  2. Universidad de Salamanca, Instituto de Biología Funcional y Genómica, CSIC, Salamanca, Spain

    Pilar Perez

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Onishi, M., Pringle, J.R. (2016). Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis. In: Sanchez-Diaz, A., Perez, P. (eds) Yeast Cytokinesis. Methods in Molecular Biology, vol 1369. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3145-3_15

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  • DOI: https://doi.org/10.1007/978-1-4939-3145-3_15

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3144-6

  • Online ISBN: 978-1-4939-3145-3

  • eBook Packages: Springer Protocols

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