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Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis.

Publication ,  Chapter
Onishi, M; Pringle, JR
January 2016

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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DOI

Publication Date

January 2016

Volume

1369

Start / End Page

205 / 218

Related Subject Headings

  • rho GTP-Binding Proteins
  • Saccharomycetales
  • Recombinant Fusion Proteins
  • Protein Binding
  • Developmental Biology
  • Cytokinesis
  • 3404 Medicinal and biomolecular chemistry
  • 3101 Biochemistry and cell biology
  • 0601 Biochemistry and Cell Biology
  • 0399 Other Chemical Sciences
 

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Onishi, M., & Pringle, J. R. (2016). Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis. (Vol. 1369, pp. 205–218). https://doi.org/10.1007/978-1-4939-3145-3_15
Onishi, Masayuki, and John R. Pringle. “Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis.,” 1369:205–18, 2016. https://doi.org/10.1007/978-1-4939-3145-3_15.
Onishi M, Pringle JR. Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis. In 2016. p. 205–18.
Onishi, Masayuki, and John R. Pringle. Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis. Vol. 1369, 2016, pp. 205–18. Epmc, doi:10.1007/978-1-4939-3145-3_15.

DOI

Publication Date

January 2016

Volume

1369

Start / End Page

205 / 218

Related Subject Headings

  • rho GTP-Binding Proteins
  • Saccharomycetales
  • Recombinant Fusion Proteins
  • Protein Binding
  • Developmental Biology
  • Cytokinesis
  • 3404 Medicinal and biomolecular chemistry
  • 3101 Biochemistry and cell biology
  • 0601 Biochemistry and Cell Biology
  • 0399 Other Chemical Sciences