In Vitro Analysis of Tem1 GTPase Activity and Regulation by the Bfa1/Bub2 GAP

  • Marco GeymonatEmail author
  • Adonis Spanos
  • Katrin Rittinger
Part of the Methods in Molecular Biology book series (MIMB, volume 1505)


Tem1 is a small GTPase that controls the mitotic progression of Saccharomyces cerevisiae through the Mitotic Exit Network. Tem1 activity is tightly controlled in mitosis by Bub2 and Bfa1 and is also regulated by the spindle orientation checkpoint that monitors the correct alignment of the mitotic spindle with the mother–daughter axis. In this chapter we describe the purification of Tem1, Bfa1, and Bub2 and a detailed radioactive filter-binding assay to study the nucleotide binding properties of Tem1 and the role of its regulators Bfa1 and Bub2.

Key words

Protein purification GTPase GAP Nucleotide binding assay 


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Marco Geymonat
    • 1
    Email author
  • Adonis Spanos
    • 2
  • Katrin Rittinger
    • 2
  1. 1.Department of GeneticsUniversity of CambridgeCambridgeUK
  2. 2.The Francis Crick InstituteLondonUnited Kingdom

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