Advertisement

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

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

Abstract

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 

References

  1. 1.
    Rock JM, Amon A (2011) Cdc15 integrates Tem1 GTPase-mediated spatial signals with Polo kinase-mediated temporal cues to activate mitotic exit. Genes Dev 25(18):1943–1954. doi: 10.1101/gad.17257711 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Geymonat M, Spanos A, Smith SJ, Wheatley E, Rittinger K, Johnston LH, Sedgwick SG (2002) Control of mitotic exit in budding yeast. In vitro regulation of Tem1 GTPase by Bub2 and Bfa1. J Biol Chem 277(32):28439–28445CrossRefPubMedGoogle Scholar
  3. 3.
    Kabcenell AK, Goud B, Northup JK, Novick PJ (1990) Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic. J Biol Chem 265(16):9366–9372PubMedGoogle Scholar
  4. 4.
    Leonard DA, Evans T, Hart M, Cerione RA, Manor D (1994) Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry 33(40):12323–12328CrossRefPubMedGoogle Scholar
  5. 5.
    Neal SE, Eccleston JF, Hall A, Webb MR (1988) Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism. J Biol Chem 263(36):19718–19722PubMedGoogle Scholar
  6. 6.
    Neuwald AF (1997) A shared domain between a spindle assembly checkpoint protein and Ypt/Rab-specific GTPase-activators. Trends Biochem Sci 22(7):243–244CrossRefPubMedGoogle Scholar
  7. 7.
    Geymonat M, Spanos A, de Bettignies G, Sedgwick SG (2009) Lte1 contributes to Bfa1 localization rather than stimulating nucleotide exchange by Tem1. J Cell Biol 187(4):497–511CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Caydasi AK, Pereira G (2012) SPOC alert—when chromosomes get the wrong direction. Exp Cell Res 318(12):1421–1427. doi: 10.1016/j.yexcr.2012.03.031 CrossRefPubMedGoogle Scholar
  9. 9.
    Geymonat M, Spanos A, Walker PA, Johnston LH, Sedgwick SG (2003) In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5. J Biol Chem 278(17):14591–14594. doi: 10.1074/jbc.C300059200C300059200[pii] CrossRefPubMedGoogle Scholar
  10. 10.
    Hu F, Wang Y, Liu D, Li Y, Qin J, Elledge SJ (2001) Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints. Cell 107(5):655–665CrossRefPubMedGoogle Scholar
  11. 11.
    Kim J, Luo G, Bahk YY, Song K (2012) Cdc5-dependent asymmetric localization of bfa1 fine-tunes timely mitotic exit. PLoS Genet 8(1):e1002450. doi: 10.1371/journal.pgen.1002450 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Maekawa H, Priest C, Lechner J, Pereira G, Schiebel E (2007) The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal. J Cell Biol 179(3):423–436CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Caydasi AK, Pereira G (2009) Spindle alignment regulates the dynamic association of checkpoint proteins with yeast spindle pole bodies. Dev Cell 16(1):146–156CrossRefPubMedGoogle Scholar
  14. 14.
    Fraschini R, D’Ambrosio C, Venturetti M, Lucchini G, Piatti S (2006) Disappearance of the budding yeast Bub2-Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit. J Cell Biol 172(3):335–346CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Valerio-Santiago M, Monje-Casas F (2011) Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function. J Cell Biol. doi: jcb.201007044 [pii]Google Scholar
  16. 16.
    Scarfone I, Venturetti M, Hotz M, Lengefeld J, Barral Y, Piatti S (2015) Asymmetry of the budding yeast Tem1 GTPase at spindle poles is required for spindle positioning but not for mitotic exit. PLoS Genet 11(2):e1004938. doi: 10.1371/journal.pgen.1004938 CrossRefPubMedPubMedCentralGoogle Scholar

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

Personalised recommendations