In Vitro Analysis of Tem1 GTPase Activity and Regulation by the Bfa1/Bub2 GAP.
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.
Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK. mg604@cam.ac.uk.
Marco
M
Geymonat
The Francis Crick Institute, 1 Midland Road, London, NW1 1BF, United Kingdom.
Adonis
A
Spanos
The Francis Crick Institute, 1 Midland Road, London, NW1 1BF, United Kingdom.
Katrin
K
Rittinger
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D008938:Mitosis; D020559:Monomeric GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*
C119117:BFA1 protein, S cerevisiae; C070351:BUB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D009711:Nucleotides; D029701:Saccharomyces cerevisiae Proteins; C089965:TEM1 protein, S cerevisiae; D020559:Monomeric GTP-Binding Proteins
GAP; GTPase; Nucleotide binding assay; Protein purification