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.
Keywords:
GAP; GTPase; Nucleotide binding assay; Protein purification.
MeSH terms
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Cell Cycle
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Cell Cycle Proteins / isolation & purification
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Cell Cycle Proteins / metabolism*
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Cytoskeletal Proteins / isolation & purification
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Cytoskeletal Proteins / metabolism*
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Enzyme Assays / methods
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Mitosis
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Monomeric GTP-Binding Proteins / isolation & purification
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Monomeric GTP-Binding Proteins / metabolism*
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Nucleotides / metabolism
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Protein Binding
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Saccharomyces cerevisiae / cytology*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / isolation & purification
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Saccharomyces cerevisiae Proteins / metabolism*
Substances
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BFA1 protein, S cerevisiae
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BUB2 protein, S cerevisiae
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Cell Cycle Proteins
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Cytoskeletal Proteins
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Nucleotides
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Saccharomyces cerevisiae Proteins
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TEM1 protein, S cerevisiae
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Monomeric GTP-Binding Proteins