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Cell Cycle Oscillators

Volume 1342 of the series Methods in Molecular Biology pp 237-257

Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the γ-Tubulin Complex

  • Po Hien EarAffiliated withDépartement de Biochimie, Université de MontréalDepartment of Genetics, Harvard Medical School
  • , Jacqueline KowarzykAffiliated withDépartement de Biochimie, Université de Montréal
  • , Michael J. BoothAffiliated withDépartement de Biochimie, Université de MontréalMerton College, University of Oxford
  • , Diala Abd-RabboAffiliated withDépartement de Biochimie, Université de MontréalCentre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal
  • , Kristian ShulistAffiliated withDepartment of Biology, McGill University
  • , Conrad HallAffiliated withDepartment of Biology, McGill University
  • , Jackie VogelAffiliated withDepartment of Biology, McGill University Email author 
  • , Stephen W. MichnickAffiliated withDépartement de Biochimie, Université de MontréalCentre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal Email author 

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Abstract

Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the γ-tubulin complex as a substrate.

Key words

Yeast cyclin-dependent kinase (Cdc28 or Cdk1) Cyclin Optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA) Budding yeast γ-tubulin (Tub4)