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)
The original version of this chapter was revised. The erratum to this chapter is available at: DOI 10.1007/978-1-4939-2957-3_22
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Ear, P.H. et al. (2016). 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. In: Coutts, A., Weston, L. (eds) Cell Cycle Oscillators. Methods in Molecular Biology, vol 1342. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2957-3_14
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DOI: https://doi.org/10.1007/978-1-4939-2957-3_14
Publisher Name: Humana Press, New York, NY
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