Using the Semi-synthetic Epitope System to Identify Direct Substrates of the Meiosis-Specific Budding Yeast Kinase, Mek1
Recent studies have shown that the meiosis-specific kinase, Mek1, plays a key role in promoting recombination between homologous chromosomes during meiosis in budding yeast by suppressing recombination between sister chromatids, as well as playing a role in the meiotic recombination checkpoint. Understanding how Mek1 regulates recombination requires the identification of direct substrates of the kinase. We have applied the semi-synthetic epitope method developed by Shokat and colleagues to Mek1. This method uses an analog-sensitive version of Mek1, GST-Mek1-as, in conjunction with an ATPγS analog, for kinase assays that detect only those proteins that are directly phosphorylated by Mek1. This method may be applicable to any kinase for which an analog-sensitive version is available. In addition, it provides a non-radioactive alternative for kinase assays with wild-type kinases.
Key wordsMeiosis Mek1 kinase assays Rad54 semi-synthetic epitope yeast
We thank Jasmina Allen, Kevan Shokat, and Beatrice Wang for help with ideas and reagents in the early development of this protocol. Patrick Sung generously provided bacterially purified Rad54 protein. Aaron Neiman provided helpful comments on the manuscript. This work was supported by an NIH grant to N. M. H. (R01 GM50717).
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