IMPROV METHODS:STRUCT ANALYS OF PROTEIN PRENYL GROUPS & ASSOCD C TERMINAL MODIFI

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We would like to look at proteins associated with various cyclin-Cdc28 complexes, starting with Clb2-Cdc28. We have made and tested a tagging cassette that allows efficient affinity purification and immuno-precipitation of tagged proteins. We have tagged Clb2, and after affinity purification of Clb2 from less than 300 ml of cells, we can see by silver staining Clb2, Cdc28, two other unknown but specifically copurifying sub-stochiometric proteins, and just one non-specific protein (as judged by comparison with the same purification from an untagged control strain). All these proteins are well separated on standard SDS-PAGE gels, and there is little background staining. We are presently doing the experiment on a larger scale, and we are doing it in both a CDC28 background, and in a strain that contains a kinase-dead, over-expressed Cdc28 as well as wild-type Cdc28. The idea of this is that a Clb2-(kinase dead Cdc28) complex might hold on to substrates, and there is some indirect support for this from two-hybrid experiments. Assuming we get specific bands, we would like to silver stain, cut the specific bands out of the gel, and submit them for identification by mass spectrometry. There would be three conditions: (1) control purification from an untagged strain; (2) experimental purification from a tagged Clb2, CDC28 strain; (3) experimental purification from a tagged Clb2, CDC28, cdc28-kinase dead strain. Potential substrates would presumably be specific to the third condition, while other kinds of associated proteins might be found in both the second and third conditions.