Abstract
Publisher Summary This chapter describes yeast phosphoproteome analysis using the specific example of the effects of the Reg1p regulatory subunit of Glc7p (PP-1C) on the yeast phosphoproteome. The chapter presents genomics and proteomics linked in order to determine the direct effects of a given gene product. The availability of yeast deletion mutants makes it possible to perform experiments that characterize the effects of these mutations on the proteome. The methods outlined in the chapter provide a guide in establishing the functional importance of genes and their products on the phosphoproteome of yeast. Mixed peptide Edman sequencing conclusively identified the proteins as Hxk2 and the Elα subunit of pyruvate dehydrogenase, and genetic studies demonstrated that Hxk2 dephosphorylation by Glc7p is regulated by Reg1p. This approach can easily be extended to characterizing substrates of other phosphatases (or kinases) in yeast, and demonstrates the utility of a functional proteomics approach coupled with functional genetics to directly evaluate the effects of a given gene/protein overall proteome. Furthermore, mixed peptide sequencing in conjunction with mass spectrometry, greatly extends the sensitivity of this method.
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