Novel Interactions of Saccharomyces cerevisiae Type 1 Protein Phosphatase Identified by Single-Step Affinity Purification and Mass Spectrometry

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• 作者：Edmund P. Walsh ; Douglas J. Lamont ; Kenneth A. Beattie ; and Michael J. R. Stark
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：February 19, 2002
• 年：2002
• 卷：41
• 期：7
• 页码：2409 - 2420
• 全文大小：245K
• 年卷期：v.41,no.7(February 19, 2002)
• ISSN：1520-4995

文摘

The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1_C) is encodedby the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potentialregulatory or targeting subunits of yeast PP1_C, we tagged Glc7p at its amino terminus with protein A andaffinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide massfingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Toconfirm the accuracy of our identifications, peptides from some of the proteins were also sequenced usinghigh-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four ofthe Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previouslybeen shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylationfactor) complex involved in messenger RNA 3'-end processing were present as major components in theGlc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used thesame approach to purify and characterize the components of the yeast CPF complex using protein A-taggedPta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified amongthe Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel componentof the CPF complex and may therefore be involved regulating mRNA 3'-end processing.