Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry.
The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing.
Walsh
Edmund P
EP
School of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH, UK.
Lamont
Douglas J
DJ
Beattie
Kenneth A
KA
Stark
Michael J R
MJ
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013205:Staphylococcal Protein A / Q000235:genetics; D039221:mRNA Cleavage and Polyadenylation Factors
D005656:Fungal Proteins; C102423:MHP1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; D039221:mRNA Cleavage and Polyadenylation Factors; D010749:Phosphoprotein Phosphatases; D010450:Endopeptidases; C099488:TEV protease