Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits. The essential gene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae. In this study, full-length GLC7(1-312), C-terminal deletion mutants, and C-terminally poly-his tagged mutants were constructed and expressed in a GLC7 knockout strain of S. cerevisiae. Viability studies of the GLC7 knockout strains carrying the plasmids expressing GLC7 C-terminal deletion mutants and their tagged forms showed that the mutants 1-295 and 1-304 were functional, whereas the mutant 1-245 was not. The C-terminally poly-his tagged Glc7p with and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni(2+) affinity chromatography and further analyzed by gel filtration and ion exchange chromatography. Phosphatase activity assays, SDS-PAGE, and Western blot analyses of the chromatographic fractions suggested that the Glc7p associated with regulatory subunits in vivo. A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedures. Monoclonal antibody against the HA tag coimmunoprecipitated the tagged Glc7p and the 40-kDa protein. This protein was further purified by a reverse phase HPLC column. Analysis by CNBr digestion, peptide sequencing, and electrospray mass spectrometry showed that this 40-kDa protein is Sds22p, one of the proteins proposed to be a regulatory subunit of Glc7. These results demonstrate that Sds22p forms a complex with Glc7p and that Sds22p:Glc7p is a stable isolatable form of yeast PP1.

• PMID: 10775415
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Hong G, Trumbly RJ, Reimann EM, Schlender KK

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