Sds22p is a subunit of a stable isolatable form of protein phosphatase 1 (Glc7p) from Saccharomyces cerevisiae

Protein phosphatase 1 (PP1) is one of the major protein phosphatases in euk aryotic cells. PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits, The essential g ene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae. In this study, full-length GLC7(1-312), C-terminal deletion mutants, and C-ter minally poly-his tagged mutants were constructed and expressed in a GLC7 kn ockout strain of S, cerevisiae, Viability studies of the GLC7 knockout stra ins carrying the plasmids expressing GLC7 C-terminal deletion mutants and t heir 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 wi th and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni2+ affinity chromatography and further analyzed by gel fil tration and ion exchange chromatography, Phosphatase activity assays, SDS-P AGE, and Western blot analyses of the chromatographic fractions suggested t hat the Glc7p associated with regulatory subunits in vivo, A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedure s, Monoclonal antibody against the HA tag coimmunoprecipitated the tagged G lc7p and the 40-kDa protein, This protein was further purified by a reverse phase HPLC column, Analysis by CNBr digestion, peptide sequencing, and ele ctrospray 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. (C) 2000 Academic Press.