Purification and characterization of type 1 protein phosphatase from Saccharomyces cerevisiae: effect of the R73C...

Type 1 protein phosphatase encoded by the GLC7 gene was purified from Saccharomyces cerevisiae as a 1:1 complex with mammalian inhibitor 2 fused to glutathione S-transferase. The complex was inactive and required treatment with Co2+ and trypsin for maximal activity. The specific activity toward phosphorylase a was about 1.8 units/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 7.3, 81, and 0.30 nM, respectively. The complex could be activated by glycogen synthase kinase-3 in the presence of Mg2+ and ATP to 20% of the activity seen with Co2+ and trypsin. Thus, the catalytic properties of the yeast type 1 phosphatase are similar to those of the mammalian protein phosphatase 1. The R73C mutant phosphatase from the glycogen-deficient strain, glc7-1, purified as a 1:1 complex with the inhibitor 2 fusion, had a specific activity toward phosphorylase a of 0.9 unit/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 13. 1, 113, and 0.37 nM, respectively. The R73C mutation slightly decreases the specific activity and sensitivity to inhibitors, suggesting that changes in biochemical properties may affect glycogen levels. However, the modest changes are consistent with our previous proposal (E. M. Reimann et al., 1993, Adv. Protein Phosphatases 7,173-182) and with the results of Stuart et al. (1994, Mol. Cell. Biol. 14, 896-905) that the mutation may selectively alter the interaction of Glc7p with regulatory proteins.