Recent experiments by the applicants have shown that the glycogen- deficient phenotype of the glc7- (ppy1-1) mutant of S. cerevisiae is a result of a mutation in the gene for the catalytic subunit of protein phosphatase 1. This gene, which has been cloned an named PPY1, shares 81% sequence identity at the amino acid level with phosphatase 1 from rabbit skeletal muscle. The first major objective of the proposed studies is to determine the DNA sequence of the mutant PPY1 gene and to compare wild type and mutant protein phosphatase 1 with respect to substrate specificity, turnover number and association with regulatory proteins. The association with regulatory proteins is of particular interest since preliminary experiments indicate that chromatographic properties of the enzyme are affected by the mutation, a finding which is most readily explained by altered association with regulatory subunits. In rabbit skeletal muscle, protein phosphatase 1 exists only in association with one of the three regulatory subunits, which are know as inhibitor 2, G-subunit and M-subunit. The second major objective of the proposed studies is to characterize regulatory subunits or heat-stable inhibitor proteins that interact with the phosphatase 1 catalytic subunit and to determine if they are homologous to the regulatory proteins that have been identified in mammals. Association with regulatory subunits will be evaluated by identification of holoenzyme forms by chromatographic and immunological methods. The results of these experiments will help to elucidate the regulation of an important class of enzymes.//
