Purification and Assay of the Ptc/Tpd1 Protein Phosphatase 2C from the Yeast Saccharomyces cerevisiae

Abstract

One species of protein phosphatase 2C (PP2C) in the yeast S. cerevisiae is encoded by the gene PTC1/TPD1 (1,2). This gene encodes a protein that is highly conserved in all eukaryotes. It is 38% identical to the rat protein over the entire sequence, with identity reaching up to 80% in distinct regions. TPD1 was shown to encode protein phosphatase 2C activity based on two lines of evidence: first, Ptc1/Tpd1 protein expressed in E. coli exhibits readily detectable Mg²⁺ or Mn²⁺ dependent protein phosphatase activity with ³²P-labeled casein as a substrate. Second, this activity does not require Ca²⁺ and is resistant to okadaic acid at concentrations capable of inhibiting all the other mam families of protein phosphatases in eukaryotic organisms. These are the primary distinguishing enzymatic characteristics of mammalian PP2C (3). Yeast has at least two other PP2C species since extracts made from cells deleted for PTC1/TPD1 exhibit substantial PP2C activity (1,2). Putative genes for this activity have been cloned (1).