Advertisement

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

  • Matthew K. Robinson
  • Eric M. Phizicky
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 93)

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).

Keywords

Glass Bead Protein Phosphatase Okadaic Acid Dialysis Tubing Freeze Thaw 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Maeda, T., Tsai, A. Y., and Saito, H. (1993) Mutatins in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect in Saccharomyces cerevisiae Mol Cell Biol 13, 5408–5417.PubMedGoogle Scholar
  2. 2.
    Robinson, M K., van Zyl, W H., Phizicky, E. M., and Broach, J R (1994) TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation. Mol Cell Biol 14, 3634–3645PubMedGoogle Scholar
  3. 3.
    Cohen, P. (1989). The structure and regulation of protein phosphatases Annu Rev. Biochem 58, 453–508PubMedCrossRefGoogle Scholar
  4. 4.
    Huang, K. N and Symington, L A. (1995) Suppressors of a Saccharomyces cerevisiae pkcl mutation identify alleles of the phosphatase gene PTC1 an of a novel gene encoding a putative basic leucine zipper protein Genetics 141, 1275–1285PubMedGoogle Scholar
  5. 5.
    Mitchell, D. A., Marshal, T K., and Deschenes, R. J. (1993). Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast. Yeast 9, 715–723PubMedCrossRefGoogle Scholar
  6. 6.
    Deana, A D, Mac Gowan, C H, Cohen, P., Marchiori, F, Meyer, H E, and Pinna, L A. (1990) An investigation of the substrate specifictiy of protein phosphatase 2C using synthteic peptide substrates, comparison with protein phosphatase 2A Biochem Biophys Acta 1051, 199–202.Google Scholar

Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Matthew K. Robinson
    • 1
  • Eric M. Phizicky
    • 2
  1. 1.Program in GeneticsUniversity of Rochester School of Medicine and Dentistry
  2. 2.Department of BiochemistryUniversity of Rochester School of Medicine and Dentistry

Personalised recommendations