Characterization of an exocellular protein phosphatase with dual substrate specificity from the yeast Yarrowia lipolytica.
In previous work, the major endocellular protein phosphatase activity has been identified in the secretory yeast Yarrowia lipolytica as a PP2A. The aim of the present work was to seek the presence of one protein phosphatase excreted in the exocellular medium and to study its activity during yeast growth in media supplemented or not supplemented with inorganic phosphate. Protein phosphatase was purified and activity was assayed by following the dephosphorylation of three substrates, [32P]casein, phosphotyrosine and a synthetic tyrosine-phosphorylated peptide. Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency. After several purification steps, the enzyme preparation presents an apparent electrophoretic homogeneity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyrosyl activities. The kinetic properties exclude contamination by a copurified protein and it is concluded that the two activities are carried by the same single proteic species. It was characterized by gel filtration as a 33 kDa protein with one single subunit demonstrated by SDS-PAGE. An absolute requirement for reducing-agents is observed suggesting that the enzyme contains at least one essential reactive cysteinyl residue. Optimum pH value is 6.1, apparent K(m) for phosphotyrosine was calculated to be 760 microM and Hill coefficient 3.2 indicating a rather high cooperativity. These results showed that the involvement of alkaline and/or acid phosphatase was unlikely. In conclusion, a protein phosphatase distinct from endocellular PP2A is secreted by Yarrowia lipolytica and characterized as a phosphotyrosine protein phosphatase with associated phosphoseryl/threonyl activity.
Laboratoire de Chimie Biologique, INRA, INA PG, Centre de Biotechnologies Agro-Industrielles, Thiverval-Grignon, France. jolivet@cardere.grignon.inra.fr
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D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000254:growth & development; D002364:Caseins; D002413:Cations, Divalent / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019000:Phosphotyrosine; D013379:Substrate Specificity
D002364:Caseins; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D010455:Peptides; D019000:Phosphotyrosine; D010749:Phosphoprotein Phosphatases; D004229:Dithiothreitol