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 PP 2A. The aim of the present work was to seek the presence of one protei n phosphatase excreted in the exocellular medium and to study its acti vity during yeast growth in media supplemented or not supplemented wit h inorganic phosphate. Protein phosphatase was purified and activity w as assayed by following the dephosphorylation of three substrates, [P- 32]casein, phosphotyrosine and a synthetic tyrosine-phosphorylated pep tide. Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency. After several purification step s, the enzyme preparation presents an apparent electrophoretic homogen eity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyro syl activities. The kinetic properties exclude contamination by a copu rified protein and it is concluded that the two activities are carried by the same single proteic species. It was characterized by gel filtr ation as a 33 kDa protein with one single subunit demonstrated by SDS- PAGE. An absolute requirement for reducing-agents is observed suggesti ng 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 mu M and Hill coefficient 3.2 indicating a rathe r high cooperativity. These results showed that the involvement of alk aline and/or acid phosphatase was unlikely. In conclusion, a protein p hosphatase distinct from endocellular PP2A is secreted by Yarrowia lip olytica and characterized as a phosphotyrosine protein phosphatase wit h associated phosphoseryl/threonyl activity. (C) 1998 Elsevier Science Ltd. All rights reserved.