CLONING AND CHARACTERIZATION OF A SACCHAROMYCES-CEREVISIAE GENE ENCODING THE LOW-MOLECULAR-WEIGHT PROTEIN-TYROSINE-PHOSPHATASE

The low molecular weight protein tyrosine phosphatase (low M(r) PTPase ) is an 18-kDa cytoplasmic enzyme of unknown function that has been pr eviously found in several vertebrates, Using an oligonucleotide probe derived from the active site sequence of the mammalian low M(r) PTPase s, a Saccharomyces cerevisiae gene that encodes a homolog of this enzy me was cloned by low stringency hybridization, This gene, LTP1, togeth er with a neighboring gene, TKL1, is shown to be located on the right arm of chromosome XVI. The deduced amino acid sequence of its 161-amin o acid residue product shows a 39% average identity with that of the m ammalian enzymes. The yeast Ltp1 protein was expressed in Escherichia coli, purified to homogeneity, and shown to possess PTPase activity. T he recombinant Ltp1 efficiently hydrolyzes phosphotyrosine and a phosp hotyrosine-containing peptide, Tyr(531)-fyn, but it shows low activity toward phosphoserine and phosphothreonine. The catalytic activity of Ltp1 toward a number of substrates was approximately 30-fold lower tha n the corresponding values measured for the bovine low M(r) PTPase. Ho wever, the yeast enzyme was markedly activated by adenine and some pur ine nucleosides and nucleotides, including cAMP and cGMP. In the case of adenine, the activity of Ltp1 was increased by approximately 30-fol d. The high degree of evolutionary conservation of the low M(r) PTPase s implies a significant role for this enzyme. However, neither the dis ruption of the LTP1 gene nor an approximately 10-fold overexpression o f its product in S. cerevisiae caused any apparent phenotypic changes under the conditions tested. No proteins related to Ltp1 could be dete cted in extracts of the ltp1 null mutant, either by immunoblotting or by gelfiltration analysis accompanied by extended kinetic assays, cons istent with the conclusion that LTP1 is the only low M(r) PTPase-encod ing gene in S. cerevisiae.