Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance.
The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.
Posas
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Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, Spain.
Bollen
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Stalmans
W
W
Ariño
J
J
D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride / Q000494:pharmacology
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride; C075119:PPZ1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1