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Characterizing purified Siw14 from Saccharomyces cerevisiae (LB261)
Author(s) -
Steidle Elizabeth,
Rolfes Ronda
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb261
Subject(s) - saccharomyces cerevisiae , subfamily , phosphatase , biochemistry , yeast , arabidopsis , biology , homology (biology) , pi , recombinant dna , phosphorylation , chemistry , gene , mutant
Yeast strains with mutations in siw14Δ are defective in endocytosis, stress responses and linking nutrient status to cell cycle progression. Siw14 is a member of a family of dual‐specificity phosphatases found in fungi, protists, and plants. A model of the structure of the protein demonstrated that the active site is basic and shallow, and contains the consensus sequence HCxxGxxR, which includes the signature catalytic residues C214 and R220 as well as two residues, H213 and G217, conserved in the subfamily. Siw14 shows extensive homology with the Arabidopsis protein At1g5000 that dephosphorylates PI(3)P and PI(5)P, and has limited homology with the mammalian protein PTEN which dephosphorylates PI(3,4,5)P3. To test the hypothesis that Siw14 dephosphorylates one or more of the phosphorylated phosphatidylinositides (PIPn), a GST‐tagged form of the protein was purified from E. coli and assayed with the generic substrate p‐nitrophenylphosphate and each of the PIP¬ns. Recombinant GST‐Siw14 exhibited the highest activity in a buffer containing Tris‐HCl, pH 6.0, 10 mM DTT at 37°C, and activity was linear over 90 min. The enzyme exhibits the highest activity with PI(3,5)P2 but shows activity with other PIPs.