Functional characterization of Schizosaccharomyces pombe neutral trehalase altered in phosphorylatable serine residues.
The activation of neutral trehalase (Ntp1) by metabolic and physical stresses in Schizosaccharomyces pombe is dependent on protein kinases Pka1 or Sck1. Mutant ntp1 alleles altered for potentially phosphorylatable serine residues within the regulatory domain of the enzyme were integrated under the control of the native promoter in an ntp1-deleted background. The trehalase variants were expressed to a level similar to that of wild type trehalase from control cells. Wild type trehalase protein accumulated and became activated upon stress while a single change in the evolutionary conserved perfect consensus site for Pka1-dependent phosphorylation (Ser71), as well as point mutations in two other putative phosphorylation sites (Ser6, Ser51), produced inactive trehalases unresponsive to stress. Trehalose content in the trehalase mutated strains increased upon salt stress to a level comparable to that shown by an ntp1-deleted mutant. When exposed to heat shock, trehalose hyperaccumulated in the ntp1-null strain lacking trehalase protein and this phenotype was shown by some (Ser71), but not all, strains with serine mutated trehalases. The mutant trehalases retained the ability to form complexes with trehalose-6-phosphate synthase. These data support a role of potentially phosphorylated specific sites for the activation of S. pombe neutral trehalase and for the heat shock-induced accumulation of trehalose.
Franco
Alejandro
A
Department of Genetics and Microbiology, University of Murcia, 30071 Murcia, Spain.
Soto
Teresa
T
Madrid
Marisa
M
Vicente-Soler
Jero
J
Gacto
Mariano
M
Cansado
José
J
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D014199:Trehalose / Q000378:metabolism
D029702:Schizosaccharomyces pombe Proteins; D012694:Serine; D014199:Trehalose; D011494:Protein Kinases; C107804:Sck1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C464880:Ntp1p protein, S pombe; D010744:Phosphoric Monoester Hydrolases