Purification and Characterization of Neutral Trehalase from the Yeast ABYS1 Mutant

Abstract

Neutral trehalase was purified from stationary yeast ABYSl mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and s.The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodi.umdodecyl sulfategel electrophoresis.Maximal activity (1 14 rcmol of trehalose min-l X mg" at 37 "C) was observed at pH 6.8-7.0.The apparent K,,, for trehalose was 34.5 RIM.Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose.Neutral trehalase is located in the cytosol.A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase.Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by 290% inactivation.Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da).The phosphorylated amino a'cid residue was identified as phosphoserine.In yeast, the trehalose-hydrolyzing enzyme trehalase was first described by Ernil Fischer (1).An inactive (zymogen) form of trehalase, which is activated by cyclic AMP-dependent phosphorylation, was reported by van Solingen and van der Plaat (2).In 1982, Wiemken and co-workers (3, 4) demonstrated that the phosphorylatable trehalase was localized in the cytosol, whereas a second, permanently active, trehalase was found in the vacuoles.Londesborough and Varimo ( 5 ) separated these two a.ctivities and determined different pH optima of the two enzymes.The permanently active vacuolar trehalase, which has its maximal activity at pH 4.5 and which was therefore designated "acid trehalase," was purified and characterized in a previous paper from this laboratory (6).Partial purification of the cytosolic "neutral trehalase," which, when phosphorylated, has its maximal activity at pH 7.0, has been worked out by several groups ( 5 , 7, 8).In this paper, purification to homogeneity of the phosphorylated neutral trehalase and characterization of the purified enzyme are presented.In accordance with the recommendations of the