MOLECULAR-CLONING OF THE NEUTRAL TREHALASE GENE FROM KLUYVEROMYCES-LACTIS AND THE DISTINCTION BETWEEN NEUTRAL AND ACID TREHALASES

We cloned the Kluyveromyces lactis KINTH1 gene, which encodes neutral trehalase. It showed 65.2% and 68.5% identity at nucleotide and amino acid sequence level, respectively, with the Saccharomyces cerevisiae N TH1 gene. Multiple alignment of the predicted trehalase protein sequen ces from yeasts, bacteria, insects, and mammals revealed two major dom ains of conservation. Only the yeast trehalases displayed in an N-term inal extension two consensus sites for cAMP-dependent protein phosphor ylation and a putative Ca2+-binding sequence. Gene disruption of the K INTH1 gene abolished neutral trehalase activity and clearly revealed a trehalase activity with an acid pH optimum. It also resulted In a hig h constitutive trehalose level. Expression of the KINTH1 gene in an S. cerevisiae nth1 Delta mutant resulted in rapid activation of the hete rologous trehalase upon addition of glucose to cells growing on a nonf ermentable carbon source and upon addition elf a nitrogen source to ce lls starved for nitrogen in a glucose-containing medium. In ii. lactis , the same responses were observed except that rapid activation by glu cose was observed only in early-exponential-phase cells. Inactivation of K. lactis neutral trehalase by alkaline phosphatase and activation by cAMP in cell extracts are consistent with control of the enzyme by cAMP-dependent protein phosphorylation.