Stability of neutral trehalase during heat stress in Saccharomyces cerevisiae is dependent on the activity of the catalytic subunits of cAMP-dependent protein kinase, Tpk1 and Tpk2.

Abstract

In Saccharomyces cerevisiae cAMP-dependent protein kinase (cAPK) is involved in nutrient sensing and growth regulation via the Ras/cAMP pathway. Target enzymes, e.g. neutral trehalase, are activated or inactivated rapidly by cAPK-mediated phosphorylation. In addition, stress-induced transcription of genes of the general stress-response, e.g. HSP12, is negatively regulated via cAPK. We have investigated the effect of low cAPK activity on the stress-induced expression of neutral trehalase Nth1p. For this purpose we used mutants (tpk1tpk2TPK3, tpk1TPK2tpk3 and TPK1tpk2tpk3) with double knockouts of the three TPK genes encoding catalytic subunits of cAPK. It is shown that the tpk1tpk2TPK3 mutant, which has very low cAPK activity, exhibits a heat-stress-induced inactivation of neutral trehalase that is not observed in tpk1TPK2tpk3, TPK1tpk2tpk3 mutants and wild-type cells. However, heat stress induces an increase in NTH1 mRNA in the tpk1tpk2TPK3 mutant. Introduction of a plasmid carrying the TPK1 or TPK2 gene into tpk1tpk2TPK3 cells restores the heat-induced increase of neutral trehalase activity. In vitro and in vivo results suggest that the heat induced inactivation of neutral trehalase is due to a reversible inactivation of Nth1p. Our data indicate that a certain level of phosphorylation is essential for maintenance of neutral trehalase activity during heat shock in S. cerevisiae. Two identical putative cAPK phosphorylation sites have been found in the sequence predicted for the Nth1p. Stabilization and activation of neutral trehalase may be regulated by these sites. Furthermore, our data suggest that the heat-stress-induced transcription of the NTH1 gene is not negatively regulated by cAPK, that the TPK genes have no effect on the glucose repression of the NTH1 gene, and that non-detectable neutral trehalase activity in derepressed tpk1tpk2TPK3 cells is correlated with the reduced thermotolerance observed in this strain, similar to the heat-shock-recovery defect reported for the nth1delta mutant.