Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2

In the yeast Saccharomyces cerevisiae, glucose or fructose represses the ex pression of a large number of genes. The phosphorylation of glucose or fruc tose is catalysed by hexokinase PI (Hxk1), hexokinase PII (Hxk2) and a spec ific glucokinase (Glk1). The authors have shown previously that either Hxk1 or Hxk2 is sufficient for a rapid, sugar-induced disappearance of cataboli te-repressible mRNAs (short-term catabolite repression). Hxk2 is specifical ly required and sufficient for long-term glucose repression and either Hxk1 or Hxk2 is sufficient for long-term repression by fructose. Mutants lackin g the TPS1 gene, which encodes trehalose 6-phosphate synthase, can not grow on glucose or fructose. In this study, suppressor mutations of the growth defect of a tps1 Delta hxk1 Delta double mutant on fructose were isolated a nd identified as novel HXK2 alleles, All six alleles studied have single am ino acid substitutions. The mutations affected glucose and fructose phospho rylation to a different extent, indicating that Hxk2 binds glucose and fruc tose via distinct mechanisms. The mutations conferred different effects on long- and short-term repression. Two of the mutants showed very similar def ects in catabolite repression, despite large differences in residual sugar- phosphorylation activity. The data show that the long- and short-term phase s of catabolite repression can be dissected using different hexokinase muta tions. The lack of correlation between in vitro catalytic hexokinase activi ty, in vivo sugar phosphate accumulation and the establishment of catabolit e repression suggests that the production of sugar phosphate is not the sol e role of hexokinase in repression. Using the set of six hxk2 mutants it wa s shown that there is a good correlation between the glucose-induced cAMP s ignal and in vivo hexokinase activity. There was no correlation between the cAMP signal and the short- or long-term repression of SUC2, arguing agains t an involvement of cAMP in either stage of catabolite repression.