Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase H is correlated with glucose repression.Hexokinase II is one of the two hexokinase isoenzymes of the budding yeast Saccharomyces cerevisiae. It functions in vivo both in glycolysis (16,17) and in catabolite repression (6,20). Among the mutations that cause failures in glucose repression (25), some have reduced levels of hexokinase activity; these mutations were determined to be alleles of the structural gene of hexokinase II, HXK2 (6). Biochemical analysis of one of the mutants (10) indicated that hexokinase II was indeed defective. The synthesis of invertase from the SUC3 gene (reviewed in references 3 and 4) was derepressed in these mutants even when cells were grown in medium with a high glucose concentration. These results suggest that hexokinase II mediates glucose repression in yeast cells. Mutations in another gene, HEX2 (which may be the same as REG] [22]), also cause failures in glucose repression, but hex2 strains have levels of hexokinase II activity higher than the wild-type level (7).It is difficult to interpret these results because mutations that either reduce (hxk2) or increase (hex2) hexokinase II catalytic activity can cause failures of glucose repression. To determine whether hexokinase II protein is required for glucose repression or derepression, we constructed null mutations in the HXKI (encoding hexokinase I) and HXK2 genes and studied their effects on glucose repression (20). The result that hxk2 null mutants do not show glucose repression indicates that hexokinase II is indeed required for glucose repression. Although hexokinase I is normally not required, it can partially suppress the hxk2 defect in glucose repression if overproduced. Entian and Frohlich (8) isolated mutations in the HXK2gene that cause defects in glucose repression yet still retain hexose phosphorylation activity. On the basis of these * Corresponding author. results and the conformational change observed in yeast hexokinases upon binding ...
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