CARBON SOURCE-DEPENDENT PHOSPHORYLATION OF HEXOKINASE PII AND ITS ROLE IN THE GLUCOSE-SIGNALING RESPONSE IN YEAST

The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism in Saccharomyces cerevisi ae. However, the molecular basis of the specific role of Hxk2p in thes e effects is still unclear. One important feature in order to understa nd the physiological function of hexokinase PII is that it is a phosph oprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exi sts in vivo in a dimeric-monomeric equilibrium which is affected by ph osphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in gluco se repression mutants cat80/grr1, hex2/reg1, and cid1/glc7. Treatment of a glucose crude extract from cid1-226 (glc7-T152K) mutant cells wit h lambda-phosphatase drastically reduces the presence of phosphoprotei n, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p m onomer. An HXK2 mutation encoding a serine-to-alanine change at positi on 15 [HXK2 (S15A)] was to clarify the in vivo function of the phospho rylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction of HXT gene expression is also affected in cells expressing the mutated enzyme. Although we cann ot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of he xokinase is essential in vivo for glucose signal transduction.