GAL80 PROTEINS OF KLUYVEROMYCES-LACTIS AND SACCHAROMYCES-CEREVISIAE ARE HIGHLY CONSERVED BUT CONTRIBUTE DIFFERENTLY TO GLUCOSE REPRESSION OF THE GALACTOSE REGULON

We cloned the GAL80 gene encoding the negative regulator of the transc riptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. T he deduced amino acid sequence of K. lactis GAL80 revealed a strong st ructural conservation between K. lactis Gal80 and the homologous Sacch aromyces cerevisiae protein, with an overall identity of 60% and two c onserved blocks with over 80% identical residues. K. lactis gal80 disr uption mutants show constitutive expression of the lactose/galactose m etabolic genes, confirming that K. lactis Gal80 functions in essential ly in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Ga l4-dependent activation is strongly inhibited by glucose even in a gal 80 mutant, glucose repressibility is almost completely lost in ga180 m utants of K. lactis. Indirect evidence suggests that this difference i n phenotype is due to a higher activator concentration in K. lactis wh ich is able to overcome glucose repression. Expression of the K. lacti s GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Ga180. Gal80 in turn not only controls Lac 9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and ne gative regulators. By mutating the Lac9 binding sites of the GAL80 pro moter, we could show that induction of GAL80 is required to prevent ac tivation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to comp letely block Lac9 function in glucose.