MOLECULAR-GENETICS OF PHOSPHOFRUCTOKINASE IN THE YEAST KLUYVEROMYCES-LACTIS

We have undertaken a study of phosphofructokinase (PFK; E.C. 2.7.1.11) in the yeast Kluyveromyces lactis. Like other eukaryotic PFKs, the K. lactis enzyme is activated by the allosteric effectors AMP and fructo se-2,6-bisphosphate. PFK activity is induced in cells grown on glucose as compared to ethanol-grown cells, in contrast to the constitutive e xpression of PFK in Saccharomyces cerevisiae. We show here that phosph ofructokinase of the yeast K. lactis is composed of two non-identical types of subunits, encoded by the genes KIPFK1 and KIPFK2. We have clo ned and sequenced both genes. KIPFK1 and KIPFK2 encode the a- and the beta-PFK subunits with deduced molecular weights of 109.336 Da and 104 .074 Da, respectively. Sequence analysis indicates that the genes evol ved from a double duplication event. Null mutants in either of the gen es lack detectable PFK activity in vitro and the respective subunits c annot be detected on Western blots. In contrast to the situation in S. cerevisiae, Klpfk1 Klpfk2 double mutants retain the ability to grow o n glucose. However, Klpfk2 mutants and the double mutants do not grow on glucose, when respiration is blocked. These data suggest that the p entose phosphate pathway and respiration play a substantial role in gl ucose utilization by K. lactis. The K. lactis PFK genes can be express ed independently in S. cerevisiae and each of them complements the glu cose-negative phenotype of pfk1 pfk2 double deletion mutants in this y east. Expression of both K. lactis PFK genes simultaneously in S. cere visiae pfk double deletion mutants complements for PFK activity. Howev er, expression of a combination of PFK genes from K. lactis and S. cer evisiae does not lead to the production of a functional enzyme.