Construction and physiological characterization of mutants disrupted in the phosphofructokinase genes of Saccharomyces cerevisiae

Summary

The structural genes coding for the two kinds of subunits of phosphofructokinase in yeast have been cloned previously (Heinisch 1986). The coding regions were defined by S1-mapping. They were disrupted in vitro by insertion of a LEU2-marker. These constructions were then used for substitution of the respective chromosomal copies. That the disruption of the PFK-genes had in fact occured was confirmed by Southern blot analysis. Furthermore, in Northern blots shorter transcripts were detected in the respective disruption mutants. Using polyclonal antibodies the α-subunits were not detectable in pfk1-disruptions whereas the β-subunits were undetectable in pfk2-disruptions.

Physiological characterization showed that the single disruption mutants still fermented glucose to ethanol and CO₂. They accumulated fructose-6-phosphate and glucose-6-phosphate over wild type levels and showed decreased levels of fructose- 1,6-bisphosphate. In addition an accumulation of sedoheptulose-7-phosphate was observed, a metabolite not detectable in wild type cells. A haploid yeast strain containing both disrupted copies of the PFK-genes is not capable of growing on rich medium containing 2% glucose. The accumulation of glucose-6-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate is much more pronounced in such mutants, whereas the fructose- 1,6-bisphosphate concentration decreases below the level of detection.