The GAL induction response in yeasts with impaired galactokinase Gal1p activity

The galactose-regulated (GAL) genes of yeast Saccharomyces cerevisiae are rapidly and efficiently expressed when cells are switched to a medium containing galactose as a sole carbon source. This GAL induction response is tightly controlled by interplay of three regulatory proteins: transcriptional activator, Gal4p; repressor, Gal80p; and sensor molecule, Gal3p. Galactokinase, Gal1p, the first enzyme of Leloir pathway catalyzes formation of galactose-1-phospate from galactose and ATP. Here we presented that S171 and A172 in conserved region III are required for catabolic activity of Gal1p. Using in vivo site-directed mutagenesis, strains gal1S171-Delta 172 Delta, gal1S171GA172G and gal1S171A with impaired galactokinase function were obtained. Transcriptional activation of GAL genes was determined by measuring fluorescence intensity of GFP under condition in which galactose utilization was limited. The expression levels were similar in all mutant strains, but significantly reduced in comparison to Gal1p knockout environment. Compared to wild type, only moderately increase of GAL induction response was determined. Results from single-cell analysis matched the general trends observed in the population-average assay. In this work we segregated enzymatic and regulatory functions of Gal1p and showed that the depletion of inducer is not the main limiting factor affecting GAL expression level.