Distinct roles of galactose-1P in galactose-mediated growth arrest of yeast deficient in galactose-1P uridylyltransferase (GALT) and UDP-galactose 4′-epimerase (GALE)

Abstract

Galactose is metabolized in humans and other species by the three-enzyme Leloir pathway comprised of galactokinase (GALK), galactose 1-P uridylyltransferase (GALT), and UDP-galactose 4′-epimerase (GALE). Impairment of GALT or GALE in humans results in the potentially lethal disorder galactosemia, and loss of either enzyme in yeast results in galactose-dependent growth arrest of cultures despite the availability of an alternate carbon source. In contrast, loss of GALK in humans is not life-threatening, and in yeast has no impact on the growth of cultures challenged with galactose. Further, the growth of both GALT-null and GALE-null yeast challenged with galactose is rescued by loss of GALK, thereby implicating the GALK reaction product, gal-1P, for a role in the galactose-sensitivity of both strains. However, the nature of that relationship has remained unclear. Here we have developed and applied a doxycycline-repressible allele of galactokinase to define the quantitative relationship between galactokinase activity, gal-1P accumulation, and growth arrest of galactose-challenged GALT or GALE-deficient yeast. Our results demonstrate a clear threshold relationship between gal-1P accumulation and galactose-mediated growth arrest in both GALT-null and GALE-null yeast, however, the threshold for the two strains is distinct. Further, we tested the galactose-sensitivity of yeast double-null for GALT and GALE, and found that although loss of GALT barely changed accumulation of gal-1P, it significantly lowered the accumulation of UDP-gal, and also dramatically rescued growth of the GALE-null cells. Together, these data suggest that while gal-1P alone may account for the galactose-sensitivity of GALT-null cells, other factors, likely to include UDP-gal accumulation, must contribute to the galactose-sensitivity of GALE-null cells.