A genetic approach to study ubiquitin function

Abstract

Alanine-scanning mutagenesis was performed with ubiquitin and the resulting alleles were expressed as the sole source of ubiquitin in yeast. Mutant ubiquitin alleles that were found to be severely deficient for growth on galactose media (gal-) were used for unbiased suppressor screens and GAL3 was identified as a suppressor of the gal- phenotype of the H10-D58A ubiquitin mutant. The antagonist of Gal3, Gal80 was subsequently investigated and the deletion of GAL80 was able to fully suppress the gal- phenotype of the H10-Ub D58A mutant strain. Gal80 was also found to be differentially degraded in glucose as compared to galactose and the increased stability of Gal80 was correlated with a lack of induction of GAL1 in the cells. The results presented here suggest that contrary to previous findings arguing that the degradation of Gal4 is necessary for the activation of the GAL genes, it is the degradation of the inhibitor Gal80 that is instead necessary for the activation of transcription.