Engineering of the yeast ubiquitin ligase Rsp5: isolation of a new variant that induces constitutive inactivation of the general amino acid permease Gap1

Rsp5 is an essential ubiquitin-protein ligase in Saccharomyces cerevisiae . We found previously that the Ala401Glu rsp 5 mutant is hypersensitive to various stresses that induce protein misfolding, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. To isolate new Rsp5 variants as suppressors of the A401E mutant, PCR random mutagenesis was used in the rsp5 ^A401E gene, and the mutagenized plasmid library was introduced into rsp5 ^A401E cells. As a phenotypic suppressor of rsp5 ^A401E cells, we isolated a quadruple variant (Thr357Ala/Glu401Gly/Lys764Glu/Glu767Gly) on a minimal medium containing the toxic proline analogue azetidine-2-carboxylate (AZC). Site-directed mutagenesis experiments showed that the rsp5 ^T357A/K764E cells were much more tolerant to AZC than the wild-type cells, due to the smaller amounts of intracellular AZC. However, the T357A/K764E variant Rsp5 did not reverse the hypersensitivity of rsp5 ^A401E cells to other stresses such as high growth temperature, ethanol, and freezing treatment. Interestingly, immunoblot and localization analyses indicated that the general amino acid permease Gap1, which is involved in AZC uptake, was absent on the plasma membrane and degraded in the vacuole of rsp5 ^T357A/K764E cells before the addition of ammonium ions. These results suggest that the T357A/K764E variant Rsp5 induces constitutive inactivation of Gap1.