UGA4 and YBR062C Regulate Oxidative Stress Tolerance Under Heavy Metal Toxicity

Heavy metals impose a major cellular stress by promoting protein dysfunction and the accumulation of reactive oxygen species (ROS). In Saccharomyces cerevisiae, the glutathione S-transferase Ure2p is a key determinant of resistance to metal-induced oxidative stress and can be synthesized through a stress-responsive, cap-independent internal ribosome entry site (IRES) in the URE2 5' UTR. Much has been learned about the cellular responses to heavy metal exposure; however, the mechanistic details of how response genes are regulated in response to stress require further investigation. Here, we identify two previously unrecognized contributors to heavy metal tolerance, UGA4, a 4-aminobutyric acid GABA permease, and YBR062C, a poorly characterized gene previously linked to filamentous growth. Deletion of UGA4 or YBR062C caused notable sensitivity when cells were exposed to sub-inhibitory concentrations of cadmium (Cd), arsenite (As(III)), and nickel (Ni), with colony-forming units reduced by approximately 50%-80% relative to the wild-type strain. Genetic analysis positioned both genes in the URE2 pathway. Double mutants ure2Δ uga4Δ and ure2Δ ybr062cΔ were not more sensitive than the single mutant ure2Δ. URE2 overexpression restored metal resistance in uga4Δ and ybr062cΔ backgrounds, whereas UGA4 or YBR062C overexpression did not rescue ure2Δ. Mechanistically, UGA4 and YBR062C acted post-transcriptionally. URE2 mRNA abundance was unchanged, but Ure2p protein levels were reduced, particularly under Cd stress. Polysome profiling revealed decreased ribosomal association of URE2 mRNA in both mutants, and a URE2-IRES-dependent β-galactosidase reporter showed approximately 80% lower activity without affecting cap-dependent translation or altering reporter mRNA levels. Together, these data demonstrate that UGA4 and YBR062C promote heavy metal tolerance by enabling IRES-mediated translation of URE2 mRNA.