An upstream open reading frame regulates expression of the mitochondrial protein Slm35 and mitophagy flux.

Mitochondrial protein Slm35 is linked to TOR1 signaling, mitophagy, and stress response in Saccharomyces cerevisiae. Nonetheless, little is known about its regulation or how it affects stress adaptation. In this work, we identified stress-related transcription factor binding sites and two upstream open reading frames (uORFs) in the 5'-UTR of SLM35. Using transcriptional reporters, we showed that the transcription factor Gis1 represses SLM35 transcription; however, Slm35 protein levels increased under oxidative stress and in early stationary phase, suggesting post-transcriptional regulation. Site-directed mutagenesis revealed that one uORF negatively regulates translation, with its disruption leading to altered Slm35 levels and a reproducible increase in mitophagy flux. These findings reveal multilayered control of SLM35 expression and underscore the role of uORF-mediated translation in mitochondrial stress responses. Impact statement This study shows that SLM35, encoding a mitochondrial protein, is controlled through multiple regulatory layers, combining transcriptional repression by stress-responsive factors with uORF-mediated translational regulation. By linking these mechanisms to mitophagy, the work provides new insight into mitochondrial quality control under stress.