In Saccharomyces cerevisiae, the selective autophagic degradation of mitochondria, termed mitophagy, is critically regulated by the adapter protein, Atg32. Despite our knowledge about the molecular mechanisms by which Atg32 controls mitophagy, its physiological roles on yeast survival and fitness remains less clear. Here, we demonstrate a requirement for Atg32 in promoting spermidine production during respiratory growth and heat-induced mitochondrial stress. During respiratory growth, mitophagy-deficient yeast exhibit profound heat-stress induced defects in growth and viability due to impaired biosynthesis of spermidine and its biosynthetic precursor S-Adenosyl-Methionine (SAM). Moreover, spermidine production is crucial for the induction of cytoprotective nitric oxide (NO) during heat stress. Hence, the re-addition of spermidine to Atg32 mutant yeast is sufficient to both enhance NO production and restore respiratory growth during heat stress. Our findings uncover a previously unrecognized physiological role for yeast mitophagy in spermidine metabolism and illuminate new interconnections between mitophagy, polyamine biosynthesis and NO signaling.

Yeast cells were cultivated in liquid SD media at 30˚C and grown to A₆₀₀ of 0.8-1.2 followed by passage into SGE with/without 0.5mM spermidine starting A₆₀₀ of 0.2 and grown at 30˚C until an OD A₆₀₀ of 0.5, then fresh SGE media with/without spermidine was replenished and yeast were allowed to continue growing until and OD of 1 at 30˚C. Subsequently, 15 μM DAF-FM-DA (Cayman Chemical Cat No. 18767) (diluted in DMSO) was added and cells were incubated in SGE with/without spermidine for an additional two and a half hours at 37˚C. Cells were next rinsed with respective media lacking DAF-FM-DA and were imaged by fluorescence microscopy using the Delta Vision Elite microscope (Applied Precision GE Healthcare Life Sciences) fitted with a 1.4 -NA 100X objective, PCO Edge cMOS camera, and run by softWoRx Resolve3D software. Capture and post-capture image processing were done using softWoRx software (Applied Precision GE Healthcare Life Sciences) and Fiji [39]. The average fluorescence across each cell was quantified using the line tool and Analyze-Measure functions in Fiji. For each experiment, calculated intensities were normalized by dividing all values by the average intensity calculated from WT 30˚C samples. For each experiment 200 cells per condition were quantified and three biological replicates (n=600 cells/condition) are represented as a violin plot with S.E.M generated in RStudio.