Atg32 dependent mitophagy sustains spermidine and nitric oxide required for heat stress tolerance in S. cerevisiae

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 in 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.

The pathogenic m.8993 T > G mutation in mitochondrial ATP6 gene prevents proton release from the subunit c-ring rotor of ATP synthase

The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K, and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G, and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionnary conservation.

Publisher Correction: Autophagy facilitates adaptation of budding yeast to respiratory growth by recycling serine for one-carbon metabolism

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20648-5

Autophagy facilitates adaptation of budding yeast to respiratory growth by recycling serine for one-carbon metabolism

The mechanism and function of autophagy as a highly-conserved bulk degradation pathway are well studied, but the physiological role of autophagy remains poorly understood. We show that autophagy is involved in the adaptation of Saccharomyces cerevisiae to respiratory growth through its recycling of serine. On respiratory media, growth onset, mitochondrial initiator tRNA modification and mitochondrial protein expression are delayed in autophagy defective cells, suggesting that mitochondrial one-carbon metabolism is perturbed in these cells. The supplementation of serine, which is a key one-carbon metabolite, is able to restore mitochondrial protein expression and alleviate delayed respiratory growth. These results indicate that autophagy-derived serine feeds into mitochondrial one-carbon metabolism, supporting the initiation of mitochondrial protein synthesis and allowing rapid adaptation to respiratory growth.

Fluoxetine Inhibits Respiratory Growth of Candida glabrata and has Cytocidal Activity

Background: Fluoxetine is a widely prescribed antidepressant that has associated antifungal activity. Methods: This study examined the growth inhibitory and cytotoxic effects of fluoxetine on Candida glabrata. Result: Fluoxetine inhibited growth of C. glabrata on media containing glucose as a carbon source: the inhibition was much greater when ethanol was the carbon source. In hypoosmotic conditions fluoxetine caused death of C. glabrata in one hour. Conclusion: Fluoxetine inhibits fermentative and mitochondrial function of C. glabrata. It rapidly kills C. glabrata in hypoosmotic conditions.

A Role for COX20 in Tolerance to Oxidative Stress and Programmed Cell Death in Saccharomyces cerevisiae

Industrial production of bioethanol from lignocellulosic materials (LCM′s) is reliant on a microorganism being tolerant to the stresses inherent to fermentation. Previous work has highlighted the importance of a cytochrome oxidase chaperone gene (COX20) in improving yeast tolerance to acetic acid, a common inhibitory compound produced during pre-treatment of LCM’s. The presence of acetic acid has been shown to induce oxidative stress and programmed cell death, so the role of COX20 in oxidative stress was determined. Analysis using flow cytometry revealed that COX20 expression was associated with reduced levels of reactive oxygen species (ROS) in hydrogen peroxide and metal-induced stress, and there was a reduction in apoptotic and necrotic cells when compared with a strain without COX20. Results on the functionality of COX20 have revealed that overexpression of COX20 induced respiratory growth in Δimp1 and Δcox18, two genes whose presence is essential for yeast respiratory growth. COX20 also has a role in protecting the yeast cell against programmed cell death.

Reverse genetic analysis of yeast YPR099C/MRPL51 reveals a critical role of both overlapping ORFs in respiratory growth and MRPL51 in mitochondrial DNA maintenance

ABSTRACT The Saccharomyces cerevisiae genome contains 6572 ORFs, of which 680 ORFs are classified as dubious ORFs. A dubious ORF is a small, noncoding, nonconserved ORF that overlaps with another ORF of the complementary strand. Our study characterizes a dubious/nondubious ORF pair, YPR099C/MRPL51, and shows the transcript and protein level expression of YPR099C. Its subcellular localization was observed in the mitochondria. The overlapping ORF, MRPL51, encodes a mitochondrial ribosomal protein of large subunit. Deletion of any ORF from YPR099C/MRPL51 pair induces common phenotypes, i.e. loss of mtDNA, lack of mitochondrial fusion and lack of respiratory growth, due to the double deletion (ypr099cΔ/Δmrpl51Δ/Δ) caused by sequence overlap. Hence, we created the single deletions of each ORF of the YPR099C/MRPL51 pair by an alternative approach to distinguish their phenotypes and identify the specific functions. Both the ORFs were found essential for the functional mitochondria and respiratory growth, but MRPL51 showed its specific requirement in mtDNA stability. The mechanism of mtDNA maintenance by Mrpl51 is probably Mhr1 dependent that physically interacts with Mrpl51 and also regulates mtDNA repair. Overall, our study provides strong evidence for the protein level expression of a dubious ORF YPR099C and the bifunctional role of Mrpl51 in mtDNA maintenance.

S. cerevisiae Strain Lacking Mitochondrial IF3 Shows Increased Levels of Tma19p during Adaptation to Respiratory Growth

After billions of years of evolution, mitochondrion retains its own genome, which gets expressed in mitochondrial matrix. Mitochondrial translation machinery rather differs from modern bacterial and eukaryotic cytosolic systems. Any disturbance in mitochondrial translation drastically impairs mitochondrial function. In budding yeast Saccharomyces cerevisiae, deletion of the gene coding for mitochondrial translation initiation factor 3 - AIM23, leads to an imbalance in mitochondrial protein synthesis and significantly delays growth after shifting from fermentable to non-fermentable carbon sources. Molecular mechanism underlying this adaptation to respiratory growth was unknown. Here, we demonstrate that slow adaptation from glycolysis to respiration in the absence of Aim23p is accompanied by a gradual increase of cytochrome c oxidase activity and by increased levels of Tma19p protein, which protects mitochondria from oxidative stress.