Autophagy determines mtDNA copy number dynamics during starvation

ABSTRACT

Derived from bacterial ancestors, mitochondria have maintained their own albeit strongly reduced genome, mitochondrial DNA (mtDNA), which encodes for a small and highly specialized set of genes. MtDNA exists in tens to thousands of copies packaged in numerous nucleoprotein complexes, termed nucleoids, distributed throughout the dynamic mitochondrial network. Our understanding of the mechanisms of how cells regulate the copy number of mitochondrial genomes has been limited. Here, we summarize and discuss our recent findings that Mip1/POLG (mitochondrial DNA polymerase gamma) critically controls mtDNA copy number by operating in 2 opposing modes, synthesis and, unexpectedly, degradation of mtDNA, when yeast cells face nutrient starvation. The balance of the 2 modes of Mip1/POLG and thus mtDNA copy number dynamics depends on the integrity of macroautophagy/autophagy, which sustains continuous synthesis and maintenance of mtDNA. In autophagy-deficient cells, a combination of nucleotide insufficiency and elevated mitochondrial ROS production impairs mtDNA synthesis and drives mtDNA degradation by the 3ʹ-5ʹ-exonuclease activity of Mip1/POLG resulting in mitochondrial genome depletion and irreversible respiratory deficiency.

Abbrivations: mtDNA: mitochondrial DNA; mtDCN: mitochondrial DNA copy number.

In our recently published work [1], we analyzed the relationship between autophagy and the copy number of mitochondrial genomes during starvation in budding yeast. We asked whether mtDNA synthesis, which generally occurs asynchronously from nuclear DNA replication, is maintained when yeast cells enter cell cycle arrest upon nitrogen depletion. Monitoring DNA synthesis by incorporation of the thymidine analog 5-ethynyl-2ʹ-deoxyuridine (EdU) in vivo, we found that wild-type yeast cells initially continue to synthesize mtDNA during the first day of starvation with a subsequent decline during prolonged starvation. In comparison, autophagy-deficient cells do not show any detectable mtDNA synthesis, indicating that starving cells require autophagy in order to sustain the synthesis of mitochondrial genomes. Consistent with ongoing mtDNA synthesis in nondividing cells, we discovered that starving wild-type cells display an initial 5-fold increase in mtDNA content after 1 d of starvation using real-time quantitative PCR. Interestingly, mtDNA copy number (mtDCN) declines after 2 d of starvation suggesting adjustment of the mtDNA content and revealing strikingly dynamic changes in mtDCN in response to metabolic changes. Defects in autophagy drastically alter the dynamics of mtDCN; in agreement with the observed defect in mtDNA synthesis, the mtDNA content in autophagy-deficient cells increases to a lesser extent than in wild-type cells and then continuously declines after 1 d of starvation. Cytological analysis confirmed the loss of mtDNA in autophagy mutants during starvation causing irreversible respiratory deficiency. In combination, these data revealed severe mitochondrial genome instability in autophagy-deficient cells during long-term starvation.

To identify the physiological basis, we tested whether an insufficient pool of nucleotides might be linked to impaired synthesis and instability of mtDNA in autophagy-deficient cells. Indeed, increasing the levels of deoxynucleotides genetically by deletion of SML1 (ribonucleotide reductase inhibitor), or by supplementing starvation medium with all 4 nucleobases stabilizes mtDNA copy number in starving cells deficient for autophagy. However, neither of the alterations is sufficient to restore mtDNA synthesis, pointing towards the existence of additional factors that prevent mitochondrial genome replication. Autophagy-deficient cells start to produce elevated levels of mitochondria-derived reactive oxygen species (ROS) upon starvation. Thus, we hypothesized that ROS production inhibits mtDNA synthesis. To test this notion, we deleted CBS1, a translation initiation factor for mitochondrially-encoded Cob/Cob1, which is required for the formation of a functional respiratory chain complex III. In the absence of Cbs1, autophagy mutants generate only low levels of ROS. However, dampening ROS production alone is also not sufficient to allow for mtDNA synthesis in starving autophagy-deficient cells. Strikingly, we found that supplementation of nucleobases in combination with suppressed ROS production fully restores mtDNA synthesis. Thus, autophagy plays a critical role in maintaining the metabolic homeostasis of mitochondria in providing sufficient metabolites to replenish nucleotide pools and in preventing elevated ROS production, which are both crucial physiological factors that together impinge upon mitochondrial genome replication and stability.

Given that wild-type and autophagy-deficient cells entered a non-dividing state, the reduction in mtDCN during starvation must have been caused by mtDNA degradation. Mechanisms for mtDNA turnover have been elusive and constitute an open question in cell biology. Thus, we hypothesized that intramitochondrial nucleases degraded mtDNA in starving cells. As the absence of the conserved mitochondrial nuclease Nuc1/EndoG by itself or upon autophagy deficiency does not affect mtDCN or stability, we turned to Mip1/POLG (mitochondrial DNA polymerase gamma). In addition to a 5ʹ-3ʹ DNA polymerase activity, Mip1/POLG contains an inherent 3ʹ-5ʹ exonuclease with critical proofreading function. To test a potential role of Mip1/POLG in mtDNA degradation, we expressed an exonuclease-deficient allele of Mip1/POLG in otherwise wild-type or autophagy-deficient cells. Notably, inhibition of the exonuclease activity of Mip1/POLG stabilizes mtDNA in the absence of autophagy during starvation and, moreover, it also prevents the decrease in mtDCN that we observed in wild-type cells upon prolonged starvation. Thus far, the exonuclease activity of Mip1/POLG has been linked to replication fidelity by the means of proofreading. Based on our data, we propose that, in addition, it enables Mip1/POLG to mediate the degradation of mtDNA in vivo. Hence, our work positions Mip1/POLG at the center of mtDCN control by operating in 2 opposing modes: mtDNA synthesis and degradation. The balance of these 2 functions of Mip1/POLG is critically determined by mitochondrial homeostasis, namely nucleotide metabolism and ROS production, which requires functional autophagy during nutrient stress. In summary, our data provide new insights into the role of autophagy for maintaining mitochondrial function by metabolically regulating intramitochondrial mechanisms, which are clearly distinct from mitochondrial quality control by selective turnover by mitophagy.

Funding Statement

This work was supported by the Deutsche Forschungsgemeinschaft [SFB1218/TP A04];Max-Planck-Gesellschaft [n/a].

Disclosure statement

No potential conflict of interest was reported by the authors.