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[Figure: see text].

[Figure: see text].

Mitochondrial genomes (mtDNA) encode essential subunits of the mitochondrial respiratory chain. Mutations in mtDNA can cause a shortage in cellular energy supply, which can lead to numerous mitochondrial diseases. How cells secure mtDNA integrity over generations has remained unanswered. Here, we show that the single-celled yeast Saccharomyces cerevisiae can intracellularly distinguish between functional and defective mtDNA and promote generation of daughter cells with increasingly healthy mtDNA content. Purifying selection for functional mtDNA occurs in a continuous mitochondrial network and does not require mitochondrial fission but necessitates stable mitochondrial subdomains that depend on intact cristae morphology. Our findings support a model in which cristae-dependent proximity between mtDNA and the proteins it encodes creates a spatial “sphere of influence,” which links a lack of functional fitness to clearance of defective mtDNA.

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin-dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function.

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd 1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (135)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondria! PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondria! PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function.

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function.

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd 1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (135)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondria! PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondria! PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function.

Prohibitins comprise an evolutionary conserved and ubiquitously expressed family of membrane proteins that is essential for development in higher eukaryotes. Large ring complexes formed in the inner mitochondrial membrane by prohibitins regulate mitochondrial dynamics and function. Roles of prohibitins in cell signaling events across the plasma membrane and transcriptional regulation in the nucleus have been proposed as well. The molecular mechanism of prohibitin function, however, remains elusive. In contrast to higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life-span. To investigate the functional role of prohibitins in yeast and to identify redundant processes that fulfill the functions of prohibitins in their absence, an unbiased genetic approach was chosen. Synthetic genetic arrays were applied to identify genes showing synthetic lethal interactions with prohibitins. This approach revealed 35 genes required for cell survival in the absence of prohibitins. The assembly of the FO-particle of the F1FO-ATP synthase was identified as one process essential in prohibitin-deficient cells. Atp23 was characterized as a novel processing peptidase with a dual function in maturation of the mitochondrially encoded subunit Atp6 and its assembly into the functional F1FO-ATP synthase. ~50% of the genes required in prohibitin-deficient cells, including the strongest genetic interactions, are demonstrated for the first time to be required for mitochondrial phospholipid homeostasis. Evidence is provided that members of a conserved protein family, Ups1 and Gep1, coordinately regulate the levels of the non-bilayer forming phospholipids cardiolipin and phosphatidyl-ethanolamine in mitochondria. Additionally, an uncharacterized putative phosphatase was identified that is required for cardiolipin biosynthesis and might represent the last missing enzyme in the biosynthesis pathway of cardiolipin in yeast. The genetic interactome of prohi

Prohibitins comprise an evolutionary conserved and ubiquitously expressed family of membrane proteins that is essential for development in higher eukaryotes. Large ring complexes formed in the inner mitochondrial membrane by prohibitins regulate mitochondrial dynamics and function. Roles of prohibitins in cell signaling events across the plasma membrane and transcriptional regulation in the nucleus have been proposed as well. The molecular mechanism of prohibitin function, however, remains elusive. In contrast to higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life-span. To investigate the functional role of prohibitins in yeast and to identify redundant processes that fulfill the functions of prohibitins in their absence, an unbiased genetic approach was chosen. Synthetic genetic arrays were applied to identify genes showing synthetic lethal interactions with prohibitins. This approach revealed 35 genes required for cell survival in the absence of prohibitins. The assembly of the FO-particle of the F1FO-ATP synthase was identified as one process essential in prohibitin-deficient cells. Atp23 was characterized as a novel processing peptidase with a dual function in maturation of the mitochondrially encoded subunit Atp6 and its assembly into the functional F1FO-ATP synthase. ~50% of the genes required in prohibitin-deficient cells, including the strongest genetic interactions, are demonstrated for the first time to be required for mitochondrial phospholipid homeostasis. Evidence is provided that members of a conserved protein family, Ups1 and Gep1, coordinately regulate the levels of the non-bilayer forming phospholipids cardiolipin and phosphatidyl-ethanolamine in mitochondria. Additionally, an uncharacterized putative phosphatase was identified that is required for cardiolipin biosynthesis and might represent the last missing enzyme in the biosynthesis pathway of cardiolipin in yeast. The genetic interactome of prohi