Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5′-3′exonuclease MGME1, elimination of the 3′-5′exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.

Steady‐state concentrations of mitochondrial Mg2+ previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains. While its structural and functional similarity to the bacterial Mg2+ transport protein CorA suggested a role for Mrs2p in Mg2+ influx into the organelle, other functions in cation homeostasis could not be excluded. Making use of the fluorescent dye mag‐fura 2 to measure free Mg2+ concentrations continuously, we describe here a high capacity, rapid Mg2+ influx system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Δψ and inhibited by cobalt(III)hexaammine. Overexpression of Mrs2p increases influx rates 5‐fold, while the deletion of the MRS2 gene abolishes this high capacity Mg2+ influx. Mg2+ efflux from isolated mitochondria, observed with low Δψ only, also requires the presence of Mrs2p. Cross‐linking experiments revealed the presence of Mrs2p‐containing complexes in the mitochondrial membrane, probably constituting Mrs2p homo‐ oligomers. Taken together, these findings characterize Mrs2p as the first molecularly identified metal ion channel protein in the inner mitochondrial membrane.

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027Δ mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027Δ mitochondria have increased cation contents, particularly K⁺ and low-membrane-potential Δψ. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K⁺/H⁺ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K⁺ homeostasis and organelle volume control.