The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F 1 -catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ 0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.human mitochondria | ATP synthase | permeability transition pore | subunit c | ATP5G

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.