Edited by Ruma BanerjeeCopper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2. Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.Mitochondrial dysfunction contributes to the pathogenesis of heart failure, neurodegenerative disorders, myopathies, and diabetes (1). Mitochondria are dynamic, double membranebound organelles with a semi-permeable outer membrane that allows exchange of metabolites between the cytosol and the intermembrane space (IMS).5 In contrast, the inner membrane (IM) that separates the IMS and the matrix is tightly sealed. Thus, numerous transporters are required to provide the matrix with a diverse range of substrates that are necessary to support metabolism, the biogenesis of iron-sulfur clusters, and the assembly of the electron transport chain (ETC) required for oxidative phosphorylation (2).Cytochrome c oxidase (COX) is the terminal electron-accepting complex of the ETC. Mammalian COX contains 14 major subunits, two of which bind three redox centers required for electron transfer (3). The catalytic core consists of the mitochondrially-encoded subunits COX1, COX2, and COX3. COX2 binds the binuclear Cu A site required for accepting electrons from cytochrome c. These electrons are then transferred to the cofactors of COX1, first to heme a and then to the heme a 3 -Cu B site where oxygen is bound. COX biogenesis requires Ͼ25 accessory proteins known as COX assembly factors (1). The overwhelming majority of ETC defects that underlie mitochondrial dysfunction and human disease is caused by pathogenic mutations in accessory factors (1). At least nine of these factors facilitate the insertion of the copper cofactors that are essential for the catalytic competence of the COX holoenzyme (4). In yeast, it has been demonstrated that the copper used for COX assembly comes from the matrix, and this matrix copper pool is conserved in mammals (5, 6). Howe...

In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix.

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