Mitochondria accumulate copper in their matrix for the eventual maturation of the cuproenzymes cytochrome c oxidase and superoxide dismutase. Transport into the matrix is achieved by mitochondrial ...carrier family (MCF) proteins. The major copper transporting MCF described to date in yeast is Pic2, which imports the metal ion into the matrix. Pic2 is one of ~30 MCFs that move numerous metabolites, nucleotides and co-factors across the inner membrane for use in the matrix. Genetic and biochemical experiments showed that Pic2 is required for cytochrome c oxidase activity under copper stress, and that it is capable of transporting ionic and complexed forms of copper. The Pic2 ortholog SLC25A3, one of 53 mammalian MCFs, functions as both a copper and a phosphate transporter. Depletion of SLC25A3 results in decreased accumulation of copper in the matrix, a cytochrome c oxidase defect and a modulation of cytosolic superoxide dismutase abundance. The regulatory roles for copper and cuproproteins resident to the mitochondrion continue to expand beyond the organelle. Mitochondrial copper chaperones have been linked to the modulation of cellular copper uptake and export and the facilitation of inter-organ communication. Recently, a role for matrix copper has also been proposed in a novel cell death pathway termed cuproptosis. This review will detail our understanding of the maturation of mitochondrial copper enzymes, the roles of mitochondrial signals in regulating cellular copper content, the proposed mechanisms of copper transport into the organelle and explore the evolutionary origins of copper homeostasis pathways.
•The mitochondrial carrier family protein Pic2/SLC25A3 transports copper to the matrix•The matrix copper pool is used for the maturation of cytochrome c oxidase and superoxide dismutase•The mitochondrial copper handling machinery modulates cellular copper homeostasis and facilitates inter-organ communication•The roles of mitochondrial signals are explored in the context of the evolutionary origins of copper homeostasis pathways
All known eukaryotes require copper for their development and survival. The essentiality of copper reflects its widespread use as a co-factor in conserved enzymes that catalyze biochemical reactions ...critical to energy production, free radical detoxification, collagen deposition, neurotransmitter biosynthesis and iron homeostasis. However, the prioritized use of copper poses an organism with a considerable challenge because, in its unbound form, copper can potentiate free radical production and displace iron-sulphur clusters to disrupt protein function. Protective mechanisms therefore evolved to mitigate this challenge and tightly regulate the acquisition, trafficking and storage of copper such that the metal ion is rarely found in its free form in the cell. Findings by a number of groups over the last ten years emphasize that this regulatory framework forms the foundation of a system that is capable of monitoring copper status and reprioritizing copper usage at both the cellular and systemic levels of organization. While the identification of relevant molecular mechanisms and signaling pathways has proven to be difficult and remains a barrier to our full understanding of the regulation of copper homeostasis, mounting evidence points to the mitochondrion as a pivotal hub in this regard in both healthy and diseased states. Here, we review our current understanding of copper handling pathways contained within the organelle and consider plausible mechanisms that may serve to functionally couple their activity to that of other cellular copper handling machinery to maintain copper homeostasis.
Synchrotron-based X-ray fluorescence microscopy (XFM) has become an important imaging technique to investigate elemental concentrations and distributions in biological specimens. Advances in ...technology now permit imaging at resolutions rivaling that of electron microscopy, and researchers can now visualize elemental concentrations in subcellular organelles when using appropriate correlative methods. XFM is an especially valuable tool to determine the distribution of endogenous trace metals that are involved in neurodegenerative diseases. Here, we discuss the latest research on the unusual copper (Cu) storage vesicles that were originally identified in mouse brains and the involvement of Cu in Alzheimer's disease. Finally, we provide an outlook of how future improvements to XFM will drive current trace element research forward.
Reversible changes in the redox state of cysteine residues represent an important mechanism with which to regulate protein function. In mitochondria, such redox reactions modulate the localization or ...activity of a group of proteins, most of which function in poorly defined pathways with essential roles in copper delivery to cytochrome c oxidase (COX) during holoenzyme biogenesis. To date, a total of 8 soluble (COX17, COX19, COX23, PET191, CMC1-4) and 3 integral membrane (COX11, SCO1, SCO2) accessory proteins with cysteine-containing domains that reside within the mitochondrial intermembrane space (IMS) have been identified in yeast, all of which have human orthologues. Compelling evidence from studies of COX17, SCO1, and SCO2 argues that regulation of the redox state of their cysteines is integral to their metallochaperone function. Redox also appears to be crucial to the regulation of a SCO-dependent, mitochondrial signaling pathway that modulates the rate of copper efflux from the cell. Here, I review our understanding of redox-dependent modulation of copper delivery to COX and IMS-localized copper-zinc superoxide dismutase (SOD1) during the maturation of each enzyme, and discuss how this in turn may serve to functionally couple mitochondrial copper handling pathways with those localized elsewhere in the cell to regulate cellular copper homeostasis.
Cytochrome c oxidase (COX) was initially purified more than 70 years ago. A tremendous amount of insight into its structure and function has since been gleaned from biochemical, biophysical, genetic, ...and molecular studies. As a result, we now appreciate that COX relies on its redox-active metal centers (heme a and a3, CuA and CuB) to reduce oxygen and pump protons in a reaction essential for most eukaryotic life. Questions persist, however, about how individual structural subunits are assembled into a functional holoenzyme. Here, we focus on what is known and what remains to be learned about the accessory proteins that facilitate CuA site maturation.
Cytochrome c oxidase (CIV) deficiency is one of the most common respiratory chain defects in patients presenting with mitochondrial encephalocardiomyopathies. CIV biogenesis is complicated by the ...dual genetic origin of its structural subunits, and assembly of a functional holoenzyme complex requires a large number of nucleus-encoded assembly factors. In general, the functions of these assembly factors remain poorly understood, and mechanistic investigations of human CIV biogenesis have been limited by the availability of model cell lines. Here, we have used small interference RNA and transcription activator-like effector nucleases (TALENs) technology to create knockdown and knockout human cell lines, respectively, to study the function of the CIV assembly factor COX20 (FAM36A). These cell lines exhibit a severe, isolated CIV deficiency due to instability of COX2, a mitochondrion-encoded CIV subunit. Mitochondria lacking COX20 accumulate CIV subassemblies containing COX1 and COX4, similar to those detected in fibroblasts from patients carrying mutations in the COX2 copper chaperones SCO1 and SCO2. These results imply that in the absence of COX20, COX2 is inefficiently incorporated into early CIV subassemblies. Immunoprecipitation assays using a stable COX20 knockout cell line expressing functional COX20-FLAG allowed us to identify an interaction between COX20 and newly synthesized COX2. Additionally, we show that SCO1 and SCO2 act on COX20-bound COX2. We propose that COX20 acts as a chaperone in the early steps of COX2 maturation, stabilizing the newly synthesized protein and presenting COX2 to its metallochaperone module, which in turn facilitates the incorporation of mature COX2 into the CIV assembly line.
Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are ...integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency.
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•COA6 is required for COX2 stability•COA6 interacts transiently with the catalytic domain of newly synthesized COX2•COA6 interacts with the copper metallochaperone SCO2•Mutations in SCO2 or COA6 link defects in COX2 metallation to COX deficiency in heart
Numerous assembly and chaperone proteins are involved in the biogenesis of cytochrome c oxidase (Complex IV), including COX2, which houses its redox-active copper center. Pacheu-Grau et al. show that cytochrome c oxidase assembly factor 6 (COA6) is part of the COX2 copper relay system involving the copper metallochaperone SCO2. Mutations preventing SCO2/COA6 interactions underlie patient mitochondrial cardiomyopathies.
Human SCO1 and SCO2 code for essential metallochaperones with ill-defined functions in the biogenesis of the CuA site of cytochrome c oxidase subunit II (CO II). Here, we have used patient cell lines ...to investigate the specific roles of each SCO protein in this pathway. By pulse-labeling mitochondrial translation products, we demonstrate that the synthesis of CO II is reduced in SCO2, but not in SCO1, cells. Despite this biosynthetic defect, newly synthesized CO II is more stable in SCO2 cells than in control cells. RNAi-mediated knockdown of mutant SCO2 abolishes CO II labeling in the translation assay, whereas knockdown of mutant SCO1 does not affect CO II synthesis. These results indicate that SCO2 acts upstream of SCO1, and that it is indispensable for CO II synthesis. The subsequent maturation of CO II is contingent upon the formation of a complex that includes both SCO proteins, each with a functional CxxxC copper-coordinating motif. In control cells, the cysteines in this motif in SCO1 exist as a mixed population comprised of oxidized disulphides and reduced thiols; however, the relative ratio of oxidized to reduced cysteines in SCO1 is perturbed in cells from both SCO backgrounds. Overexpression of wild-type SCO2, or knockdown of mutant SCO2, in SCO2 cells alters the ratio of oxidized to reduced cysteines in SCO1, suggesting that SCO2 acts as a thiol-disulphide oxidoreductase to oxidize the copper-coordinating cysteines in SCO1 during CO II maturation. Based on these data we present a model in which each SCO protein fulfills distinct, stage-specific functions during CO II synthesis and CuA site maturation.
We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable ...mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu+-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu+ in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metalation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a global copper deficiency at the whole cell level, total copper and exchangeable mitochondrial Cu+ pools in SCO1 and SCO2 patient fibroblasts are largely unaltered relative to wild-type controls. Our findings reveal that the cell maintains copper homeostasis in mitochondria even in situations of copper deficiency and mitochondrial metallochaperone malfunction, illustrating the importance of regulating copper stores in this energy-producing organelle.
Copper 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.
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