Nodule-specific Cu+ -chaperone NCC1 is required for symbiotic nitrogen fixation in Medicago truncatula root nodules.

Cu+ -chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+ -chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. MtNCC1 is able to interact with nodule-specific Cu+ -importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu+ -loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

Amyotrophic Lateral Sclerosis and swim training affect copper metabolism in skeletal muscle in a mouse model of disease.

INTRODUCTION/AIMS: Swim training and regulation of copper metabolism result in clinical benefits in amyotrophic lateral sclerosis (ALS) mice. Therefore, the study aimed to determine whether swim training improves copper metabolism by modifying copper metabolism in the skeletal muscles of ALS mice. METHODS: SOD1G93A mice (n = 6 per group) were used as the ALS model, and wild-type B6SJL (WT) mice as controls (n = 6). Mice with ALS were analyzed before the onset of ALS (ALS BEFORE), at baseline ALS (first disease symptoms, trained and untrained, ALS ONSET), and at the end of ALS (last stage disease, trained and untrained, ALS TERMINAL). Copper concentrations and the level of copper metabolism proteins in the skeletal muscles of the lower leg were determined. RESULTS: ALS disease caused a reduction in the copper concentration in ALS TERMINAL untrained mice compared with the ALS BEFORE (10.43 ± 1.81 and 38.67 ± 11.50 µg/mg, respectively, p = .0213). The copper chaperon for SOD1 protein, which supplies copper to SOD1, and ATPase7a protein (copper exporter), increased at the terminal stage of disease by 57% (p = .0021) and 34% (p = .0372), while the CTR1 protein (copper importer) decreased by 45% (p = .002). Swim training moderately affected the copper concentration and the concentrations of proteins responsible for copper metabolism in skeletal muscles. DISCUSSION: The results show disturbances in skeletal muscle copper metabolism associated with ALS progression, which is moderately affected by swim training. From a clinical point of view, exercise in water for ALS patients should be an essential element of rehabilitation for maintaining quality of life.

An implication of the mitochondrial carrier SLC25A3 as an oxidative stress modulator in NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a type of steatosis not associated with excessive alcohol intake and includes nonalcoholic steatohepatitis (NASH), which can progress to advanced fibrosis and hepatocellular carcinoma. Mitochondrial dysfunction causes oxidative stress, triggering hepatocyte death and inflammation; therefore, the present study aimed to explore relationship between mitochondrial carriers and oxidative stress. Firstly, we established a high fat diet (HFD)-fed ICR mouse NAFLD model characterized by obesity with insulin resistance and found transcriptional upregulation of Slc25a17 and downregulation of Slc25a3 (isoform B) and Slc25a13 in their fatty liver. A mitochondrial phosphate and Cu carrier, SLC25A3, was further studied in wild-type (wt) and SLC25A3-defective HepG2 cells (C1 and C3). SLC25A3 deficiency had insignificant effect on mitochondrial membrane potential (MtMP) and oxygen consumption rate (OCR) in untreated cells but suppressed them when cells were exposed to oleic acid. C1 and C3 cells were prone to produce reactive oxygen species (ROS), and increased ROS was associated with reduced mRNA expression of glutathione peroxidase (GPX) 1 and glutathione disulfide reductase (GSX) in these cell lines. Interestingly, cytoplasmic and mitochondrial Cu accumulation significantly reduced in C1 cells, demonstrating a predominant contribution of SLC25A3 to Cu transport into mitochondrial matrix. Cytotoxicity of free fatty acids was unchanged between wt and SLC25A3-deficient cells. These results indicate that reduced expression of SLC25A3 in fatty liver contributes to electron leak from mitochondria by limiting Cu availability, rendering hepatocytes more susceptible to oxidative stress. This study provides evidence that SLC25A3 is a novel risk factor for developing NASH.

Oenothera biennis with strong copper toxicity resistance enriches trace copper in seeds under copper pollution soil.

Excess copper (Cu) imparts negative effects on plant growth and productivity in soil. To develop the ability of O. biennis to govern pollution soil containing excessive Cu, we investigated seed germination, seedling growth, and seed yield. Furthermore, Cu content and the expression levels of Cu transport related genes in different tissues were measured under exogenous high concentration Cu. O. biennis seeds were sensitive to excess Cu, with an observed reduction in the germination rate, primary root length, fresh weight, and number of seeds germinated daily. Consecutive Cu stress did not cause fatal damage to evening primrose, yet it slowed down plant growth slightly by reducing the leaf water, chlorophyll, plant yield, and seed oil contents while increasing the soluble sugar, proline, malondialdehyde, and H2O2 contents. The Cu content in different organs of O. biennis was disrupted by excess Cu. In particular, the Cu content in O. biennis seeds and seed oil increased and subsequently decreased with the increase of exogenous Cu, reaching a peak under 600 mg·kg-1 consecutive Cu. Furthermore, the 4-month 900 mg·kg-1 Cu treatment did not induce the excessive accumulation of Cu in peels, seeds, and seed oil, maintaining the Cu content within the range required by the Chinese National Food Safety Standards. The treatment also resulted in an upregulation of Cu-uptake (ObCOPT5, ObZIP4, and ObYSL2) and vigorous efflux (ObHMA1) of transport genes, of which expression levels were significant positive correlation (p < 0.05) with the Cu content. Among all organs, the stem replaced the root as the organ exhibited the greatest ability to absorb and store Cu, and even the Cu transport genes could still function continuously in stem under excess Cu. This work identified a species that can tolerate high Cu content in soil while maintaining a high yield. Furthermore, the results revealed the enrichment of Cu to occur primarily in the O. biennis stem rather than the seeds and peel under excess Cu.

Copper Chaperone Atox1 Protected the Cochlea From Cisplatin by Regulating the Copper Transport Family and Cell Cycle.

Antioxidant 1 copper chaperone (Atox1) may contribute to preventing DDP cochlear damage by regulating copper transport family and cell cycle proteins. A rat model of cochlear damage was developed by placing gelatin sponges treated with DDP in the cochlea. HEI-OC1 cells were treated with 133 µM DDP as a cell model. DDP-induced ototoxicity in rats was confirmed by immunofluorescence (IF) imaging. The damage of DDP to HEI-OC1 cells was assessed by using CCK-8, TUNEL, and flow cytometry. The relationship between Atox1, a member of the copper transport protein family, and the damage to in vivo/vitro models was explored by qRT-PCR, western blot, CCK-8, TUNEL, and flow cytometry. DDP had toxic and other side effects causing cochlear damage and promoted HEI-OC1 cell apoptosis and cell cycle arrest. The over-expression of Atox1 (oe-Atox1) was accomplished by transfecting lentiviral vectors into in vitro/vivo models. We found that oe-Atox1 increased the levels of Atox1, copper transporter 1 (CTR1), and SOD3 in HEI-OC1 cells and decreased the expression levels of ATPase copper transporting α (ATP7A) and ATPase copper transporting ß (ATP7B). In addition, the transfection of oe-Atox1 decreased cell apoptosis rate and the number of G2/M stage cells. Similarly, the expression of myosin VI and phalloidin of cochlea cells in vivo decreased. Atox1 ameliorated DDP-induced damage to HEI-OC1 cells or rats' cochlea by regulating the levels of members of the copper transport family.

Epidemiology of Wilson's Disease and Pathogenic Variants of the ATP7B Gene Leading to Diversified Protein Disfunctions.

Wilson's disease (WD) is an autosomal recessive disorder characterized by toxic accumulation of copper in the liver, brain, and other organs. The disease is caused by pathogenic variants in the ATP7B gene, which encodes a P-type copper transport ATPase. Diagnosing WD is associated with numerous difficulties due to the wide range of clinical manifestations and its unknown dependence on the physiological characteristics of the patient. This leads to a delay in the start of therapy and the subsequent deterioration of the patient's condition. However, in recent years, molecular genetic testing of patients using next generation sequencing (NGS) has been gaining popularity. This immediately affected the detection speed of WD. If, previously, the frequency of this disease was estimated at 1:35,000-45,000 people, now, when conducting large molecular genetic studies, the frequency is calculated as 1:7026 people. This certainly points to the problem of identifying WD patients. This review provides an update on the performance of epidemiological studies of WD and describes normal physiological functions of the protein and diversified disfunctions depending on pathogenic variants of the ATP7B gene. Future prospects in the development of WD genetic diagnostics are also discussed.

The copper-linked Escherichia coli AZY operon: Structure, metal binding, and a possible physiological role in copper delivery.

The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this study, we present the first biochemical and functional studies of the AZY proteins. Biochemical characterization and structural modeling indicate that YobA binds a single Cu2+ ion with high affinity. Bioinformatics analysis shows that YebY is widespread and encoded either in AZY operons or in other genetic contexts unrelated to copper homeostasis. We also determined the 1.8 Å resolution crystal structure of E. coli YebY, which closely resembles that of the lantibiotic self-resistance protein MlbQ. Two strictly conserved cysteine residues form a disulfide bond, consistent with the observed periplasmic localization of YebY. Upon treatment with reductants, YebY binds Cu+ and Cu2+ with low affinity, as demonstrated by metal-binding analysis and tryptophan fluorescence. Finally, genetic manipulations show that the AZY operon is not involved in copper tolerance or antioxidant defense. Instead, YebY and YobA are required for the activity of the copper-related NADH dehydrogenase II. These results are consistent with a potential role of the AZY operon in copper delivery to membrane proteins.

The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease.

Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.

Ligand Induced CuII Transport Restricts Cancer and Mycobacterial Growth: Towards a Plug-and-Select Ion Channel Scaffold.

Synthetic channels with high ion selectivity are attractive drug targets for diseases involving ion dysregulation. Achieving selective transport of divalent ions is highly challenging due their high hydration energies. A small tripeptide amphiphilic scaffold installed with a pybox ligand selectively transports CuII ions across membranes. The peptide forms stable dimeric pores in the membrane and transports ions by a Cu2+ /H+ antiport mechanism. The ligand-induced excellent CuII selectivity as well as high membrane permeability of the peptide is exploited to promote cancer cell death. The peptide's ability to restrict mycobacterial growth serves as seeds to evolve antibacterial strategies centred on selectively modulating ion homeostasis in pathogens. This simple peptide can potentially function as a universal, yet versatile, scaffold wherein the ion selectivity can be precisely controlled by modifying the ligand at the C terminus.

PCuAC domains from methane-oxidizing bacteria use a histidine brace to bind copper.

Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is copper-dependent. In addition to pMMO, many other copper proteins are encoded in the genomes of methanotrophs, including proteins that contain periplasmic copper-Achaperone (PCuAC) domains. Using bioinformatics analyses, we identified three distinct classes of PCuAC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCuAC domains from other types of bacteria bind a single Cu(I) ion via an HXnMX21/22HXM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCuAC domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCuAC domain. Crystal structures of the PmoF1 and PmoF2 PCuAC domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HX10H motif. This binding site is distinct from those of previously characterized PCuAC domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCuAC family reveals previously unappreciated diversity, including sequences that contain both the HXnMX21/22HXM and HX10H motifs, and sequences that lack either set of copper-binding ligands. These findings provide the first characterization of an additional class of copper proteins from methanotrophs, further expand the PCuAC family, and afford new insight into the biological significance of histidine brace-mediated copper coordination.

Zinc excess increases cellular demand for iron and decreases tolerance to copper in Escherichia coli.

Transition metals serve as an important class of micronutrients that are indispensable for bacterial physiology but are cytotoxic when they are in excess. Bacteria have developed exquisite homeostatic systems to control the uptake, storage, and efflux of each of biological metals and maintain a thermodynamically balanced metal quota. However, whether the pathways that control the homeostasis of different biological metals cross-talk and render cross-resistance or sensitivity in the host-pathogen interface remains largely unknown. Here, we report that zinc (Zn) excess perturbs iron (Fe) and copper (Cu) homeostasis in Escherichia coli, resulting in increased Fe and decreased Cu levels in the cell. Gene expression analysis revealed that Zn excess transiently up-regulates Fe-uptake genes and down-regulates Fe-storage genes and thereby increases the cellular Fe quota. In vitro and in vivo protein-DNA binding assays revealed that the elevated intracellular Fe poisons the primary Cu detoxification transcription regulator CueR, resulting in dysregulation of its target genes copA and cueO and activation of the secondary Cu detoxification system CusSR-cusCFBA Supplementation with the Fe chelator 2,2'-dipyridyl (DIP) or with the reducing agent GSH abolished the induction of cusCFBA during Zn excess. Consistent with the importance of this metal homeostatic network in cell physiology, combined metal treatment, including simultaneously overloading cells with both Zn (0.25 mm) and Cu (0.25 mm) and sequestering Fe with DIP (50 µm), substantially inhibited E. coli growth. These results advance our understanding of bacterial metallobiology and may inform the development of metal-based antimicrobial regimens to manage infectious diseases.

The yeast copper chaperone for copper-zinc superoxide dismutase (CCS1) is a multifunctional chaperone promoting all levels of SOD1 maturation.

Copper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O2). However, Cu's chemical properties also make it toxic, requiring specific cellular mechanisms for Cu uptake and handling, mediated by Cu chaperones. CCS1, the budding yeast (S. cerevisiae) Cu chaperone for Cu-zinc (Zn) superoxide dismutase (SOD1) activates by directly promoting both Cu delivery and disulfide formation in SOD1. The complete mechanistic details of this transaction along with recently proposed molecular chaperone-like functions for CCS1 remain undefined. Here, we present combined structural, spectroscopic, kinetic, and thermodynamic data that suggest a multifunctional chaperoning role(s) for CCS1 during SOD1 activation. We observed that CCS1 preferentially binds a completely immature form of SOD1 and that the SOD1·CCS1 interaction promotes high-affinity Zn(II) binding in SOD1. Conserved aromatic residues within the CCS1 C-terminal domain are integral in these processes. Previously, we have shown that CCS1 delivers Cu(I) to an entry site at the SOD1·CCS1 interface upon binding. We show here that Cu(I) is transferred from CCS1 to the entry site and then to the SOD1 active site by a thermodynamically driven affinity gradient. We also noted that efficient transfer from the entry site to the active site is entirely dependent upon the oxidation of the conserved intrasubunit disulfide bond in SOD1. Our results herein provide a solid foundation for proposing a complete molecular mechanism for CCS1 activity and reclassification as a first-of-its-kind "dual chaperone."

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration.

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.

Copper physiology in ruminants: trafficking of systemic copper, adaptations to variation in nutritional supply and thiomolybdate challenge.

Ruminants are recognised to suffer from Cu-responsive disorders. Present understanding of Cu transport and metabolism is limited and inconsistent across vets and veterinary professionals. There has been much progress from the studies of the 1980s and early 1990s in cellular Cu transport and liver metabolism which has not been translated into agricultural practice. Cu metabolism operates in regulated pathways of Cu trafficking rather than in pools of Cu lability. Cu in the cell is chaperoned to enzyme production, retention within metallothionein or excretion via the Golgi into the blood. The hepatocyte differs in that Cu-containing caeruloplasmin can be synthesised to provide systemic Cu supply and excess Cu is excreted via bile. The aim of the present review is to improve understanding and highlight the relevant progress in relation to ruminants through the translation of newer findings from medicine and non-ruminant animal models into ruminants.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis.

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

MtCOPT2 is a Cu+ transporter specifically expressed in Medicago truncatula mycorrhizal roots.

Arbuscular mycorrhizal fungi are critical participants in plant nutrition in natural ecosystems and in sustainable agriculture. A large proportion of the phosphorus, nitrogen, sulfur, and transition metal elements that the host plant requires are obtained from the soil by the fungal mycelium and released at the arbuscules in exchange for photosynthates. While many of the plant transporters responsible for obtaining macronutrients at the periarbuscular space have been characterized, the identities of those mediating transition metal uptake remain unknown. In this work, MtCOPT2 has been identified as the only member of the copper transporter family COPT in the model legume Medicago truncatula to be specifically expressed in mycorrhizal roots. Fusing a C-terminal GFP tag to MtCOPT2 expressed under its own promoter showed a distribution pattern that corresponds with arbuscule distribution in the roots. When expressed in tobacco leaves, MtCOPT2-GFP co-localizes with a plasma membrane marker. MtCOPT2 is intimately related to the rhizobial nodule-specific MtCOPT1, which is suggestive of a shared evolutionary lineage that links transition metal nutrition in the two main root endosymbioses in legumes.

Novel interaction of antioxidant-1 with TRAF4: role in inflammatory responses in endothelial cells.

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and copper (Cu), an essential micronutrient, have been implicated in vascular inflammatory diseases. We reported that in proinflammatory cytokine TNF-α-stimulated endothelial cells (ECs), cytosolic Cu chaperone antioxidant-1 (Atox1) functions as a Cu-dependent transcription factor for the NOX organizer p47phox, thereby increasing ROS-dependent inflammatory gene expression. However, the role and mechanism of Atox1 nuclear translocation in inflamed ECs remain unclear. Using enface staining and nuclear fractionation, here we show that Atox1 was localized in the nucleus in inflamed aortas from ApoE-/- mice with angiotensin II infusion on a high-fat diet, while it was found in cytosol in those from control mice. In cultured human ECs, TNF-α stimulation promoted Atox1 nuclear translocation within 15 min, which was associated with Atox1 binding to TNF-α receptor-associated factor 4 (TRAF4) in a Cu-dependent manner. TRAF4 depletion by siRNA significantly inhibited Atox1 nuclear translocation, p47phox expression, and ROS production as well as its downstream VCAM1/ICAM1 expression and monocyte adhesion to inflamed ECs, which were rescued by overexpression of nuclear targeted Atox1. Furthermore, Atox1 colocalized with TRAF4 at the nucleus in TNF-α-stimulated inflamed ECs and vessels. In summary, Cu-dependent Atox1 binding to TRAF4 plays an important role in Atox1 nuclear translocation and ROS-dependent inflammatory responses in TNF-α-stimulated ECs. Thus the Atox1-TRAF4 axis is a novel therapeutic target for vascular inflammatory disease such as atherosclerosis.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis.

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.

Methanobactins: Maintaining copper homeostasis in methanotrophs and beyond.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified natural products that bind copper with high affinity and specificity. Originally identified in methanotrophic bacteria, which have a high need for copper, operons encoding these compounds have also been found in many non-methanotrophic bacteria. The proteins responsible for Mbn biosynthesis include several novel enzymes. Mbn transport involves export through a multidrug efflux pump and re-internalization via a TonB-dependent transporter. Release of copper from Mbn and the molecular basis for copper regulation of Mbn production remain to be elucidated. Future work is likely to result in the identification of new enzymatic chemistry, opportunities for bioengineering and drug targeting of copper metabolism, and an expanded understanding of microbial metal homeostasis.

Reconstitution of a thermophilic Cu+ importer in vitro reveals intrinsic high-affinity slow transport driving accumulation of an essential metal ion.

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu+ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu+ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu+ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu+ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag+ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu+ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu+ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu+ pools.