Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease.

Kidneys play an especial role in copper redistribution in the organism. The epithelial cells of proximal tubules perform the functions of both copper uptake from the primary urine and release to the blood. These cells are equipped on their apical and basal membrane with copper transporters CTR1 and ATP7A. Mosaic mutant mice displaying a functional dysfunction of ATP7A are an established model of Menkes disease. These mice exhibit systemic copper deficiency despite renal copper overload, enhanced by copper therapy, which is indispensable for their life span extension. The aim of this study was to analyze the expression of Slc31a1 and Slc31a2 genes (encoding CTR1/CTR2 proteins) and the cellular localization of the CTR1 protein in suckling, young and adult mosaic mutants. Our results indicate that in the kidney of both intact and copper-injected 14-day-old mutants showing high renal copper content, CTR1 mRNA level is not up-regulated compared to wild-type mice given a copper injection. The expression of the Slc31a1 gene in 45-day-old mice is even reduced compared with intact wild-type animals. In suckling and young copper-injected mutants, the CTR1 protein is relocalized from the apical membrane to the cytoplasm of epithelial cells of proximal tubules, the process which prevents copper transport from the primary urine and, thus, protects cells against copper toxicity.

Ubiquitin-independent proteasome system is required for degradation of Arabidopsis COPPER TRANSPORTER 2.

Plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients. Membrane transport proteins play a central role in nutrient uptake from soil. In Arabidopsis thaliana, the COPPER TRANSPORTOR (COPT) family encodes high-affinity copper transporters. COPT2 is transcriptionally regulated in response to changing levels of cellular copper. However, little is known about whether COPT2 activity is subject to multiple levels of regulation. Here, we showed that the plasma membrane-/endoplasmic reticulum-resident COPT2 protein is degraded in response to excess copper. Confocal microscopy analysis together with pharmacological treatment with a vesicle trafficking inhibitor or vacuolar ATPase inhibitor indicated that copper-mediated downregulation of COPT2 is unlikely to be controlled by endosomal recycling and vacuolar system. However, COPT2 protein is stabilized by proteasome inhibition. Through site-directed mutagenesis, we found that COPT2 cannot be ubiquitinated, and lysine residues at the C-terminus is dispensable for copper-induced degradation of COPT2 but required for copper acquisition. Altogether, our findings reveal that unlike many metal transporters in Arabidopsis, COPT2 is a substrate of ubiquitin-independent proteasomal degradation but not of vacuolar proteases. These findings highlight the mechanistic diversity and complexity of plasma membrane transporter degradation.

Altered copper homeostasis underlies sensitivity of hepatocellular carcinoma to copper chelation.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, of which ∼800 000 new cases will be diagnosed worldwide this year, portends a five-year survival rate of merely 17% in patients with unresectable disease. This dismal prognosis is due, at least in part, from the late stage of diagnosis and the limited efficacy of systemic therapies. As a result, there is an urgent need to identify risk factors that contribute to HCC initiation and provide targetable vulnerabilities to improve patient survival. While myriad risk factors are known, elevated copper (Cu) levels in HCC patients and the incidence of hepatobiliary malignancies in Wilson disease patients, which exhibit hereditary liver Cu overload, suggests the possibility that metal accumulation promotes malignant transformation. Here we found that expression of the Cu transporter genes ATP7A, ATP7B, SLC31A1, and SLC31A2 was significantly altered in liver cancer samples and were associated with elevated Cu levels in liver cancer tissue and cells. Further analysis of genomic copy number data revealed that alterations in Cu transporter gene loci correlate with poorer survival in HCC patients. Genetic loss of the Cu importer SLC31A1 (CTR1) or pharmacologic suppression of Cu decreased the viability, clonogenic survival, and anchorage-independent growth of human HCC cell lines. Mechanistically, CTR1 knockdown or Cu chelation decreased glycolytic gene expression and downstream metabolite utilization and as a result forestalled tumor cell survival after exposure to hypoxia, which mimics oxygen deprivation elicited by transarterial embolization, a standard-of-care therapy used for patients with unresectable HCC. Taken together, these findings established an association between altered Cu homeostasis and HCC and suggest that limiting Cu bioavailability may provide a new treatment strategy for HCC by restricting the metabolic reprogramming necessary for cancer cell survival.

Designing yeast as plant-like hyperaccumulators for heavy metals.

Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators are promising candidates for bioremediation applications. However, compared to bacteria-based bioremediation systems, plant life cycle is long and growing conditions are difficult to maintain hindering their adoption. Herein, we combine the robust growth and engineerability of bacteria with the unique waste management mechanisms of plants by using a more tractable platform-the common baker's yeast-to create plant-like hyperaccumulators. Through overexpression of metal transporters and engineering metal trafficking pathways, engineered yeast strains are able to sequester metals at concentrations 10-100 times more than established hyperaccumulator thresholds for chromium, arsenic, and cadmium. Strains are further engineered to be selective for either cadmium or strontium removal, specifically for radioactive Sr90. Overall, this work presents a systematic approach for transforming yeast into metal hyperaccumulators that are as effective as their plant counterparts.

Cu(II) Binding to the N-Terminal Model Peptide of the Human Ctr2 Transporter at Lysosomal and Extracellular pH.

It was shown that His3 of human copper transporter 1 (hCtr1) prompts the ATCUN-like Cu(II) coordination for model peptides of the hCtr1 N-terminus. Its high Cu(II) affinity is a potential driving force for the transfer of Cu(II) from extracellular Cu(II) carriers to hCtr1. Having a sequence similar to that of hCtr1, hCtr2 has been proposed as another human copper transporter. However, the N-terminal domain of hCtr2 is much shorter than that of hCtr1, with different copper binding motifs at its N-terminus. Employing a model peptide of the hCtr2 N-terminus, MAMHF-am, we demonstrated that His4 provides a unique pattern of Cu(II) complexes, involving Met sulfurs in their Cu(II) coordination sphere. The affinity of Cu(II) for MAMHF-am is a few orders of magnitude lower than that reported for the hCtr1 model peptides at the extracellular pH of 7.4, suggesting a maximal complementary role of Cu(II) binding to hCtr2 in the import of copper from the extracellular space to the cytoplasm. On the other hand, the ability of the hCtr2 model peptide to capture Cu(II) from amino acids and short peptides (potential degradation products of proteins) at pH 5.0 and the known predominant lysosomal localization of hCtr2 support an important potential role of the Cu(II)-hCtr2 interaction in the recovery of copper from lysosomes.

Copper transporter COPT5 participates in the crosstalk between vacuolar copper and iron pools mobilisation.

Copper (Cu) deficiency affects iron (Fe) homeostasis in several plant processes, including the increased Fe requirements due to cuproprotein substitutions for the corresponding Fe counterpart. Loss-of-function mutants from Arabidopsis thaliana high affinity copper transporter COPT5 and Fe transporters NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 3/4 (NRAMP3 and NRAMP4) were used to study the interaction between metals internal pools. A physiological characterisation showed that the copt5 mutant is sensitive to Fe deficiency, and that nramp3nramp4 mutant growth was severely affected under limiting Cu. By a transcriptomic analysis, we observed that NRAMP4 expression was highly induced in the copt5 mutant under Cu deficiency, while COPT5 was overexpressed in the nramp3nramp4 mutant. As a result, an enhanced mobilisation of the vacuolar Cu or Fe pools, when the other metal export through the tonoplast is impaired in the mutants, has been postulated. However, metals coming from internal pools are not used to accomplish the increased requirements that derive from metalloprotein substitution under metal deficiencies. Instead, the metal concentrations present in aerial parts of the copt5 and nramp3nramp4 mutants conversely show compensated levels of these two metals. Together, our data uncover an interconnection between Cu and Fe vacuolar pools, whose aim is to fulfil interorgan metal translocation.

COPT2, a plasma membrane located copper transporter, is involved in the uptake of Au in Arabidopsis.

The mechanism of gold nanoparticle formation and genes involved in such processes, especially Au transport in plants are not understood. Previous reports pointed to the probable role of COPT2 in Au transport based on the transcript accumulation of COPT2 under Au exposure. Here, we provide evidence revealing the additional role of COPT2 for Au mobilization in yeast and Arabidopsis. The COPT2 transcripts significantly accumulated in the root of Arabidopsis under Au exposure. The expression of COPT2 restores Cu uptake ability in ctr1Δctr3Δ mutants and leads to Au sensitivity in yeast, which is comparable to Cu in growth kinetics experiments. The metal measurement data showed that the Au level was increased in COPT2, expressing yeast cells compared to vector transformed control. The copt2 mutant of Arabidopsis displayed a similar growth pattern to that of Col-0 under Au treatment. However, a notable phenotypic difference was noticed in three-week-old plants treated with and without Au. Consistent with yeast, Au uptake was reduced in the copt2 mutant of Arabidopsis. Together, these results clearly reveal the Au uptake capability of COPT2 in yeast and Arabidopsis. This is the first report showing the potential role of any transporter towards uptake and accumulation of Au in plants.

Association between polymorphisms in CTR1, CTR2, ATP7A, and ATP7B and platinum resistance in epithelial ovarian cancer.

The copper transporters CTR1, CTR2, ATP7A, and ATP7B regulate intracellular concentration of platinum by mediating its uptake and efflux in cells. We sought to explore the effect of genetic polymorphisms in CTR1, CTR2, ATP7A, and ATP7B on platinum resistance in patients suffering from epithelial ovarian cancer (EOC). A total of 152 Chinese EOC patients were enrolled in this study, all of whom underwent adjuvant chemotherapy using platinum and taxane after maximal debulking surgery. In total, 11 single-nucleotide polymorphisms (SNPs) in CTR1, CTR2, ATP7A, and ATP7B were genotyped in these patients. The CTR1 rs10981694 polymorphism was observed to be associated with carboplatin resistance, while patients with the rs10981694 G allele showed a significantly higher rate of carboplatin resistance (OR = 4.00, 95% CI 1.309 - 12.23, p < 0.01). In addition, we found that ATP7A rs2227291 was associated with cisplatin resistance and that carriers of the C allele were more sensitive to cisplatin (OR = 0.40, 95% CI: 0.17 - 0.94, p = 0.03). Our findings suggest that the CTR1 and ATP7A genetic polymorphisms could affect platinum resistance. The CTR1 and ATP7A genes might be considered a predictive marker for carboplatin and cisplatin resistance, respectively.
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Cell-surface copper transporters and superoxide dismutase 1 are essential for outgrowth during fungal spore germination.

During fungal spore germination, a resting spore returns to a conventional mode of cell division and resumes vegetative growth, but the requirements for spore germination are incompletely understood. Here, we show that copper is essential for spore germination in Schizosaccharomyces pombe Germinating spores develop a single germ tube that emerges from the outer spore wall in a process called outgrowth. Under low-copper conditions, the copper transporters Ctr4 and Ctr5 are maximally expressed at the onset of outgrowth. In the case of Ctr6, its expression is broader, taking place before and during outgrowth. Spores lacking Ctr4, Ctr5, and the copper sensor Cuf1 exhibit complete germination arrest at outgrowth. In contrast, ctr6 deletion only partially interferes with formation of outgrowing spores. At outgrowth, Ctr4-GFP and Ctr5-Cherry first co-localize at the spore contour, followed by re-location to a middle peripheral spore region. Subsequently, they move away from the spore body to occupy the periphery of the nascent cell. After breaking of spore dormancy, Ctr6 localizes to the vacuole membranes that are enriched in the spore body relative to the germ tube. Using a copper-binding tracker, results showed that labile copper is preferentially localized to the spore body. Further analysis showed that Ctr4 and Ctr6 are required for copper-dependent activation of the superoxide dismutase 1 (SOD1) during spore germination. This activation is critical because the loss of SOD1 activity blocked spore germination at outgrowth. Taken together, these results indicate that cell-surface copper transporters and SOD1 are required for completion of the spore germination program.

Gene duplication and neo-functionalization in the evolutionary and functional divergence of the metazoan copper transporters Ctr1 and Ctr2.

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu+ across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu+ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu+ transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1-/- mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins.

Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity.

The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus.

Copper(I) stabilization by cysteine/tryptophan motif in the extracellular domain of Ctr4.

Copper transporter Ctr4 of fission yeast has a quasi-palindromic sequence rich in cysteine and aromatic amino acid residues, CX4YWNWYX4C (where X represents any amino acid), in the N-terminal extracellular domain. A 24-mer peptide comprising this sequence is bound to Cu(I) through the cysteine thiolate coordination. Luminescence, UV absorption and resonance Raman spectra of the Cu(I)-peptide complex show that at least one of the two tryptophan side chains is located in close proximity to the thiolate-Cu(I) center and interacts with the Cu(I) ion via π-electrons of the indole ring. Although the thiolates and Cu(I) are oxidized to disulfide and Cu(II), respectively, only very slowly in air-saturated solutions, replacements of the tryptophan residues to phenylalanine significantly accelerate the oxidation reactions. The results obtained indicate that the interaction between Cu(I) and tryptophan via π-electrons plays a significant role in protecting the thiolate-Cu(I) center against the oxidation. The cysteine- and tryptophan-rich quasi-palindromic sequence may be a metal binding motif that stabilizes Cu(I) in the oxidizing extracellular environment.

Daily rhythmicity of high affinity copper transport.

A differential demand for copper (Cu) of essential cupro-proteins that act within the mitochondrial and chloroplastal electronic transport chains occurs along the daily light/dark cycles. This requires a fine-tuned spatiotemporal regulation of Cu delivery, becoming especially relevant under non-optimal growth conditions. When scarce, Cu is imported through plasma membrane-bound high affinity Cu transporters (COPTs) whose coding genes are transcriptionally induced by the SPL7 transcription factor. Temporal homeostatic mechanisms are evidenced by the presence of multiple light- and clock-responsive regulatory cis elements in the promoters of both SPL7 and its COPT targets. A model is presented here for such temporal regulation that is based on the synchrony between the basal oscillatory pattern of SPL7 and its targets, such as COPT2. Conversely, Cu feeds back to coordinate intracellular Cu availability on the SPL7-dependent regulation of further Cu acquisition. This occurs via regulation at COPT transporters. Moreover, exogenous Cu affects several circadian-clock components, such as the timing of GIGANTEA transcript abundance. Together we propose that there is a dynamic response to Cu that is integrated over diurnal time to maximize metabolic efficiency under challenging conditions.

Modulation of copper deficiency responses by diurnal and circadian rhythms in Arabidopsis thaliana.

Copper homeostasis under deficiency is regulated by the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) transcription factor. The daily oscillating expression of two SPL7-dependent copper deficiency markers, COPPER TRANSPORTER (COPT2) and IRON SUPEROXIDE DISMUTASE (FSD1), has been followed by quantitative PCR and in promoter:LUCIFERASE transgenic plants. Both genes showed circadian and diurnal regulation. Under copper deficiency, their expression decreased drastically in continuous darkness. Accordingly, total copper content was slightly reduced in etiolated seedlings under copper deficiency. The expression of SPL7 and its targets COPT2 and FSD1 was differently regulated in various light signalling mutants. On the other hand, increased copper levels reduced the amplitude of nuclear circadian clock components, such as GIGANTEA (GI). The alteration of copper homeostasis in the COPT1 overexpression line and spl7 mutants also modified the amplitude of a classical clock output, namely the circadian oscillation of cotyledon movements. In the spl7 mutant, the period of the oscillation remained constant. These results suggest a feedback of copper transport on the circadian clock and the integration of rhythmic copper homeostasis into the central oscillator of plants.

Decreased expression of CTR2 predicts poor prognosis of patients with clear cell renal cell carcinoma.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is well known for its hypervascularity due to the Von Hippel-Lindau/hypoxia-inducible factor dysregulation. Recent findings suggested that copper transporter 2 (CTR2) is also associated with angiogenesis through copper׳s modulation of the hypoxia-inducible factor pathway. Our group thus explored the prognostic role of CTR2 in patients with ccRCC. MATERIALS AND METHODS: A total of 331 patients with ccRCC who underwent nephrectomy were enrolled between February 2005 and June 2007 at a single institution. The median follow-up was 98.97 months (2.63-120.47mo). Patients׳ samples were collected and stained for CTR2 by immunohistochemistry. The staining intensity was analyzed quantitatively and defined as high/low expression using X-tile software. Stage, Size, Grade, and Necrosis score and University of California Los Angeles Integrated Staging System score were applied to stratify patients׳ risks. Survival analyses were performed through the Kaplan-Meier method and Cox proportional hazard model. After integrating tumoral CTR2 expression with other clinical parameters, 2 nomograms were generated for overall survival (OS) and disease-free survival (DFS) prediction. RESULTS: CTR2 expression in ccRCC was decreased compared with that in the peritumoral tissue (P<0.001) and negatively correlated with many other clinical parameters. In survival analyses using the Kaplan-Meier method, low tumoral CTR2 expression displayed a dismal prognostic effect both in OS and DFS prediction (P<0.001). Multivariate analyses also revealed the same result after adjusted with other clinical parameters (P<0.001). Stratifying patients into 3 risk levels using the Stage, Size, Grade, and Necrosis score and University of California Los Angeles Integrated Staging System score, decreased CTR2 expression associated with shorter OS and DFS in the low- and intermediate-risk groups. Moreover, the generated nomogram integrating tumoral CTR2 expression performed better in predicting patients׳ OS than using TNM stages alone (c-index = 0.799; 95% CI: 0.752-0.846 vs. 0.691; 95% CI: 0.637-0.745). CONCLUSIONS: CTR2 is a novel prognostic marker for patients with ccRCC both in OS and DFS prediction, and could be incorporated with other clinical parameters for better patient risk stratification.

The Multi-allelic Genetic Architecture of a Variance-Heterogeneity Locus for Molybdenum Concentration in Leaves Acts as a Source of Unexplained Additive Genetic Variance.

Genome-wide association (GWA) analyses have generally been used to detect individual loci contributing to the phenotypic diversity in a population by the effects of these loci on the trait mean. More rarely, loci have also been detected based on variance differences between genotypes. Several hypotheses have been proposed to explain the possible genetic mechanisms leading to such variance signals. However, little is known about what causes these signals, or whether this genetic variance-heterogeneity reflects mechanisms of importance in natural populations. Previously, we identified a variance-heterogeneity GWA (vGWA) signal for leaf molybdenum concentrations in Arabidopsis thaliana. Here, fine-mapping of this association reveals that the vGWA emerges from the effects of three independent genetic polymorphisms that all are in strong LD with the markers displaying the genetic variance-heterogeneity. By revealing the genetic architecture underlying this vGWA signal, we uncovered the molecular source of a significant amount of hidden additive genetic variation or "missing heritability". Two of the three polymorphisms underlying the genetic variance-heterogeneity are promoter variants for Molybdate transporter 1 (MOT1), and the third a variant located ~25 kb downstream of this gene. A fourth independent association was also detected ~600 kb upstream of MOT1. Use of a T-DNA knockout allele highlights Copper Transporter 6; COPT6 (AT2G26975) as a strong candidate gene for this association. Our results show that an extended LD across a complex locus including multiple functional alleles can lead to a variance-heterogeneity between genotypes in natural populations. Further, they provide novel insights into the genetic regulation of ion homeostasis in A. thaliana, and empirically confirm that variance-heterogeneity based GWA methods are a valuable tool to detect novel associations of biological importance in natural populations.

Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

The copper transporter 1 (CTR1) is required to maintain the stability of copper transporter 2 (CTR2).

Mammalian cells have two influx Cu transporters that form trimers in membranes. CTR1 is the high affinity transporter that resides largely in the plasma membrane, and CTR2 is the low affinity transporter that is primarily associated with vesicular structures inside the cell. The major differences between CTR1 and CTR2 are that CTR1 contains a HIS/MET-rich domain N-terminal of the METS that participate in the first two stacked rings that form the pore, and a longer C-terminal tail that includes a Cu binding HIS-CYS-HIS (HCH) motif right at the end. It has been reported that CTR1 and CTR2 are physically associated with each other in the cell. We used the CRISPR-Cas9 technology to knock out either CTR1 or CTR2 in fully malignant HEK293T and OVCAR8 human ovarian cancer cells to investigate the interaction of CTR1 and CTR2. We report here that the level of CTR2 protein is markedly decreased in CTR1 knockout clones while the CTR2 transcript level remains unchanged. CTR2 was found to be highly ubiquitinated in the CTR1 knock out cells, and inhibition of the proteasome prevented the degradation of CTR2 when CTR1 was not present while inhibition of autophagy had no effect. Re-expression of CTR1 rescued CTR2 from degradation in the CTR1 knockout cells. We conclude that CTR1 is essential to maintain the stability of CTR2 and that in the absence of CTR1 CTR2 is degraded by the proteasome. This reinforces the concept that the functions of CTR1 and CTR2 are inter-dependent within the Cu homeostasis system.

Defective copper transport in the copt5 mutant affects cadmium tolerance.

Cadmium toxicity interferes with essential metal homeostasis, which is a problem for both plant nutrition and the consumption of healthy food by humans. Copper uptake is performed by the members of the Arabidopsis high affinity copper transporter (COPT) family. One of the members, COPT5, is involved in copper recycling from the vacuole toward the cytosolic compartment. We show herein that copt5 mutants are more sensitive to cadmium stress than wild-type plants, as indicated by reduced growth. Exacerbated cadmium toxicity in copt5 mutants is due specifically to altered copper traffic through the COPT5 transporter. Three different processes which have been shown to affect cadmium tolerance are altered in copt5 mutants. First, ethylene biosynthesis diminishes under copper deficiency and, in the presence of cadmium, ethylene production diminishes further. Copper deficiency responses are also attenuated under cadmium treatment. Remarkably, while copt5 roots present higher oxidative stress toxicity symptoms than controls, aerial copt5 parts display lower oxidative stress, as seen by reduced cadmium delivery to shoots. Taken together, these results demonstrate that copper transport plays a key role in cadmium resistance, and suggest that oxidative stress triggers an NADPH oxidase-mediated signaling pathway, which contributes to cadmium translocation and basal plant resistance. The slightly lower cadmium levels that reach aerial parts in the copt5 mutants, irrespective of the copper content in the media, suggest a new biotechnological approach to minimize toxic cadmium entry into food chains.

The role of Ctr1 and Ctr2 in mammalian copper homeostasis and platinum-based chemotherapy.

Copper (Cu) is an essential metal for growth and development that has the potential to be toxic if levels accumulate beyond the ability of cells to homeostatically balance uptake with detoxification. One system for Cu acquisition is the integral membrane Cu(+) transporter, Ctr1, which has been quite well characterized in terms of its function and physiology. The mammalian Ctr2 protein has been a conundrum for the copper field, as it is structurally closely related to the high affinity Cu transporter Ctr1, sharing important motifs for Cu transport activity. However, in contrast to mammalian Ctr1, Ctr2 fails to suppress the Cu-dependent growth phenotype of yeast cells defective in Cu(+) import, nor does it appreciably stimulate Cu acquisition when over-expressed in mammalian cells, underscoring important functional dissimilarities between the two proteins. Several roles for the mammalian Ctr2 have been suggested both in vitro and in vivo. Here, we summarize and discuss current insights into the Ctr2 protein and its interaction with Ctr1, its functions in mammalian Cu homeostasis and platinum-based chemotherapy.