The Conformational Plasticity of the Selectivity Filter Methionines Controls the In-Cell Cu(I) Uptake through the CTR1 transporter

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity Copper Transporter 1 (CTR1), being the main in-cell copper (Cu(I)) entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two Methionine triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake-rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import-rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, these outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake.

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Mammalian copper homeostasis requires retromer-dependent recycling of the high-affinity copper transporter 1

Journal of Cell Science ◽

10.1242/jcs.249201 ◽

2020 ◽

Vol 133 (16) ◽

pp. jcs249201 ◽

Cited By ~ 1

Author(s):

Rachel Curnock ◽

Peter J. Cullen

Keyword(s):

Cell Surface ◽

Nutrient Balance ◽

Copper Homeostasis ◽

Copper Exposure ◽

Copper Uptake ◽

Copper Transporter ◽

High Affinity ◽

Cellular Copper ◽

Copper Transporter 1 ◽

Toxicity Effects

ABSTRACTThe concentration of essential micronutrients, such as copper (used here to describe both Cu+ and Cu2+), within the cell is tightly regulated to avoid their adverse deficiency and toxicity effects. Retromer-mediated sorting and recycling of nutrient transporters within the endo-lysosomal network is an essential process in regulating nutrient balance. Cellular copper homeostasis is regulated primarily by two transporters: the copper influx transporter copper transporter 1 (CTR1; also known as SLC31A1), which controls the uptake of copper, and the copper-extruding ATPase ATP7A, a recognised retromer cargo. Here, we show that in response to fluctuating extracellular copper, retromer controls the delivery of CTR1 to the cell surface. Following copper exposure, CTR1 is endocytosed to prevent excessive copper uptake. We reveal that internalised CTR1 localises on retromer-positive endosomes and, in response to decreased extracellular copper, retromer controls the recycling of CTR1 back to the cell surface to maintain copper homeostasis. In addition to copper, CTR1 plays a central role in the trafficking of platinum. The efficacy of platinum-based cancer drugs has been correlated with CTR1 expression. Consistent with this, we demonstrate that retromer-deficient cells show reduced sensitivity to the platinum-based drug cisplatin.

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The CaCTR1 gene is required for high-affinity iron uptake and is transcriptionally controlled by a copper-sensing transactivator encoded by CaMAC1

Microbiology ◽

10.1099/mic.0.27004-0 ◽

2004 ◽

Vol 150 (7) ◽

pp. 2197-2208 ◽

Cited By ~ 36

Author(s):

Marcus E. Marvin ◽

Robert P. Mason ◽

Annette M. Cashmore

Keyword(s):

Saccharomyces Cerevisiae ◽

Iron Uptake ◽

Invasive Growth ◽

Hostile Environment ◽

Null Mutant ◽

Copper Uptake ◽

Similar System ◽

Copper Transporter ◽

High Affinity ◽

Null Strain

The ability of Candida albicans to acquire iron from the hostile environment of the host is known to be necessary for virulence and appears to be achieved using a similar system to that described for Saccharomyces cerevisiae. In S. cerevisiae, high-affinity iron uptake is dependent upon the acquisition of copper. The authors have previously identified a C. albicans gene (CaCTR1) that encodes a copper transporter. Deletion of this gene results in a mutant strain that grows predominantly as pseudohyphae and displays aberrant morphology in low-copper conditions. This paper demonstrates that invasive growth by C. albicans is induced by low-copper conditions and that this is augmented in a Cactr1-null strain. It also shows that deletion of CaCTR1 results in defective iron uptake. In S. cerevisiae, genes that facilitate high-affinity copper uptake are controlled by a copper-sensing transactivator, ScMac1p. The authors have now identified a C. albicans gene (CaMAC1) that encodes a copper-sensing transactivator. A Camac1-null mutant displays phenotypes similar to those of a Cactr1-null mutant and has no detectable CaCTR1 transcripts in low-copper conditions. It is proposed that high-affinity copper uptake by C. albicans is necessary for reductive iron uptake and is transcriptionally controlled by CaMac1p in a similar manner to that in S. cerevisiae.

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The Fe(II) permease Fet4p functions as a low affinity copper transporter and supports normal copper trafficking in Saccharomyces cerevisiae

Biochemical Journal ◽

10.1042/bj3510477 ◽

2000 ◽

Vol 351 (2) ◽

pp. 477-484 ◽

Cited By ~ 50

Author(s):

Richard HASSETT ◽

David R. DIX ◽

David J. EIDE ◽

Daniel J. KOSMAN

Keyword(s):

Metal Ion ◽

Competitive Inhibitor ◽

Yeast Cells ◽

Amino Acid Residues ◽

Copper Uptake ◽

Copper Transporter ◽

Copper Trafficking ◽

High Affinity ◽

Uptake Pathway ◽

Mutant Forms

The plasma-membrane of Saccharomycescerevisiae contains high affinity permeases for Cu(I) and Fe(II). A low affinity Fe(II) permease has also been identified, designated Fet4p. A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion. We demonstrate in the present study that Fet4p can function as a low affinity copper permease. Copper is a non-competitive inhibitor of 55Fe uptake through Fet4p (Ki = 22µM). Fet4p-dependent 67Cu uptake was kinetically characterized, with Km and Vmax values of 35µM and 8pmol of copper/min per 106 cells respectively. A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p. Mutant forms of Fet4p that exhibited decreased efficiency in 55/59Fe uptake were similarly compromised in 67Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes. The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p. This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast. Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p. Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx. 10µM caused a 50% reduction in the transcriptional activity of Mac1p. The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small ‘holding’pool prior to its partitioning within the cell.

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Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1)

Biochemical Journal ◽

10.1042/bj20011803 ◽

2002 ◽

Vol 364 (2) ◽

pp. 497-505 ◽

Cited By ~ 131

Author(s):

Adriana E.M. KLOMP ◽

Bastiaan B.J. TOPS ◽

Inge E.T. vandenBERG ◽

Ruud BERGER ◽

Leo W.J. KLOMP

Keyword(s):

Subcellular Localization ◽

Molecular Mechanisms ◽

Biochemical Characterization ◽

Copper Toxicity ◽

Menkes Disease ◽

Cell Types ◽

Polyclonal Antiserum ◽

Copper Uptake ◽

Copper Transporter ◽

Copper Transporter 1

The human copper transporter 1 gene (hCTR1) was previously identified by functional complementation in ctr1-deficient yeast. Overexpression of hCTR1 in wild-type yeast leads to increased sensitivity to copper toxicity, and mice with a homozygous disruption at the Ctr1 locus die early during embryogenesis. It is proposed that hCTR1 is responsible for high-affinity copper uptake into human cells, but the underlying molecular mechanisms are unknown. To begin to investigate the biochemical characteristics of hCTR1, a polyclonal antiserum was raised against recombinant hCTR1-fusion peptides. Biosynthetic studies using this antiserum revealed that hCTR1 was synthesized as a precursor protein of 28kDa containing N-linked oligosaccharides, and is then converted to a mature protein of approx. 35kDa, which is ubiquitously expressed. Immunofluorescence studies showed that subcellular hCTR1 localization differed markedly between cell types. In some cell lines, hCTR1 was located predominantly in an intracellular vesicular perinuclear compartment, and in others hCTR1 was located predominantly at the plasma membrane. In contrast with the copper export P-type ATPases mutated in Wilson disease and Menkes disease, the localization of hCTR1 was not influenced by copper concentrations. Inhibition of endocytosis by methyl-β-cyclodextrin caused a partial redistribution of hCTR1 to the cell surface of HeLa cells. Taken together, the results in this study suggest a cell-specific control of copper uptake, which involves subcellular localization of the hCTR1 protein.

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The Candida albicans CTR1 gene encodes a functional copper transporter

Microbiology ◽

10.1099/mic.0.26172-0 ◽

2003 ◽

Vol 149 (6) ◽

pp. 1461-1474 ◽

Cited By ~ 42

Author(s):

Marcus E. Marvin ◽

Peter H. Williams ◽

Annette M. Cashmore

Keyword(s):

Candida Albicans ◽

Amino Acid ◽

Protein Product ◽

Uptake System ◽

Copper Binding ◽

Null Mutant ◽

Copper Uptake ◽

Similar System ◽

Copper Transporter ◽

High Affinity

Copper and iron uptake in Saccharomyces cerevisiae are linked through a high-affinity ferric/cupric-reductive uptake system. Evidence suggests that a similar system operates in Candida albicans. The authors have identified a C. albicans gene that is able to rescue a S. cerevisiae ctr1/ctr3-null mutant defective in high-affinity copper uptake. The 756 bp ORF, designated CaCTR1, encodes a 251 amino acid protein with a molecular mass of 27·8 kDa. Comparisons between the deduced amino acid sequence of the C. albicans Ctr1p and S. cerevisiae Ctr1p indicated that they share 39·6 % similarity and 33·0 % identity over their entire length. Within the predicted protein product of CaCTR1 there are putative transmembrane regions and sequences that resemble copper-binding motifs. The promoter region of CaCTR1 contains four sequences with significant identity to S. cerevisiae copper response elements. CaCTR1 is transcriptionally regulated in S. cerevisiae in response to copper availability by the copper-sensing transactivator Mac1p. Transcription of CaCTR1 in C. albicans is also regulated in a copper-responsive manner. This raises the possibility that CaCTR1 may be regulated in C. albicans by a Mac1p-like transactivator. A C. albicans ctr1-null mutant displays phenotypes consistent with the lack of copper uptake including growth defects in low-copper and low-iron conditions, a respiratory deficiency and sensitivity to oxidative stress. Furthermore, changes in morphology were observed in the C. albicans ctr1-null mutant. It is proposed that CaCTR1 facilitates transport of copper into the cell.

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The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1

Metallomics ◽

10.1039/c8mt00274f ◽

2018 ◽

Vol 10 (12) ◽

pp. 1723-1727 ◽

Cited By ~ 15

Author(s):

Ewelina Stefaniak ◽

Dawid Płonka ◽

Simon C. Drew ◽

Karolina Bossak-Ahmad ◽

Kathryn L. Haas ◽

...

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Copper Uptake ◽

Model Peptide ◽

Copper Transporter ◽

Copper Transporter 1

The superior Cu(ii) affinity of human copper transporter 1 (hCtr1) drives copper acquisition from human serum albumin (HSA).

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Cloning, expression pattern and essentiality of the high-affinity copper transporter 1 (ctr1) gene in zebrafish

Gene ◽

10.1016/j.gene.2003.11.019 ◽

2004 ◽

Vol 328 ◽

pp. 113-120 ◽

Cited By ~ 56

Author(s):

Natalia C. Mackenzie ◽

Mónica Brito ◽

Ariel E. Reyes ◽

Miguel L. Allende

Keyword(s):

Expression Pattern ◽

Copper Transporter ◽

High Affinity ◽

Copper Transporter 1

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Mechanistic Comparison of Human High-Affinity Copper Transporter 1-Mediated Transport between Copper Ion and Cisplatin

Molecular Pharmacology ◽

10.1124/mol.109.056416 ◽

2009 ◽

Vol 76 (4) ◽

pp. 843-853 ◽

Cited By ~ 37

Author(s):

Zheng D. Liang ◽

David Stockton ◽

Niramol Savaraj ◽

Macus Tien Kuo

Keyword(s):

Copper Ion ◽

Copper Transporter ◽

High Affinity ◽

Copper Transporter 1

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Deletion of hepatic Ctr1 reveals its function in copper acquisition and compensatory mechanisms for copper homeostasis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90632.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. G356-G364 ◽

Cited By ~ 49

Author(s):

Heejeong Kim ◽

Hwa-Young Son ◽

Sarah M. Bailey ◽

Jaekwon Lee

Keyword(s):

Critical Role ◽

Copper Metabolism ◽

Copper Homeostasis ◽

Normal Growth ◽

Copper Uptake ◽

Compensatory Mechanisms ◽

Copper Transporter ◽

Hepatic Copper ◽

Urinary Copper ◽

Copper Transporter 1

Copper is a vital trace element required for normal growth and development of many organisms. To determine the roles for copper transporter 1 (Ctr1) in hepatic copper metabolism and the contribution of the liver to systemic copper homeostasis, we have generated and characterized mice in which Ctr1 is deleted specifically in the liver. These mice express less than 10% residual Ctr1 protein in the liver and exhibit a small but significant growth retardation, which disappears with age. Hepatic copper concentrations and the activities of copper-requiring enzymes are reduced; however, mild copper deficiency relative to Ctr1 protein deficit indicates compensatory mechanisms for copper metabolism. Copper concentrations of other organs did not alter despite the defect in hepatic copper uptake. Whereas biliary copper excretion is reduced, urinary copper concentration in these mice is higher than that of control mice. Our data indicate that Ctr1 plays a critical role in copper acquisition in the liver, and, when Ctr1 expression is compromised, compensatory mechanisms facilitate copper uptake and/or retention in the liver and excretion of copper via urine.

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