Isolation of a murine copper transporter gene, tissue specific expression and functional complementation of a yeast copper transport mutant
Introduction
Copper (Cu) is an essential cofactor for many enzymes including Cu, Zn superoxide dismutase (Cu, Zn SOD), cytochrome c oxidase, lysyl oxidase, dopamine β-monooxygenase, peptidylglycine-amidating monooxygenase and ceruloplasmin (Linder, 1991, Pena et al., 1999). Although Cu is an essential trace element, uncontrolled Cu accumulation readily facilitates the production of hydroxyl radical, which causes lipid peroxidation, oxidation of proteins and cleavage of DNA and RNA (Halliwell and Gutteridge, 1984, Stadtman, 1992). The importance of maintaining Cu homeostasis between essential nutritional levels and toxic levels is underscored by the existence of human genetic disorders of Cu homeostasis such as Menkes syndrome and Wilson disease (Bull and Cox, 1994, DiDonato and Sarkar, 1997, Schaefer and Gitlin, 1999). The entrapment of Cu in intestinal cells and vascular endothelial cells in the blood–brain barrier in Menkes patients results in a severe deficiency in the activity of Cu-dependent enzymes that is eventually lethal. Wilson disease is characterized by excessive accumulation of Cu in the liver and brain that leads to devastating hepatic dysfunction and neurological defects. Therefore, given the critical role Cu plays in crucial biochemical reactions, and the consequences of abnormal copper homeostasis, it is important to understand the identity, mechanisms of action and regulation of cellular components responsible for the acquisition, distribution and detoxification of Cu.
The baker's yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have provided excellent model systems for the identification and characterization of proteins that play key roles in cellular Cu homeostasis (Eide, 1998, Labbé and Thiele, 1999, Pena et al., 1999). High affinity Cu transport in S. cerevisiae is mediated by two functionally redundant yet structurally distinct integral membrane proteins Ctr1 and Ctr3. Indeed, the Ctr1 and Ctr3 proteins may have fused during the course of evolution to generate the Ctr4 high affinity Cu transporter in S. pombe and a putative human Cu transporter, hCtr1 (Labbé and Thiele, 1999, Labbé et al., 1999, Zhou and Gitschier, 1997). As a consequence of the inability to provide Cu to the Cu-dependent high affinity Fe uptake system, to cytochrome oxidase and to Cu, Zn superoxide dismutase, yeast cells defective in high affinity Cu uptake are iron starved, unable to carry out mitochondrial respiration and sensitive to superoxide generating compounds (Dancis et al., 1994a, Labbé et al., 1999).
Important steps in understanding Cu metabolism in humans have been accomplished by the cloning of genes and through studies of the proteins defective in Wilson and Menkes diseases. The Menkes (ATP7A) and Wilson (ATP7B) disease genes encode P-type ATPases localized in trans-Golgi network (TGN) where they function to deliver Cu to proteins that traverse the secretory pathway (Bull et al., 1993, Chelly et al., 1993, Mercer et al., 1993, Tanzi et al., 1993, Vulpe et al., 1993, Yamaguchi et al., 1993). ATP7B expression is almost exclusive to hepatic and brain tissue, consistent with its requirement for incorporation of Cu into ceruloplasmin and for biliary excretion of Cu. The identification of mouse and rat models of Menkes and Wilson disease, and the isolation of the corresponding cDNAs and genes, have greatly facilitated comprehensive investigations of the role and mechanisms of action of ATP7A and ATP7B in Cu homeostasis.
Genes encoding additional components involved in Cu uptake and intracellular distribution in mammals have been identified due to the remarkable conservation of structure and function with yeast counterparts. Recently, a human cDNA (hCtr1) has been identified by functional complementation of the respiratory defect associated with S. cerevisiae cells with mutations that inactivate the Ctr1 and Ctr3 high affinity Cu transport genes (Zhou and Gitschier, 1997). Based on both functional complementation in yeast, and structural similarity to the S. cerevisiae and S. pombe high affinity Cu transport proteins, hCtr1 has been postulated to encode a mammalian high affinity Cu transporter. Although the role and physiological importance of hCtr1 in mammalian Cu acquisition have not been demonstrated, the isolation of the murine Ctr1 (mCtr1) cDNA and gene will provide important tools for biochemical and genetic investigations. We report here the isolation and characterization of the mCtr1 cDNA and gene, functional complementation in yeast, exon–intron organization and chromosomal localization of the mCtr1 gene, tissue specific and developmental expression patterns, and steady state mRNA levels in cultured cells and rodents in response to dietary Cu deficiency or sufficiency. These studies provide fundamental information for further investigations of the function and regulation of mCtr1 in mammalian Cu acquisition.
Section snippets
Yeast growth conditions
The yeast strain used in this study was MPY17 (genotype MATa gal1 trp1-1 his3Δ200 ura3-52 ctr1::ura3::Knr ctr3::TRP1 his3 lys2-801 CUP1r) (Pena et al., 1998). Yeast cells were grown in rich medium (YPD: 1% yeast extract, 2% bactopeptone, and 2% dextrose) or YPEG medium (1% yeast extract, 2% bactopeptone, 2% ethanol, and 3% glycerol) in which cell growth requires functional cytochrome oxidase activity for mitochondrial respiration. Synthetic complete (SC) medium lacking specific nutrients was used for
Cloning of hCtr1 and mCtr1 cDNAs
A previous report described the isolation of a human cDNA encoding a putative Cu transport protein, hCtr1, identified by functional complementation of the respiratory defect in yeast cells lacking both high affinity Cu transporters (Zhou and Gitschier, 1997). Using a similar approach, we identified 26 independent human cDNA isolates which, when expressed in ctr1Δ ctr3Δ yeast cells (strain MPY17), complemented the respiratory defect and allowed growth on YPEG medium. Restriction enzyme mapping
Discussion
Cu acquisition from dietary sources and its subsequent distribution to organs and tissues is critical to provide adequate Cu to an array of important Cu-dependent enzymes. Recent investigations have begun to shed light on the identity and mechanisms of action of proteins involved in the distribution, compartmentalization and efflux of Cu ions in yeast and mammalian systems, and clearly demonstrate remarkable conservation of the structure and function of Cu homeostasis proteins in eukaryotes.
Acknowledgements
We thank Marj Peña for comments on the manuscript, and Bruce Brokate and Chen Kuang for skilled technical assistance. This work was supported by grants from the American Heart Association Postdoctoral Fellowship 9920536Z (J.L.), USDA NRICGP 96-35200-3138 (J.R.P.) and the National Institutes of Health GM41840 (D.J.T.) and DK44130 (T.G.).
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