Characterization of the hCTR1 gene: Genomic organization, functional expression, and identification of a highly homologous processed gene
Introduction
The cellular copper content is generally determined by a delicate balance between uptake and efflux of copper ions by transmembrane transport proteins that are remarkably conserved between different organisms (for a review, see Pena et al., 1999). For a wide range of organisms, copper efflux has been shown to be mediated by proteins belonging to a structurally distinct subgroup of the P-type ATPases (Solioz 1998). The human disorders Menkes and Wilson's diseases, are caused by the deficiency of two such ATPases, which are involved in copper efflux and in delivery of copper to secreted metallo-enzymes in the trans-Golgi network (for reviews, see DiDonato and Sarkar, 1997, Pena et al., 1999).
Uptake of copper ions has been most thoroughly studied in the yeast S. cerevisiae, where two partially redundant membrane proteins, Ctr1 and Ctr3, mediate high-affinity uptake of Cu(I) ions formed by prior reduction by a cell-surface Cu/Fe reductase, Fre1 (Dancis et al., 1994a, Knight et al., 1996). The Ctr1 protein consists of 406 aa residues and contains an extracellular N-terminal domain with 11 potential copper binding motifs of the type MXXM (Dancis et al., 1994a). The Ctr3 protein of 241 amino acid residues lacks the numerous methionine-rich motifs found in Ctr1, but it contains several cysteine residues, some of which might be involved in metal binding (Knight et al., 1996). In spite of the limited sequence homology, the two proteins are predicted to have similar membrane topologies (Labbé et al., 1999). A third yeast protein, Ctr2, with some homology to Ctr1 and Ctr3 has been proposed to be a low-affinity uptake system for copper, but over-expression of Ctr2 was not sufficient to complement a ctr1 ctr3 mutant (Kampfenkel et al., 1995).
A human protein potentially involved in high-affinity copper uptake, hCtr1, was identified by complementation of a yeast ctr1 ctr3 double mutant (Zhou and Gitschier 1997). The human hCtr1 protein is only 190 amino acids long and is predicted to contain three transmembrane segments like the yeast transporters. Interestingly, the primary structures of hCtr1 and a high-affinity copper transporter, Ctr4, from S. pombe suggest that these proteins have evolved by fusion of domains corresponding to the N-terminal domain of Ctr1 and the C-terminal domain of Ctr3 (Labbé et al., 1999). The putative metal-binding N-terminal domain of hCtr1 is considerably shorter than the Ctr1 counterpart, however, and contains only two MXXM motifs. By DNA sequence analysis Zhou and Gitschier (1997) also identified another human gene, hCTR2, which is highly homologous to hCTR1. However, the hCtr2 protein of 143 aa residues did not complement the transport-deficient yeast mutant.
So far, no human disorders of copper metabolism have been associated with the hCTR1 gene, and functional studies of hCtr1 have only been performed in yeast cells. In the present work, the exon–intron structure of the hCTR1 structural gene has been determined to facilitate screening for disease-causing mutations. Furthermore, we provide experimental evidence that hCtr1 functions in copper uptake in human cells, and we describe a highly homologous processed pseudogene, hCTR1ψ.
Section snippets
Preparation of genomic DNA, RNA and cDNA
Genomic DNA was isolated from cultured human fibroblasts by the NaCl extraction method (Grimberg et al., 1989). Total RNA was isolated from 104–106cultured fibroblasts using the QIAgen RNeasy Mini Kit (QIAgen), and the RNA was eluted in 50 μl of elution buffer. The RNA samples used for detection of hCTR1 and hCTR1ψ mRNA were treated with DNAse I (Gibco BRL) prior to the RT-PCR amplification to prevent amplification of residual genomic DNA. Single-stranded cDNA was synthesized with Superscript II
Mapping of introns in the hCTR1 structural gene
Screening for mutations can be performed by reverse transcriptase-polymerase chain reaction (RT-PCR). However, nonsense mutations frequently lead to enhanced mRNA decay (Cheng and Maquat, 1993), which may reduce the amount of the mutated allelic mRNA variant to an extent that it evades detection in such analyses. This problem can be circumvented, however, by performing PCR screenings directly on genomic DNA if the location of introns are known for the gene in question.
To be able to screen for
Involvement of hCTR1 in human copper metabolism and comparison with hCTR2
So far, no human disorders of copper metabolism have been associated with the hCTR1 gene, but the localization of the introns in the hCTR1 coding sequence and the identification of the homologous pseudogene should greatly facilitate the screening of potentially affected individuals for mutations in the hCTR1 gene. Presently, we can only speculate upon the phenotype caused by such mutations. However, patients suffering from Menkes disease are characterized by several neurological and connective
Acknowledgements
We thank Claus Hansen and Zeynep Tümer, Department of Medical Genetics, Institute of Medical Genetics and Biochemistry, The Panum Institute, University of Copenhagen, Denmark for providing help with the chromosomal localization of hCTR1ψ. We are grateful to Jean Feunteun, Laboratoire de Génétique Oncologique, U.R.A. 1967 CNRS, Institute Gustave Roussy, Villejuif, France plasmid for generously providing the plasmid carrying the SV40 large T antigen.
This research was supported by grants from the
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