Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jun 1;364(Pt 2):497–505. doi: 10.1042/BJ20011803

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

Adriana E M Klomp 1, Bastiaan B J Tops 1, Inge E T Van Denberg 1, Ruud Berger 1, Leo W J Klomp 1
PMCID: PMC1222595  PMID: 12023893

Abstract

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 28 kDa containing N-linked oligosaccharides, and is then converted to a mature protein of approx. 35 kDa, 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-beta-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.

Full Text

The Full Text of this article is available as a PDF (362.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amaravadi R., Glerum D. M., Tzagoloff A. Isolation of a cDNA encoding the human homolog of COX17, a yeast gene essential for mitochondrial copper recruitment. Hum Genet. 1997 Mar;99(3):329–333. doi: 10.1007/s004390050367. [DOI] [PubMed] [Google Scholar]
  2. Bose S., Chapin S. J., Seetharam S., Feix J., Mostov K. E., Seetharam B. Brefeldin A (BFA) inhibits basolateral membrane (BLM) delivery and dimerization of transcobalamin II receptor in human intestinal epithelial Caco-2 cells. BFA effects on BLM cholesterol content. J Biol Chem. 1998 Jun 26;273(26):16163–16169. doi: 10.1074/jbc.273.26.16163. [DOI] [PubMed] [Google Scholar]
  3. Bull P. C., Cox D. W. Wilson disease and Menkes disease: new handles on heavy-metal transport. Trends Genet. 1994 Jul;10(7):246–252. doi: 10.1016/0168-9525(94)90172-4. [DOI] [PubMed] [Google Scholar]
  4. Bull P. C., Thomas G. R., Rommens J. M., Forbes J. R., Cox D. W. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet. 1993 Dec;5(4):327–337. doi: 10.1038/ng1293-327. [DOI] [PubMed] [Google Scholar]
  5. Chege N. W., Pfeffer S. R. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J Cell Biol. 1990 Sep;111(3):893–899. doi: 10.1083/jcb.111.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chelly J., Tümer Z., Tønnesen T., Petterson A., Ishikawa-Brush Y., Tommerup N., Horn N., Monaco A. P. Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat Genet. 1993 Jan;3(1):14–19. doi: 10.1038/ng0193-14. [DOI] [PubMed] [Google Scholar]
  7. Cid-Arregui A., Parton R. G., Simons K., Dotti C. G. Nocodazole-dependent transport, and brefeldin A--sensitive processing and sorting, of newly synthesized membrane proteins in cultured neurons. J Neurosci. 1995 Jun;15(6):4259–4269. doi: 10.1523/JNEUROSCI.15-06-04259.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ciechanover A., Schwartz A. L., Lodish H. F. Sorting and recycling of cell surface receptors and endocytosed ligands: the asialoglycoprotein and transferrin receptors. J Cell Biochem. 1983;23(1-4):107–130. doi: 10.1002/jcb.240230111. [DOI] [PubMed] [Google Scholar]
  9. Culotta V. C., Klomp L. W., Strain J., Casareno R. L., Krems B., Gitlin J. D. The copper chaperone for superoxide dismutase. J Biol Chem. 1997 Sep 19;272(38):23469–23472. doi: 10.1074/jbc.272.38.23469. [DOI] [PubMed] [Google Scholar]
  10. Dancis A., Haile D., Yuan D. S., Klausner R. D. The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem. 1994 Oct 14;269(41):25660–25667. [PubMed] [Google Scholar]
  11. Dancis A., Yuan D. S., Haile D., Askwith C., Eide D., Moehle C., Kaplan J., Klausner R. D. Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell. 1994 Jan 28;76(2):393–402. doi: 10.1016/0092-8674(94)90345-x. [DOI] [PubMed] [Google Scholar]
  12. De Brabander M. J., Van de Veire R. M., Aerts F. E., Borgers M., Janssen P. A. The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer Res. 1976 Mar;36(3):905–916. [PubMed] [Google Scholar]
  13. Erlich R., Gleeson P. A., Campbell P., Dietzsch E., Toh B. H. Molecular characterization of trans-Golgi p230. A human peripheral membrane protein encoded by a gene on chromosome 6p12-22 contains extensive coiled-coil alpha-helical domains and a granin motif. J Biol Chem. 1996 Apr 5;271(14):8328–8337. doi: 10.1074/jbc.271.14.8328. [DOI] [PubMed] [Google Scholar]
  14. Fridovich I. The biology of oxygen radicals. Science. 1978 Sep 8;201(4359):875–880. doi: 10.1126/science.210504. [DOI] [PubMed] [Google Scholar]
  15. Glerum D. M., Shtanko A., Tzagoloff A. Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase. J Biol Chem. 1996 Jun 14;271(24):14504–14509. doi: 10.1074/jbc.271.24.14504. [DOI] [PubMed] [Google Scholar]
  16. Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med. 1991 Sep 30;91(3C):14S–22S. doi: 10.1016/0002-9343(91)90279-7. [DOI] [PubMed] [Google Scholar]
  17. Hammond C., Helenius A. Quality control in the secretory pathway. Curr Opin Cell Biol. 1995 Aug;7(4):523–529. doi: 10.1016/0955-0674(95)80009-3. [DOI] [PubMed] [Google Scholar]
  18. Horn M., Banting G. Okadaic acid treatment leads to a fragmentation of the trans-Golgi network and an increase in expression of TGN38 at the cell surface. Biochem J. 1994 Jul 1;301(Pt 1):69–73. doi: 10.1042/bj3010069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hung I. H., Suzuki M., Yamaguchi Y., Yuan D. S., Klausner R. D., Gitlin J. D. Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae. J Biol Chem. 1997 Aug 22;272(34):21461–21466. doi: 10.1074/jbc.272.34.21461. [DOI] [PubMed] [Google Scholar]
  20. James D. E., Strube M., Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature. 1989 Mar 2;338(6210):83–87. doi: 10.1038/338083a0. [DOI] [PubMed] [Google Scholar]
  21. Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klomp L. W., Lin S. J., Yuan D. S., Klausner R. D., Culotta V. C., Gitlin J. D. Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis. J Biol Chem. 1997 Apr 4;272(14):9221–9226. doi: 10.1074/jbc.272.14.9221. [DOI] [PubMed] [Google Scholar]
  23. Knight S. A., Labbé S., Kwon L. F., Kosman D. J., Thiele D. J. A widespread transposable element masks expression of a yeast copper transport gene. Genes Dev. 1996 Aug 1;10(15):1917–1929. doi: 10.1101/gad.10.15.1917. [DOI] [PubMed] [Google Scholar]
  24. Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kuo S. C., Lampen O. Tunicamycin inhibition of (3H) glucosamine incorporation into yeast glycoproteins: binding of tunicamycin and interaction with phospholipids. Arch Biochem Biophys. 1976 Feb;172(2):574–581. doi: 10.1016/0003-9861(76)90110-7. [DOI] [PubMed] [Google Scholar]
  26. Kuo Y. M., Zhou B., Cosco D., Gitschier J. The copper transporter CTR1 provides an essential function in mammalian embryonic development. Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6836–6841. doi: 10.1073/pnas.111057298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ladinsky M. S., Howell K. E. The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A. Eur J Cell Biol. 1992 Oct;59(1):92–105. [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Lahtinen U., Hellman U., Wernstedt C., Saraste J., Pettersson R. F. Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein. J Biol Chem. 1996 Feb 23;271(8):4031–4037. doi: 10.1074/jbc.271.8.4031. [DOI] [PubMed] [Google Scholar]
  30. Lee J., Prohaska J. R., Dagenais S. L., Glover T. W., Thiele D. J. Isolation of a murine copper transporter gene, tissue specific expression and functional complementation of a yeast copper transport mutant. Gene. 2000 Aug 22;254(1-2):87–96. doi: 10.1016/s0378-1119(00)00287-0. [DOI] [PubMed] [Google Scholar]
  31. Lee J., Prohaska J. R., Thiele D. J. Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development. Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6842–6847. doi: 10.1073/pnas.111058698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lin S. J., Pufahl R. A., Dancis A., O'Halloran T. V., Culotta V. C. A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport. J Biol Chem. 1997 Apr 4;272(14):9215–9220. [PubMed] [Google Scholar]
  33. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mercer J. F., Livingston J., Hall B., Paynter J. A., Begy C., Chandrasekharappa S., Lockhart P., Grimes A., Bhave M., Siemieniak D. Isolation of a partial candidate gene for Menkes disease by positional cloning. Nat Genet. 1993 Jan;3(1):20–25. doi: 10.1038/ng0193-20. [DOI] [PubMed] [Google Scholar]
  35. Møller L. B., Petersen C., Lund C., Horn N. Characterization of the hCTR1 gene: genomic organization, functional expression, and identification of a highly homologous processed gene. Gene. 2000 Oct 17;257(1):13–22. doi: 10.1016/s0378-1119(00)00394-2. [DOI] [PubMed] [Google Scholar]
  36. Ooi C. E., Rabinovich E., Dancis A., Bonifacino J. S., Klausner R. D. Copper-dependent degradation of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p in the apparent absence of endocytosis. EMBO J. 1996 Jul 15;15(14):3515–3523. [PMC free article] [PubMed] [Google Scholar]
  37. Orlean P., Kuranda M. J., Albright C. F. Analysis of glycoproteins from Saccharomyces cerevisiae. Methods Enzymol. 1991;194:682–697. doi: 10.1016/0076-6879(91)94050-m. [DOI] [PubMed] [Google Scholar]
  38. Petris M. J., Mercer J. F., Culvenor J. G., Lockhart P., Gleeson P. A., Camakaris J. Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J. 1996 Nov 15;15(22):6084–6095. [PMC free article] [PubMed] [Google Scholar]
  39. Roelofsen H., Wolters H., Van Luyn M. J., Miura N., Kuipers F., Vonk R. J. Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion. Gastroenterology. 2000 Sep;119(3):782–793. doi: 10.1053/gast.2000.17834. [DOI] [PubMed] [Google Scholar]
  40. Ronin C., Stannard B. S., Rosenbloom I. L., Magner J. A., Weintraub B. D. Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins. Biochemistry. 1984 Sep 25;23(20):4503–4510. doi: 10.1021/bi00315a001. [DOI] [PubMed] [Google Scholar]
  41. Rudd P. M., Dwek R. A. Glycosylation: heterogeneity and the 3D structure of proteins. Crit Rev Biochem Mol Biol. 1997;32(1):1–100. doi: 10.3109/10409239709085144. [DOI] [PubMed] [Google Scholar]
  42. Strous G. J., van Kerkhof P., van Meer G., Rijnboutt S., Stoorvogel W. Differential effects of brefeldin A on transport of secretory and lysosomal proteins. J Biol Chem. 1993 Feb 5;268(4):2341–2347. [PubMed] [Google Scholar]
  43. Tabuchi M., Yoshimori T., Yamaguchi K., Yoshida T., Kishi F. Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells. J Biol Chem. 2000 Jul 21;275(29):22220–22228. doi: 10.1074/jbc.M001478200. [DOI] [PubMed] [Google Scholar]
  44. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
  45. Vulpe C. D., Packman S. Cellular copper transport. Annu Rev Nutr. 1995;15:293–322. doi: 10.1146/annurev.nu.15.070195.001453. [DOI] [PubMed] [Google Scholar]
  46. Vulpe C., Levinson B., Whitney S., Packman S., Gitschier J. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat Genet. 1993 Jan;3(1):7–13. doi: 10.1038/ng0193-7. [DOI] [PubMed] [Google Scholar]
  47. Yamaguchi Y., Heiny M. E., Gitlin J. D. Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem Biophys Res Commun. 1993 Nov 30;197(1):271–277. doi: 10.1006/bbrc.1993.2471. [DOI] [PubMed] [Google Scholar]
  48. Yamaguchi Y., Heiny M. E., Suzuki M., Gitlin J. D. Biochemical characterization and intracellular localization of the Menkes disease protein. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14030–14035. doi: 10.1073/pnas.93.24.14030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yancey P. G., Rodrigueza W. V., Kilsdonk E. P., Stoudt G. W., Johnson W. J., Phillips M. C., Rothblat G. H. Cellular cholesterol efflux mediated by cyclodextrins. Demonstration Of kinetic pools and mechanism of efflux. J Biol Chem. 1996 Jul 5;271(27):16026–16034. doi: 10.1074/jbc.271.27.16026. [DOI] [PubMed] [Google Scholar]
  50. Yang W., Storrie B. Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins. Mol Biol Cell. 1998 Jan;9(1):191–207. doi: 10.1091/mbc.9.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou B., Gitschier J. hCTR1: a human gene for copper uptake identified by complementation in yeast. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7481–7486. doi: 10.1073/pnas.94.14.7481. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES