The transition metal copper is an essential trace element required for a variety of cellular processes such as oxidative metabolism, neurotransmitter synthesis, free radical detoxification, iron uptake, and maturation of connective tissue. However, at elevated levels copper can be toxic to the cell. To balance the beneficial and toxic effects of this essential ion, all organisms possess homeostatic mechanisms that properly control the cellular accumulation, distribution, and detoxification of copper.
It is proposed that copper is imported into the cell by the high-affinity copper transporter hCTR1, and the low-affinity copper import protein hCTR2. Subsequently copper is carried to subcellular compartments or copper-dependent enzymes through the action of target-specific copper chaperon proteins (Fig 1). hATOX1 delivers copper to the trans-golgi network where ATP7A/B, the proteins mutated in the two copper-related diseases (Menkes and Wilson disease respectively), are localized. hCCS donates copper to Cu,Zn superoxide-dismutase, and hCOX17 shuttles copper to the mitochondria, where it is used as a cofactor in cytochrome C oxidase.
Our study focuses on cellular copper import. Mice with a homozygous disruption at the mCtr1 locus have severe developmental defects and die in utero, presumably of copper deficiency. Taken together with the ubiquitous expression of mCtr1 during development, these findings suggest a crucial role for CTR1 in mammalian copper homeostasis and embryonic development. Using a polyclonal antiserum against the N-terminus of hCTR1 we reveal that hCTR1 is heavily glycosylated and is ubiquitously expressed. Between different cell types subcellular hCTR1 localization differs markedly from an intracellular vesicular perinuclear compartment in HeLa cells, to the plasma membrane in Caco2 cells (Fig 2). This steady state localization seems to be the result of a dynamic process, which involves constituve endocytosis of hCTR1 from the plasma membrane.
To characterize the topology of hCTR1, we used indirect immunofluorescense studies and glycosylation site insertion and deletion mapping. Both approaches indicate that hCTR1 contains three transmembrane domains and that the N-terminus of hCTR1, which contains several putative copper binding sites, is localized extracellularly, whereas the C-terminus is exposed to the cytosol. There are some indications that exogenous epitope-tagged hCTR1 associates to higher molecular weight protein complexes. Using yeast two-hybrid studies we have shown that interaction of yeast, mouse, rat and human CTR1 occurs at the sites of their N-terminal domains, and is not dependent on the copper concentration in the growth media. Taken together, these results suggest that CTR1 spans the membrane at least six times, permitting formation of a channel, which is consistent with its proposed role as a copper transporter (Fig 3).
Theses: Diana Klomp - Mechanisms of cellular copper homeostasis. 9 December 2003.
Peter van den Berghe - Posttranslational regulation of copper transporters. 28 April 2009
last update: 11 November 2004