Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1).
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.
Klomp
Adriana E M
AE
Department of Metabolic Diseases, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands.
Tops
Bastiaan B J
BB
Van Denberg
Inge E T
IE
Berger
Ruud
R
Klomp
Leo W J
LW
D001483:Base Sequence; D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D017931:DNA Primers; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism*
D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D017931:DNA Primers; D008565:Membrane Proteins; D011994:Recombinant Proteins; C490079:SLC31A1 protein, human