title: The N-terminus of the human copper transporter 1 (hCTR1) is localized extracellularly, and interacts with itself.
abstract: We have used indirect immunofluorescense studies and glycosylation-site insertion and deletion mapping to characterize the topology of human copper transporter 1 (hCTR1), the putative human high-affinity copper-import protein. Both approaches indicated 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. Based on previous observations that CTR1 proteins form high-molecular-mass complexes, we investigated directly whether CTR1 proteins interact with themselves. Yeast two-hybrid studies showed 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. Analysis of deletion constructs indicated that multiple regions in the N-terminus are essential for this self-interaction. In contrast, the N-terminal tail of the presumed low-affinity copper transporter, hCTR2, does not interact with itself. 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.
authors.affiliation: Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Room KC.02.069.1, Lundlaan 6, 3584 EA Utrecht, The Netherlands.
authors.forename: Adriana E M
authors.initials: AE
authors.lastname: Klomp
authors.forename: Jenneke A
authors.initials: JA
authors.lastname: Juijn
authors.forename: Linda T M
authors.initials: LT
authors.lastname: van der Gun
authors.forename: Inge E T
authors.initials: IE
authors.lastname: van den Berg
authors.forename: Ruud
authors.initials: R
authors.lastname: Berger
authors.forename: Leo W J
authors.initials: LW
authors.lastname: Klomp
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D027682:Cation Transport Proteins; D002460:Cell Line; D003300:Copper / Q000378:metabolism; D000079923:Copper Transporter 1; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D020798:Two-Hybrid System Techniques
chemical_list: D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D005656:Fungal Proteins; D008565:Membrane Proteins; C490079:SLC31A1 protein, human; C490077:Slc31a1 protein, mouse; D003300:Copper