Molecular characterization of hCTR1, the human copper uptake protein.
We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K(m) of approximately 3.5 microm. We also expressed functional transporters where the N-linked glycosylation sites were substituted, and we provided evidence for the extracellular location of the amino terminus. Accessibility of amino-terminal FLAG epitope to antibody prior to permeabilization and of carboxyl-terminal FLAG only after permeabilization confirmed the extracellular location of the amino terminus and established the intracellular location of the carboxyl terminus. Tryptic digestion of hCTR1 occurred within the cytoplasmic loop and generated a 10-Da carboxyl-terminal peptide; cleavage was prevented by the presence of copper. hCTR1 mutants where Cys-161 and Cys-189, the two native cysteines, were replaced with serines also mediated copper uptake, indicating that neither cysteine residue was essential for transport. However, the mutants provided evidence that these residues may stabilize hCTR1 oligomerization. Western blots of hCTR1 in Sf9 cells showed expression levels 100-fold higher than in mammalian (HepG2) cells. The high level of functional expression and the low level of endogenous copper uptake will enable future structure-function analysis of this important protein.
Eisses
John F
JF
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97201, USA.
Kaplan
Jack H
JH
D000581:Amidohydrolases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D027682:Cation Transport Proteins; D002460:Cell Line; D003300:Copper / Q000378:metabolism / Q000493:pharmacokinetics*; D000079923:Copper Transporter 1; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006031:Glycosylation; D007313:Insecta; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D018076:DNA, Complementary; D008565:Membrane Proteins; D010455:Peptides; C490079:SLC31A1 protein, human; D003300:Copper; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D003545:Cysteine