The mechanism of copper uptake mediated by human CTR1: a mutational analysis.
Cellular copper uptake is a prerequisite for the biosynthesis of many copper-dependent enzymes; disruption of copper uptake results in embryonic lethality. In humans, copper is transported into cells by hCTR1, a membrane protein, composed of 190 amino acids with only three trans-membrane segments. To provide insight into the mechanism of this unusual transporter, we characterized the functional properties of various hCTR1 mutants stably expressed in Sf9 cells. Most single amino acid substitutions involving charged and potential copper-coordinating residues have some influence on the V(max) and K(m) values for copper uptake but do not greatly alter hCTR1-mediated copper transport. However, there were two notable exceptions. Replacement of Tyr(156) with Ala greatly reduced the maximal transport rate without effect on the K(m) value for copper. Also, replacement of His(139) in the second trans-membrane segment with Arg caused a dramatic increase in the rate of copper uptake and a large increase in the K(m) value for copper. This effect was not seen with an Ala replacement, pointing to the role of a positive charge in modulating copper exit from the pathway. Truncated mutants demonstrated that the deletion of a large portion of the N-terminal domain only slightly decreased the apparent K(m) value for copper and decreased the rate of transport. Similar effects were observed with the removal of the last 11 C-terminal residues. The results suggested that the N and C termini, although nonessential for transport, may have an important role in facilitating the delivery of copper to and retrieving copper from, respectively, the translocation pathway. A model of how hCTR1 mediates copper entry into cells was proposed that included a rate-limiting site in the pore close to the intracellular exit.
Eisses
John F
JF
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Kaplan
Jack H
JH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079923:Copper Transporter 1; D004252:DNA Mutational Analysis; D006801:Humans; D007313:Insecta; D017136:Ion Transport; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid
D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D011994:Recombinant Proteins; C490079:SLC31A1 protein, human; D003300:Copper