Abstract
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.
Highlights
Copper is an essential element required for many important cellular reactions that rely on the redox properties of this metal [1,2,3]
Are there specific copperbinding sites that are alternately exposed to the outside and the inside of the cell via protein conformational changes? Are there amino acid residues that control entry of copper into a translocation pathway? Does copper migrate through copper transporter 1 (CTR1) via a chain of “essential” residues or is there a “channel-like” pathway through which copper migrates without forming strong contacts with the pore-lining amino acid residues? Does the exit of copper from the transporter represent a regulated event? What is the rate-limiting step of transport? The answers to these questions are currently unknown
In order to readily compare the effects of modifications in hCTR1, the rate of uptake of copper into Sf9 cells that did not contain transfected hCTR1 was subtracted from the total copper uptake into the transfected cells
Summary
Copper is transported into cells by hCTR1, a membrane protein, composed of 190 amino acids with only three trans-membrane segments. Replacement of His139 in the second transmembrane segment with Arg caused a dramatic increase in the rate of copper uptake and a large increase in the Km 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. Recent studies demonstrated that mutations of the methionines in the N-terminal domain (Met40–Met45) and in the second trans-membrane segment (Met150 and Met154) decrease the ability of hCTR1 to transport copper into cells [19]. Because of the sequence homology between yCtr and hCTR1, these residues may be relevant to hCTR1 These initial studies provided first glimpses into the molecular organization of CTR1, the mechanism of copper translocation by this transporter remains uncharacterized. We combine our results with those of previous studies to propose a model of how hCTR1 mediates copper entry into cells
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