Mechanism of Ion Permeation in the Eukaryotic Copper Transporter CTR

Abstract

Copper uptake in eukaryotic cells is facilitated by the CTR copper transport proteins. Crystal structure of a CTR homolog from xx S. salar shows that CTR is a homotrimer and that each protomer has 3 transmembrane helices. A central permeation pathway is formed by the three subunits and two Cu binding sites were identified, each formed by three highly conserved methionines. It remains unknown how each of the Cu binding site contribute to selective permeation of Cu, and whether Cu transport is coupled to transport of another ion. We expressed and purified CTRs from human and C. elegans, and examined their functions in cell-free transport and binding assays. Because Cu+ is not stable in solution, we measured Ag+ transport in CTR. CTR transports Ag+ at a rate at least 10 fold higher than divalent transition metal ions such as nickel (Ni2+) or cobalt (Co2+). Mutations to the conserved methionines significantly reduce Ag+ transport but have little effect on Ni2+ transport. Ni2+ transport was instead affected by mutation to a conserved glutamate residue. These results indicate that CTR favors monovalent transition metal ions and that mono- and divalent cations may interact with CTR at different sites. This conclusion is also supported by affinity measurement of different ions. We also discovered that the ion transport process is electroneutral, indicating that permeation of Ag+ or other transition metal ions is accompanied by transport of another ion. After testing all possible candidates, we arrived at the conclusion that hydroxyl ions (OH−) are co-transported with the metal ions. It is not clear if CTR transports the two ions individually or together as a neutral metal hydroxide complex ion. Our study revealed for the first time the mechanism of ion selectivity and transport in CTR.