Reconstitution of a thermophilic Cu+ importer in vitro reveals intrinsic high-affinity slow transport driving accumulation of an essential metal ion

Abstract

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu⁺ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu⁺ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu⁺ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu⁺ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag⁺ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu⁺ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu⁺ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu⁺ pools.

Keywords: Ctr1; copper; copper transport; liposome; metal homeostasis; thermophile.

Figure 1.

Bioinformatic analysis identifies C. thermophilum copper transporters. A, model showing a homotrimeric Ctr1/Ctr3 family protein complex with key Cys and Met residues involved in Cu⁺ transport connected with a dashed red circle and key Gly residues involved in trimer stability via transmembrane helical packing highlighted with a red box. B, putative C. thermophilum Ctr proteins were aligned against characterized S. cerevisiae Ctr proteins with MUSCLE and compiled into a phylogenetic tree. C, C. thermophilum Ctr2 and Ctr3 proteins possess critical functional residues that are hallmarks of Ctr family members. Residues in white type surrounded by red are strictly conserved, residues in red type surrounded by a blue line possess similar biochemical properties, and residues in black type are not conserved. Asterisks indicate strictly conserved residues that are critical for transport activity.

Figure 2.

C. thermophilum Ctr2 and Ctr3 homologues rescue growth on nonfermentable medium. A, S. cerevisiae ctr1Δctr3Δ cells (strain MPY17) were transformed with the indicated plasmids and plated on rich agar medium containing either dextrose (YPD), ethanol and glycerol (YPEG), or YPEG 50 μm CuSO₄ and photographed after 2 days (YPD) or 5 days (YPEG). B, the same cells as in A were plated on minimal agar medium containing either dextrose (SC), ethanol and glycerol (SCEG), or SCEG plus the indicated final CuSO₄ concentrations and photographed after 3 days (SC-His) or 7 days (SCEG-His). C, the same cells as in A and B were inoculated into liquid YPEG or SCEG-His medium, and growth was monitored by optical absorbance.

Figure 3.

Dependence on metal binding ectodomain for Cu transport function. A, MUSCLE alignment of the Ctr2, Ctr3a, and Ctr3b ectodomains from WT, trunc1, and trunc2 proteins with the predicted metal-binding residues Met, His, and Cys shown in red, blue, and green, respectively. Asterisks, residues predicted to be necessary for Cu transport based on prior work referenced herein. B, S. cerevisiae ctr1Δctr3Δ cells (strain MPY17) were transformed with plasmids expressing the indicated proteins, plated on the indicated media, and photographed after 3 days (SC-His) or 7 days (SCEG-His).

Figure 4.

C. thermophilum Ctr2 is a vacuolar Cu transporter. A, model depicting protein variants generated in this study. Letters in black indicate WT amino acid residues, and letters in red indicate mutated residues. TEV indicates a TEV protease cleavage site, and His 8x and StrepII indicate the respective affinity purification tags. B, S. cerevisiae ctr1Δctr3Δ cells (strain MPY17) were transformed with plasmids expressing the indicated proteins and plated on the indicated media. C, immunoblot from Triton X-100–solubilized protein extracts from selected transformants in B probed with anti-His₆ antibody or anti-PGK antibody. D, cells from B were visualized by fluorescence microscopy and photographed. DIC, differential interference contrast.

Figure 5.

C. thermophilum Ctr3a and Ctr3b are plasma membrane–localized transporters. A, S. cerevisiae ctr1Δctr3Δ cells (strain MPY17) were transformed with plasmids expressing the indicated proteins and plated on media to assay growth. B, immunoblot from Triton X-100–solubilized protein extracts from selected transformants in A probed with anti-His₆ antibody or anti-PGK antibody. C, cells from A were visualized by fluorescence microscopy and photographed. DIC, differential interference contrast.

Figure 6.

C. thermophilum Ctr3a and Ctr3b mediate cellular Cu and Ag accumulation and Ag toxicity. A, S. cerevisiae ctr1Δctr3Δ cells (strain MPY17) were transformed with plasmids expressing the indicated proteins and grown in liquid YPD medium to mid-log phase before harvest and inductively coupled plasma MS analysis. B, mid-log phase cells from A were grown in the presence of 1 μm AgNO₃ in YPD for 60 min before harvest and ICP-MS analysis. C, cells from A were transferred into a sterile 96-well plate containing the indicated final concentration of AgNO₃ and allowed to grow for 48 h before A₆₀₀ measurements. Error bars, S.E. for biological triplicates as analyzed by paired t tests. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. n.s., not significant.

Figure 7.

C. thermophilum Ctr3a purification, stability, and incorporation into proteoliposomes. A, Ctr3a tag was expressed in S. cerevisiae, extracted from solubilized membrane preparations, and purified via Ni-NTA affinity chromatography followed by size-exclusion chromatography (day 0). Peak fractions were pooled. After 7 days at 4 °C, the pooled sample was re-analyzed by size exclusion chromatography (day 7). B, the Ctr3a tag LXXXL protein was purified and analyzed as in A. C, workflow depicting steps for incorporation of purified Ctr3a proteins into unilamellar proteoliposomes. D, stain-free SDS-polyacrylamide gel of proteoliposomes without protein (Empty vesicle), with Ctr3a tag and with Ctr3a tag LXXXL. Molecular weight markers are indicated with mass in kDa.

Figure 8.

In vitro Ag⁺ transport assays in reconstituted proteoliposomes. A, model depicting the in vitro reconstituted Ctr3a-dependent Ag⁺ transport assay. Stars, fluorescent reporter molecules of PGSK that are quenched in the presence of Ag⁺. Ctr3a is incorporated into liposomes in both inward-facing and outward-facing orientations. B, traces from stopped-flow measurement of PGSK fluorescence (relative fluorescence units (RFU)) over time (in seconds) after the addition of Ag⁺, with no protein (black), Ctr3a tag (red), or Ctr3a tag LXXXL (orange). C, traces from stopped-flow measurements of PGSK fluorescence from liposomes containing no protein (black) or increasing amounts of Ctr3a tag (11.25 μm (orange), 22.5 μm (green), 45 μm (purple), and 95 μm (blue)). D, initial rate of PGSK fluorescence quenching at a fixed concentration of Ag⁺ (500 μm) as a function of Ctr3a tag protein concentration incorporated into proteoliposomes. Red dotted line, best-fit linear regression. E, traces from stopped-flow measurements of PGSK fluorescence due to increasing Ag⁺ concentrations added to proteoliposomes containing a fixed concentration of Ctr3a tag (45 μm). F, Michaelis–Menten plot of initial Ag⁺ transport velocity versus Ag⁺ concentration, with K_½ and turnover calculations. Red dotted line, best-fit nonlinear regression.