Dissection of the relative contribution of the Schizosaccharomyces pombe Ctr4 and Ctr5 proteins to the copper transport and cell surface delivery functions

Abstract

The Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Saccharomyces cerevisiae Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4-Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used S. cerevisiae mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X(3)-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4-Ctr5 complex, whereas the Met-X(3)-Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of Sch. pombe cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface.

Fig. 1.

Notable features of Sch. pombe Ctr4 and Ctr5 proteins. (a) Sequence alignment and primary structural features of Ctr4 and Ctr5 proteins. The alignment was performed with clustal w2 software. Asterisks indicate the presence of an identical amino acid in both proteins, whereas dots indicate similarity between amino acids. (b) Schematic representation of the putative topology of Ctr4 and Ctr5 proteins. According to the topology displayed by the human and S. cerevisiae Ctr1 proteins, we propose that the amino-termini of Ctr4 and Ctr5 proteins would localize to the extracellular medium and that their carboxyl-termini would face the cytosol. Grey-shaded boxes represent the predicted locations of membrane-spanning domains (TMDs). The methionine residues located in conserved positions, which are essential for the Cu transport activity in human and S. cerevisiae Ctr1 proteins, are shown in bold type. The amino acid sequence number relating to the first amino acid of the protein is indicated.

Fig. 2.

The Ctr5 Met-X₃-Met motif is not required for the function of the Ctr4–Ctr5 complex in S. cerevisiae. (a) Analysis of the role of the Met-X₃-Met motif in Ctr4 and Ctr5 function. The S. cerevisiae ctr1Δctr3Δ deletion mutant was cotransformed with combinations of vector (V), CTR4 (wild-type, left column), CTR4-M223/227A, CTR5 (wild-type, right column), CTR5-M130/134A and CTR1, as indicated. Cells were spotted onto SC-ura-his, YPD, YPD with 100 μM BPS, YPEG and YPEG with 100 μM CuSO₄, grown for 3 days at 30 °C, and photographed. (b) Dissection of the relative contribution of the Met²²³ and Met²²⁷ residues of Ctr4 to its Cu transport function. The ctr1Δctr3Δ deletion mutant was cotransformed with the indicated combinations of vector (V), CTR4 (wild-type, left column), CTR5 (wild-type, right column), CTR4-M223/227A, CTR4-M223A, CTR4-M227A and CTR1. In addition to the media used in (a), cells were also spotted onto YPEG with 100 μM BCS.

Fig. 3.

Subcellular localization of the Ctr4 and Ctr5 mutant proteins in S. cerevisiae. The ctr1Δctr3Δ deletion mutant was cotransformed with combinations of vector, CTR4, CTR4-GFP, CTR4-M223/227A, CTR4-M223/227A-GFP, CTR5, CTR5-GFP, CTR5-M130/134A and CTR5-M130/134A-GFP, as indicated in the different panels. Cells were grown to exponential phase, and then visualized for GFP by fluorescence microscopy. Representative images of four separate experiments are shown. Cell morphology was examined through Nomarski optics.

Fig. 4.

Functionality of the Ctr4 and Ctr5 protein chimeras in S. cerevisiae. (a, b) Schematic representation of Ctr4/Ctr5 chimeras. The Ctr4 and Ctr5 proteins were divided into three regions at the conserved Ctr4 Met¹²² and Met²²⁷ residues, and at the Ctr5 Met³¹ and Met¹³⁴ residues. All the possible combinations between the Ctr4 and Ctr5 protein regions were constructed, including the Ctr4-based chimeras CTR544, CTR445 and CTR545 (a), and the Ctr5-based chimeras CTR455, CTR554 and CTR454 (b). (c, d) The S. cerevisiae ctr1Δctr3Δ deletion mutant was cotransformed with combinations of vector (V), CTR4 (wild-type, left column), CTR544, CTR445, CTR545, CTR5 (wild-type, right column), CTR455, CTR554, CTR454 and CTR1, as indicated. In addition to the media assayed in Fig. 2(a), cells were spotted onto SC with 50 μM BPS. Cells were grown for 3 days at 30 °C and photographed.

Fig. 5.

Subcellular localization of the Ctr4 and Ctr5 protein chimeras in S. cerevisiae. The S. cerevisiae ctr1Δctr3Δ deletion mutant was cotransformed with combinations of vector, CTR4, CTR4-GFP, CTR544-GFP, CTR445-GFP, CTR5, CTR5-GFP, CTR545-GFP, CTR455-GFP, CTR554-GFP and CTR454-GFP, as indicated in the different panels. Representative images of four separate experiments are shown. Cells were grown and analysed as in Fig. 3.

Fig. 6.

Functionality of the Ctr4 and Ctr5 protein mutants and chimeras in Sch. pombe. (a) The Ctr5 Met-X₃-Met motif is not required for the function of the Ctr4–Ctr5 complex in Sch. pombe. The Sch. pombe ctr4Δctr5Δ deletion strain was cotransformed with combinations of vector (V), CTR4 (left column), CTR5 (right column), CTR4-M223/227A and CTR5-M130/134A, as indicated. Wild-type (FY435) and the transformed ctr4Δctr5Δ Sch. pombe isogenic cells were spotted onto YES agar media containing glycerol/ethanol without (YES-EG) or with 15 μM CuSO₄ (YES-EG+Cu). (b) Expression of a Ctr445 chimeric protein complements the ctr4Δctr5Δ respiratory defect. The Sch. pombe ctr4Δctr5Δ deletion strain was cotransformed with combinations of vector (V), CTR4, CTR4-GFP, CTR4-M223/227-GFP, CTR5, CTR5-GFP, CTR5, CTR445-GFP and CTR544-GFP, as indicated. Sch. pombe cells were grown at 30 °C and photographed.

Fig. 7.

Subcellular localization of the Ctr4 and Ctr5 protein mutants and chimeras in Sch. pombe. The Sch. pombe ctr4Δctr5Δ deletion strain was cotransformed with vector, CTR4-GFP, CTR5, CTR5-GFP, CTR4-M223/227A-GFP, CTR445-GFP and CTR544-GFP, as indicated. Transformant cells were grown and analysed as previously described (Beaudoin et al., 2006). Representative images of four separate experiments are shown.