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Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport

Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave Nord, Sherbrooke, QC, Canada J1H 5N4

Correspondence
Simon Labbé
Simon.Labbe{at}USherbrooke.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Copper uptake in the fission yeast Schizosaccharomyces pombe is carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106–289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44–173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met¹²² was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met¹²² have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.

Supplementary figures showing the fluorescence microscopy visualization of the cellular location of wild-type or mutant Ctr4–GFP proteins, and the visualization of wild-type or mutant Ctr5–MYC₁₂ proteins by indirect immunofluorescence microscopy, are available with the online version of this paper.

	INTRODUCTION

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All living organisms, from bacteria to humans, require copper (reviewed by Rees & Thiele, 2004a). Because of the ease with which it can gain and lose electrons, copper serves as an electron-transfer intermediate for a variety of cellular enzymes (reviewed by Peña et al., 1999). However, when present in excess, copper becomes toxic, reacting with hydrogen peroxide or dioxygen to produce hydroxyl radicals that can damage DNA, proteins and membrane lipids (Halliwell & Gutteridge, 1984). A critical balance must therefore be maintained by specialized cellular transport mechanisms to regulate intracellular copper content.

Biological management of copper requires uptake from the environment through the cellular membrane for delivery to copper-containing enzymes (reviewed by Puig & Thiele, 2002a). Studies in the baker's yeast Saccharomyces cerevisiae have identified genes encoding components of the high-affinity copper-transport machinery that resides at the cell surface. Extracellular Cu²⁺ is reduced by the Fre1 and Fre2 metalloreductases (Hassett & Kosman, 1995; Georgatsou et al., 1997; Martins et al., 1998). Either subsequent to, or concomitant with, reduction, Cu⁺ is taken up through two high-affinity copper transporters, Ctr1 (Dancis et al., 1994a, b; Puig et al., 2002b) and Ctr3 (Knight et al., 1996; Peña et al., 2000). Although Ctr1 and Ctr3 are functionally redundant, these two plasma-membrane proteins mediate copper uptake independently of each other (Peña et al., 2000). Based on bioinformatics and biochemical analyses, Ctr1 and Ctr3 possess three predicted transmembrane domains (reviewed by Puig & Thiele, 2002a). The N terminus of Ctr1 harbours eight copies of the sequence Met-X₂-Met-X-Met, called the Mets motif (Dancis et al., 1994a; Puig et al., 2002b). The Ctr1 Mets motifs are exposed to the extracellular face of the plasma membrane (Puig et al., 2002b). Although the eight Mets motifs present in Ctr1 play an important role in copper assimilation when cells are grown under copper-deficient conditions, the last methionine (Met¹²⁷) of the eighth Mets motif is essential for Ctr1 function (Puig et al., 2002b). Likewise, a Met-X₃-Met motif (residues 256–260) within the second transmembrane domain of Ctr1 has also been shown to be indispensable for the function of Ctr1 in high-affinity copper uptake (Puig et al., 2002b). Despite the fact that the Ctr3 protein exhibits a limited overall sequence homology to Ctr1, it has been shown that Ctr3 contains a similar Met-X₃-Met motif (residues 185–189) within its second transmembrane domain (Puig et al., 2002b). This enables Ctr3 to import copper from the plasma membrane, in conjunction with other critical residues, such as Cys¹⁶ within the N-terminal region, Cys⁴⁸ and Cys⁵¹ within the first transmembrane domain, and Cys¹⁹⁹ within the third transmembrane domain (Peña et al., 2000).

When environmental copper levels are scarce, the early molecular mechanisms of copper capture in the fission yeast Schizosaccharomyces pombe differ somewhat from those of Sac. cerevisiae. While deletion of the ctr4⁺ gene by homologous recombination in fission yeast abrogates high-affinity copper transport (Labbé et al., 1999), its heterologous expression in a ctr1 ctr3 Sac. cerevisiae strain is insufficient to complement the copper-transport defect in this strain (Zhou & Thiele, 2001). Complementation requires a second S. pombe gene, ctr5⁺ (Zhou & Thiele, 2001). In the absence of Ctr5, a Ctr4–GFP (green fluorescent protein) fusion protein is mislocalized to an intracellular compartment likely to be the endoplasmic reticulum (Zhou & Thiele, 2001). However, when co-expressed with Ctr5 in ctr1 ctr3 Sac. cerevisiae cells, Ctr4–GFP is localized to the plasma membrane (Zhou & Thiele, 2001). Likewise, when functional Ctr4–GFP and Ctr5–GFP fusion proteins are co-expressed in S. pombe cells from high-copy-number episomal plasmids, both proteins are localized in the plasma membrane. However, in the absence of Ctr5–GFP, the bulk of Ctr4–GFP fusion protein is found in a perinuclear compartment (Zhou & Thiele, 2001). Co-immunoprecipitation experiments reveal that the Ctr4 and Ctr5 proteins are found in a complex at the membrane (Zhou & Thiele, 2001). However, within this heterocomplex, the exact role of each protein remains unexplored. We have observed that both proteins have N-terminal conserved sequences that could serve as potential copper ligands. This suggests that both Ctr4 and Ctr5 may bind copper through these sequences, and that this may be an important aspect of their respective functions in copper transport. This study presents the first molecular dissection of the fission-yeast two-component copper-transporting system, which comprises the Ctr4 and Ctr5 proteins. The data reveal functional N-terminal domains whereby the heteroprotein complex transports copper as a function of copper availability.

	METHODS

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Yeast strains and media.
S. pombe strains used in this work were the wild-type FY435 (h⁺ his7-366 leu1-32 ura4-18 ade6-M210) (Bezanilla et al., 1997), and the ctr4 (isogenic to FY435 plus ctr4 : : ura4⁺), ctr5 (isogenic to FY435 plus ctr5 : : KAN^r) and ctr4 ctr5 double-mutant (isogenic to FY435 plus ctr4 : : ura4⁺ ctr5 : : KAN^r) disruption strains. Yeast strains were grown at 30 °C in yeast extract plus supplements (YES) or in selective Edinburgh minimal medium (EMM) supplemented with the appropriate auxotrophic requirements (Alfa et al., 1993). The respiratory carbon source medium YES/glycerol/ethanol (glycerol/EtOH) was prepared by replacing the glucose in YES with 3 % glycerol (v/v) and 2 % ethanol. Growth under conditions of low copper availability was carried out as described previously (Beaudoin et al., 2003), except that the cells were grown to OD₆₀₀ 1·0 and then treated with 100 µM of the copper chelator bathocuproine disulphonic acid (BCS) for 3 h instead of 1 h.

Ctr4 plasmids.
To facilitate the construction of ctr4 alleles, the plasmid pBluescript SK (Stratagene) was digested with AccI, treated with Klenow enzyme, and ligated to eliminate the AccI restriction site within the polylinker. The resulting plasmid was denoted pSKAccI. An SmaI–BspEI ctr4⁺ promoter fragment up to –737 from the start codon of the ctr4⁺ gene was isolated by PCR. BspEI–BamHI fragments of the wild-type and ctr4⁺–GFP fusion alleles were isolated by PCR from the pSPctr4⁺B-E/1.6 and pSPctr4⁺GFP plasmids (Labbé et al., 1999), respectively. Plasmids pSK-737ctr4⁺ and pSK-737ctr4⁺-GFP were constructed via three-piece ligation by simultaneously introducing the SmaI–BspEI ctr4⁺ promoter fragment and the BspEI–BamHI fragment from pSPctr4⁺B-E/1.6 and pSPctr4⁺GFP, respectively, into the SmaI/BamHI-cut pSKAccI vector. The integrative fission-yeast plasmid pBPade6⁺ was constructed by three-piece ligation by simultaneously introducing a 1727 bp Asp718–HindIII PCR-amplified fragment containing the ade6⁺ locus starting at –888 from the translational start codon up to +839 after the initiator codon, and a 1140 bp HindIII–BamHI PCR-amplified fragment containing the ade6⁺ locus starting at +840 to +1979, into the Asp718/BamHI-digested pBluescript SK vector (Stratagene). One primer was engineered to ensure the presence of the BsiWI restriction site at the end of the ade6⁺ locus. Subsequently, the pSKade6⁺ plasmid was digested with Asp718, filled-in with Klenow, and digested with BsiWI. The purified Asp718 (Klenow)–BsiWI DNA fragment was inserted into the AatII (which was blunt-ended by Mung Bean nuclease)/BsiWI-cut pJK148 plasmid (Keeney & Boeke, 1994). The resulting plasmid, named pBPade6⁺, generated a useful ade6⁺ auxotrophic marker in place of a leu1⁺ marker for targeted integration at the ade6⁺ locus of S. pombe. The integrative plasmids pBPctr4⁺ and pBPctr4⁺-GFP were constructed by subcloning the PstI–SpeI fragments from pSK-737ctr4⁺ and pSK-737ctr4⁺-GFP, containing the entire ctr4⁺ and ctr4⁺–GFP genes, respectively, into the corresponding sites of pBPade6⁺. The resulting plasmids were then used to integrate, in single copy, the wild-type and ctr4⁺–GFP genes at the ade6 locus of the ctr4 ctr5 strain. By using this strategy, we ensured that the ctr4⁺ and ctr4⁺–GFP genes were under the control of the ctr4⁺ promoter. To generate the mutation of Met¹²² to Ala in the ctr4⁺ gene, the primer CTR4M122A (5'-CATCGATAGTATACCAGTTCCAATACGCGGATAATTTACAAGAAGAAGCC-3') was made with mutations (underlined). Furthermore, the primer was designed to ensure the presence of the AccI restriction site found naturally within the ctr4⁺ gene. Using a second primer that hybridized downstream at the SmaI restriction site, a SmaI–AccI fragment was amplified by PCR and then swapped for an identical DNA region into the pSK-737ctr4⁺ or pSK-737ctr4⁺-GFP plasmid. These plasmids were digested with PstI and SpeI and cloned into the PstI/SpeI-cut pBPade6⁺ vector to generate pBPctr4M122A and pBPctr4M122A-GFP, respectively. Using either pBPctr4⁺-GFP or pBPctr4M122A-GFP, a set of deletions (53, 58, 69, 81 and 93) was created within the N-terminal portion of Ctr4. Five primers containing a BspEI site were engineered to anneal pairwise to DNA at codons 54, 59, 70, 82 and 94 of ctr4⁺, respectively. The antisense primer for amplification contained a BamHI restriction site. The DNA sequence from each respective PCR-amplified fragment was digested with BspEI and BamHI and exchanged with a DNA region into the pBPctr4⁺-GFP or pBPctr4M122A-GFP plasmid. To generate the ctr4-M6 mutant allele, the primer CTR4-M6-A (5'-GCACGCTCCGGAATGTCCAACAGCACAACATCCGCGTCTGGCGCGAATGCGACTAATACC-3') was made (underlined letters represent nucleotide substitutions that gave rise to mutations in the last Mets motif). This primer was used in conjunction with an antisense primer that hybridized immediately after the stop codon and in which a BamHI restriction site was present. Once generated, the ctr4-M6 mutant allele was used to replace the equivalent DNA segment in pBP93ctr4M122A-GFP. Plasmids pBP105ctr4-GFP, pBP135ctr4-GFP and pBP166ctr4-GFP were created as follows: three PCR fragments encompassing the ctr4⁺–GFP ORF starting at +316, +406 and +499 from the initiator codon up to the stop codon, including the gfp tag, were amplified from pSK-737ctr4⁺-GFP. This was performed using primers designed to introduce BspEI and SpeI at the termini of the upstream and downstream DNA fragments, respectively. The PCR products obtained were digested with BspEI and SpeI and cloned into the corresponding sites of pBPctr4⁺-GFP.

Ctr5 plasmids.
To generate the pSKctr5⁺ plasmid, a 1342 bp PstI–SmaI PCR-amplified DNA segment containing the S. pombe ctr5⁺ locus starting at –820 from the translational start codon up to the stop codon was inserted into the PstI and SmaI sites of pBluescript SK (Stratagene). To create a plasmid that has the ctr5⁺ gene with twelve copies of the myc epitope, the DNA fragment containing the ctr5⁺ gene and its promoter region was isolated from the pSKctr5⁺ plasmid using PstI and SmaI and inserted into the corresponding sites of pctr4⁺-X-myc₁₂ (Bellemare et al., 2001). By substituting the ctr5⁺ promoter in place of the ctr4⁺ promoter, the plasmid has the ctr5⁺ promoter driving the expression of the ctr5⁺–myc₁₂ fusion gene. Once created, the ctr5⁺–myc₁₂ fusion gene and its regulatory region were isolated using PstI and XbaI and inserted into the corresponding sites of pJK148. The resulting integrative plasmid was designated pJKctr5⁺-myc₁₂. The ctr5 mutant alleles containing either site-specific mutations (M1, M2, M3, M4, M5 and M6) or N-terminal deletions (pJK43ctr5-myc₁₂ and pJK76ctr5-myc₁₂) were created by the overlap-extension method (Ho et al., 1989). The DNA sequence of the PstI–XbaI fragment from each respective PCR-amplified fragment was used to replace the corresponding fragment from plasmid pJKctr5⁺-myc₁₂. The DNA sequence of the PstI–XbaI fragment from each respective mutant was confirmed by dideoxy sequencing.

Microscopic analysis of Ctr4 and Ctr5 localization.
ctr4 ctr5 mutant cells expressing the GFP fusion proteins were grown in YES medium to OD₆₀₀ 1·0. After incubation in the presence of 100 µM of the copper chelator BCS for 3 h, direct fluorescence microscopy was used to detect the Ctr4–GFP fusion protein or its mutant derivatives in live cells mounted in 1 % low-melt agarose. Indirect immunofluorescence microscopy was used to localize the MYC₁₂ epitope-tagged versions of the Ctr5 protein. The cells were treated identically to those described above, except that after 3 h incubation in the presence of BCS (100 µM), the cells were fixed by adding formaldehyde (methanol-free) (Polysciences) to a final concentration of 3·7 %. Fixed cells were harvested and washed with 0·1 M potassium phosphate, pH 6·5, containing 1·2 M sorbitol. Cells were spheroplasted as described previously (Bellemare et al., 2002) and adsorbed to poly-lysine-coated multiwell slides. After a 30 min block with TBS (10 mM Tris/HCl, pH 7·4, 150 mM NaCl, 1 % BSA), cells were incubated with anti-c-myc antibody (9E10) (Roche Diagnostics) diluted 1 : 200 in TBS. After an 18 h reaction, cells were washed with TBS and incubated for 4 h with goat anti-mouse Alexa Fluor 546 conjugate (Invitrogen) diluted 1 : 200 in TBS. The cells were viewed with a Nikon Eclipse E800 epifluorescent microscope equipped with a Hamamatsu ORCA-ER cooled charge-coupled device (CCD) camera.

	RESULTS

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Reciprocal effects of Ctr4 on Ctr5 localization and Ctr5 on Ctr4 localization
In S. pombe, the Ctr4-Ctr5 high-affinity copper-transport system consists of two structurally related transmembrane proteins (Fig. 1). An earlier study has shown that S. pombe strains harbouring a deletion of either ctr4⁺ or ctr5⁺ are defective in copper uptake (Zhou & Thiele, 2001). Consequently, these strains cannot grow on respiratory carbon sources such as glycerol/ethanol, presumably due to the lack of copper incorporation into mitochondrial cytochrome c oxidase (Zhou & Thiele, 2001). Because the earlier study was performed using single-deletion strains and genes expressed from plasmids present in multiple copies, we sought to examine the requirement of Ctr5 for Ctr4 activity and vice versa using a double-disruption strain, ctr4 ctr5, in which ctr4⁺ or ctr5⁺, or both genes, were returned in a low copy number by integration. As shown in Fig. 2(A), when both ctr4⁺ and ctr5⁺ were insertionally inactivated, the double-mutant strain failed to grow on respiratory carbon sources. This growth defect could be reversed by the addition of exogenous copper to the growth medium at concentrations of at least 15 µM (Fig. 2A). Expression of ctr4⁺ in ctr4 ctr5 cells under the control of its native promoter was not sufficient to restore respiratory growth. Likewise, expression of ctr5⁺ in ctr4 ctr5 cells did not correct the respiratory growth defect. In contrast, co-expression of both ctr4⁺ and ctr5⁺ restored respiratory proficiency to these cells (Fig. 2A).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Primary structural features of the Ctr4 and Ctr5 proteins. The top panel shows a schematic representation of theCtr4protein. The grey-shaded regions indicate the location of the five potential copper-binding motifs, termed Mets motifs (M-X2-M-X-M). The position of Met122 in Ctr4 is indicated. The bottom panel shows a schematic representation of the primary structure of the Ctr5 protein. Ctr5 has two copies of partially overlapping Mets motifs and a C-X-M-X-M sequence, which lies downstream of the Mets motifs. The predicted transmembrane domains (TM1–3) of both proteins are shown as black rectangles. The amino acid sequence numbers refer to the position relative to the first amino acid of each protein.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Co-expression of both the Ctr4 and the Ctr5 protein is necessary for growth on respiratory carbon sources and for localization to the plasma membrane. (A) S. pombe cells harbouring a ctr4 ctr5 double deletion were transformed with empty integrative vectors (–), a vector alone and ctr4+, a vector alone and ctr5+, or ctr4+ and ctr5+. Cultures were spotted onto YES agar media containing glucose or glycerol/ethanol(EtOH) with 0 and 15 µM CuSO4. WT, isogenic wild-type strain FY435. (B) Ctr4–GFP and Ctr5–MYC12 fusion proteins are functional. The tagged proteins were expressed into a ctr4 ctr5 mutant strain as in (A) and tested for their ability to confer growth on respiratory carbon sources (glycerol/ethanol). (C) Localization of Ctr4–GFP and Ctr5–MYC12 fusion proteins. Yeast cells disrupted for ctr4+ and ctr5+ that expressed individually, or in combination, thetagged genes were grown in YES medium containing 100 µM BCS and analysed by fluorescence microscopy. Cell morphology was examined through Nomarski optics.

The N-terminal regions of both Ctr4 and Ctr5 play an important role in copper assimilation
Although co-expression of Ctr4 and Ctr5 restores copper acquisition to an S. pombe ctr4 ctr5 strain, the exact function of each protein within this heterocomplex at the plasma membrane is currently unclear. As shown in Fig. 1, the N termini of both proteins have conserved Mets motifs that are predicted to extend into the extracellular environment. We hypothesized that both Ctr4 and Ctr5 utilize these motifs to acquire copper from the environment, making them essential components of the copper-transport complex. To test this hypothesis, we used site-directed mutagenesis to convert each individual Met and Cys residue to Ala within the two Mets motifs and the Cys-X-Met-X-Met sequence of Ctr5–MYC₁₂ (Fig. 3A). This mutant allele was denoted ctr5-M6. The importance of the Ctr4 Met-rich sequences for Ctr4-Ctr5 function was assessed by co-expressing the wild-type ctr4⁺–GFP and ctr5-M6 alleles in the ctr4 ctr5 (JSY22) strain and assaying for their ability to restore respiratory proficiency. This was compared to the parental strain FY435 or the ctr4 ctr5 mutant strain co-expressing ctr4⁺–GFP and ctr5⁺–myc₁₂. As shown in Fig. 3(A), the growth of cells co-expressing Ctr4–GFP and Ctr5-M6 on glycerol/ethanol medium was equivalent to cells expressing wild-type Ctr4–GFP and Ctr5–MYC₁₂, except when extracellular bioavailable copper was chelated by the addition of 20 or 25 µM BCS. Under these conditions of copper deprivation, no growth was observed.

View larger version (42K): [in this window] [in a new window]   Fig. 3. The N-terminal regions of both the Ctr4 and the Ctr5 protein are required for respiratory cell growth under conditions of copper limitation. (A) the strain JSY22 (ctr4 ctr5) was co-transformed with integrative plasmids alone, or integrative plasmids expressing the indicated versions of Ctr4–GFP and Ctr5–MYC12 proteins. Cells were grown either for 5 days on YES in the presence of glucose or for 9 days on YES in the presence of glycerol/ethanol. For simplicity, only the N termini of the Ctr proteins are shown. FY435, parental wild-type strain. (B) Localization of Ctr4–GFPwt+Ctr5–MYC12wt, Ctr4–GFPwt+Ctr5-M6, Ctr4-M6+Ctr5–MYC12wt, and Ctr4-M6+Ctr5-M6 in ctr4 ctr5 cells. Representative fluorescence images of GFP are shown. Indirect immunofluorescence microscopy (anti-MYC) was performed using the anti-myc monoclonal antibody. Nomarski microscopy was used to ascertain cell morphology.

The five Mets motifs of Ctr4 and Ctr4 Met¹²² are functionally redundant
The N terminus of Ctr4 contains five copies of the sequence Met-X₂-Met-X-Met. As shown above, the Ctr4-M6 mutant lacking these sequences was unable to restore respiratory growth when co-expressed with Ctr5-M6. To determine the importance of these motifs in copper acquisition, sequential deletions of this sequence were created in Ctr4 and co-expressed with Ctr5-M6 in strain JSY22. The Ctr4-M1 mutant lacks the first 53 N-terminal residues, while Ctr4-M2 lacks the 93 N-terminal residues, and the mutants harbour five and one Mets motifs, respectively. Co-expression of either ctr4-M1 or ctr4-M2 with ctr5-M6 in a ctr4 ctr5 strain allowed the cells to grow on glycerol/ethanol medium, except in the presence of 20 or 25 µM BCS (Fig. 4). These results suggested that the fifth Mets motif of Ctr4 might be required for respiratory growth. Surprisingly, however, cells expressing the ctr4-M5 allele in which the first four Mets motifs had been deleted and the fifth Mets motif substituted with Ala residues exhibited respiratory competence compared to the wild-type ctr4⁺ allele (Fig. 4). A previous study has shown that a conserved methionine found 20 amino acid residues from the beginning of the first transmembrane domain in the Sac. cerevisiae Ctr1 protein is essential for copper transport (Puig et al., 2002b). A comparison of the Sac. cerevisiae Ctr1 and S. pombe Ctr4 primary sequences suggested that the Ctr4 Met¹²², found 22 amino acid residues from the first transmembrane domain of Ctr4, might also be essential for copper acquisition. To determine the importance of the Met¹²² residue for Ctr4 function, Met¹²² was mutated to Ala, generating the Ctr4-M4 mutant. Although the Ctr4-M4 mutant protein fused with GFP was localized to the plasma membrane (see Supplementary Fig. S1), ctr4 ctr5 cells co-expressing the ctr5-M6 and ctr4-M4 alleles exhibited only a weak growth on glycerol/ethanol medium compared to those co-expressing the ctr5-M6 and ctr4⁺ alleles. Given this result, and to further delineate the role of Met¹²² in Ctr4 function, Ctr4 Met¹²² was mutated to Ala in the full-length Ctr4, creating the Ctr4-M3 mutant. Surprisingly, in the context of the full-length Ctr4 protein, the Met¹²² to Ala mutation did not interfere with growth on glycerol/ethanol medium containing 0, 5 and 10 µM BCS (Fig. 4). Moreover, even in the presence of 20 or 25 µM BCS, the cells exhibited growth, although markedly slower than wild-type Ctr4 (Fig. 4). This observation suggests that the five Mets motifs may compensate for the loss of the Met¹²² residue, giving rise to the question of how many Mets motifs are required for such a compensatory effect.

View larger version (41K): [in this window] [in a new window]   Fig. 4. The role of the Mets motifs and Met122 in Ctr4 function. The strain JSY22 (ctr4 ctr5) was co-transformed with the pJK148 and pBPade6+ vectors alone, ctr5+–myc12wt+ctr4+–GFPwt, ctr5-M6+ctr4+–GFPwt, ctr5-M6+ctr4-M1, ctr5-M6+ctr4-M2, ctr5-M6+ctr4-M3, ctr5-M6+ctr4-M4, ctr5-M6+ctr4-M5, or ctr5-M6+ctr4-M6. For simplicity, only the N-terminal regions of the indicated versions of Ctr5–MYC12 and Ctr4–GFP fusion proteins are depicted. The amino acid sequence numbers refer to the position relative to the first amino acid of the indicated protein. Cells were spotted at a density of 3000 cells (5 µl)–1 onto YES media containing glucose or glycerol/ethanol with 0, 5, 10, 20 or 25 µM BCS. FY435, parental wild-type strain.

View larger version (63K): [in this window] [in a new window]   5 Functional dissection of the fiveMets motifs in Ctr4. (A) JSY22 (ctr4 ctr5) was co-transformed with pJK148 and pBPade6+ vectors alone, ctr5+–myc12wt+ctr4+–GFPwt, ctr5-M6+ctr4-M3, ctr5-M6+ctr4-M7, ctr5-M6+ctr4-M8, ctr5-M6+ctr4-M9, ctr5-M6+ctr4-M4, or ctr5-M6+ctr4-M6. Growth was tested on fermentable (glucose) and non-fermentable (glycerol/ethanol) agar media and incubated at 30 °C for 5 and 9 days, respectively. Schematic representations (on the left) exhibit only the N terminiof the Ctr4 and Ctr5 fusion proteins or theirmutant derivatives. FY435, isogenic wild-type strain. (B) ctr4 ctr5 cells co-expressing the indicated alleles were grown to OD600 1·0. At this OD, BCS (100 µM) was added and the treated cultures were incubated for 3 h at 30 °C, and then visualized for GFP by fluorescence microscopy. The cells werealso examined by Nomarski microscopy for cell morphology.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Functional features of the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5. Cells (JSY22) harbouring a ctr4 ctr5 double deletion were co-transformed with empty integrative vectors, ctr4+–GFPwt+ctr5+–myc12wt, ctr4-M6+ctr5+–myc12wt, ctr4-M6+ctr5-M1, ctr4-M6+ctr5-M2, ctr4-M6+ctr5-M3, ctr4-M6+ctr5-M4, ctr4-M6+ctr5-M5, or ctr4-M6+ctr5-M6. The co-transformed cells were spotted on media containing 3 % glucose (fermentable) or 3 % glycerol/2 % ethanol (non-fermentable) as carbon sources. Growth rates of the co-transformed cells were compared with the parental strain (FY435). Cells were grown for 5 (glucose) or 9 (glycerol/ethanol) days at 30 °C and photographed. For simplicity, only the N-terminal regions of the indicated variants of the Ctr4–GFP and Ctr5–MYC12 fusion proteins are shown.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Minimal C-terminal regions of Ctr4 and Ctr5 required to allow growth by respiration. (A) The top panel shows ctr4ctr5 cells harbouring the wild-type ctr5+–myc12 allele and transformed with 1ctr4+289–GFP, 106ctr4+289–GFP, 136ctr4+289–GFP or 167ctr4+289–GFP alleles. The bottom panel shows ctr4 ctr5 cells containing the wild-type ctr4+–GFP allele and transformed with 1ctr5+173–myc12, 44ctr5+173–myc12 or 77ctr5+289–myc12 alleles. Cells were photographed after incubation at 30 °C for 5 days on yeast extract plus glucose plates and 9 days on yeast extract plus glycerol/ethanol plates. FY435 represents wild-type cells. (B) Cells co-expressing the indicated Ctr4 and Ctr5 fusion proteins were visualized by fluorescence microscopy. The Ctr4–GFP fusions are shown in the top panel with the GFP microscope images on the left and the Nomarski images on the right. The epitope-tagged Ctr5 proteins are shown in the bottom panel with the fluorescence (anti-MYC) microscope images on the left and the Nomarski images on the right.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we used a stable expression system utilizing integrative plasmids to return wild-type or mutant alleles of the ctr4⁺ and ctr5⁺ genes in a ctr4 ctr5 mutant strain. Using this single-copy expression system, we determined that simultaneous expression of full-length Ctr4 or Ctr4 106–289 and full-length Ctr5 or Ctr5 44–173 is required for proper localization of both proteins at the cell surface. In the absence of Ctr5, Ctr4 is mislocalized within the secretory pathway. Similarly, we found that in the absence of Ctr4, Ctr5 fails to exit an intracellular compartment likely to be the endoplasmic reticulum. Like most Ctr family members, Ctr4 and Ctr5 are rich in methionine residues within their putative N-terminal extracellular hydrophilic domains. Ctr4 harbours five almost perfectly spaced Mets motifs, whereas Ctr5 contains two copies of partially overlapping Mets motifs and a Cys-X-Met-X-Met sequence, which lies downstream of the two Mets motifs (Labbé et al., 1999; Zhou & Thiele, 2001). To begin to understand the mechanism by which these two proteins deliver copper into the cell, we performed experiments to delineate the respective roles of the Ctr4 and Ctr5 Met-rich sequences in copper assimilation. The analysis of yeast strains expressing Ctr4 and Ctr5 proteins in which the N-terminal regions have been mutated demonstrates that at least one N-terminal region provided by either protein suffices for growth on glycerol/ethanol media containing 0, 5 or 10 µM BCS. However, addition of higher concentrations of BCS (20 and 25 µM) results in an inability to grow on respiratory carbon sources, unless ctr4 ctr5 cells co-express both wild-type ctr4⁺ and wild-type ctr5⁺ genes. Accordingly, these results reveal that although the N-terminal regions of Ctr4 and Ctr5 can function independently in copper transport, the presence of both N termini is required for the most efficient copper-transport activity, especially under severe copper-limiting conditions. Therefore, yeast cells that express both full-length Ctr4 and full-length Ctr5 protein have a distinct growth advantage, when copper is limiting, over cells expressing only one N-terminal region from these copper transporters.

Recent studies have revealed that a single Met or a Met-X-Met motif located approximately 20 amino acids upstream of transmembrane domain one in the Ctr proteins is essential for copper transport (Puig et al., 2002b; Rees et al., 2004b). Ctr4 has one methionine residue (Met¹²²) 22 amino acids from its first transmembrane domain. We assessed the potential role of this residue on Ctr4 function by mutating Met¹²² to Ala. In the context of the full-length protein, no growth-defect phenotype was observed when cells were grown on respiratory carbon sources in the presence of 0, 5 or 10 µM BCS. In the absence of the five Mets motifs (Ctr4-M5), however, the Met¹²² was essential for the growth of cells on non-fermentable media in the absence or presence of BCS (5 or 10 µM). These data suggest that the Ctr4 protein has two potential ways for sequestering copper to the plasma membrane prior to its import through the membrane. One mechanism involves the five Mets motifs, while the second requires Met¹²². In the absence of Met¹²², the presence of all five Mets motifs appears to be crucial for Ctr4 function. Removal of Mets motif 1 (Ctr4-M7) drastically reduces the ability of the cells to grow on standard glycerol/ethanol medium, with no growth on medium supplemented with exogenous BCS. Furthermore, deletion of the first two or the first three Mets motifs (Ctr4-M8 or Ctr4-M9) precludes normal localization of Ctr4 at the plasma membrane. While Ctr4 Mets motif 5 (Ctr4-M4) is sufficient to complement respiratory deficiency on standard glycerol/ethanol medium to the same extent as Ctr4-M7, these cells (Ctr4-M4) fail to grow on respiratory carbon sources when extracellular copper is chelated by BCS.

Within its N terminus, Ctr5 contains two copies of partially overlapping Mets motifs and a Cys-X-Met-X-Met sequence. The last Met residue (Met³¹) of the latter sequence is located 24 amino acids from the first transmembrane domain. Mutation of this Met residue to Ala in the Ctr5-M4 mutant still allowed the transformed cells to grow on glycerol/ethanol medium when co-expressed with Ctr4-M6. This result suggests that the Ctr5 Met³¹ residue is dispensable for the function of Ctr5. Although we demonstrate that the two partially overlapping Mets motifs of Ctr5 and the Ctr5 Cys-X-Met-X-Met sequence are functionally redundant, the growth of cells lacking the Cys-X-Met-X-Met sequence was more severely limited under conditions of copper deprivation compared to those lacking the two partially overlapping Mets motifs. This may be due to the fact that a Cys rather than a Met residue is found at the first position within the Cys-X-Met-X-Met sequence. It is known that the Cys residue with its external SH group coordinates copper with more affinity than a Met residue (Puig et al., 2002b). Furthermore, the fact that the Cys-X-Met-X-Met sequence is closer to the first transmembrane domain may be important. For the mammalian CPx-type ATPases ATP7A and ATP7B, the metal-binding domains located closer to the first transmembrane domain appear to play a more important role in copper transport (Payne & Gitlin, 1998; Iida et al., 1998; Strausak et al., 1999; Forbes et al., 1999; Huster & Lutsenko, 2003). Perhaps the putative copper-binding motif closest to the first membrane-embedded region can facilitate the translocation of copper through the membrane channel for delivery into the cell.

It is intriguing that the fission yeast genome encodes two structurally related proteins that are interdependent for co-localization to the cell surface and function in copper uptake. This situation is reminiscent of the FTR1-encoded iron permease and the FET3-encoded multicopper oxidase in Sac. cerevisiae that must be co-expressed for stable assembly and function in high-affinity iron transport at the plasma membrane (Askwith et al., 1994; De Silva et al., 1995; Stearman et al., 1996; Severance et al., 2004). Interestingly, SPAC1F7.07 and SPAC1F7.08 genes encoding the Ftr1 and Fet3 homologues from S. pombe, designated fip1⁺ and fio1⁺, respectively, must also be co-expressed to restore high-affinity iron transport to a Sac. cerevisiae fet3 mutant strain (Askwith & Kaplan, 1997). Furthermore, the fip1⁺ and fio1⁺ genes lie adjacent to one another in the S. pombe genome and are transcribed divergently, suggesting coordinate regulation (Askwith & Kaplan, 1997; Labbé et al., 1999). What might be the potential advantages for cells to possess two membrane proteins such as Ctr4 and Ctr5 that form a two-component copper-transporting complex at the cell surface? Clearly, our data demonstrate that the presence of the N termini of both Ctr4 and Ctr5 is required for optimal cell growth under conditions in which copper is required, yet limiting. Because the putative extracellular N-terminal regions of both Ctr4 and Ctr5 have a direct role in copper assimilation, this may prevent a complete loss of function resulting from the inactivation of one extracellular N terminus. While the efficacy of copper transport would be diminished, it would still allow the heteroprotein complex to mediate copper acquisition and transport. It may also be that the heteromeric Ctr4–Ctr5 complex is involved in the ordered assembly of higher-order complexes at the cell surface. The possibility exists that the heteroprotein complex may play a role in protein–protein interactions with, for example, the Fe³⁺/Cu²⁺ reductases or cytosolic carrier proteins. Recently, it has been suggested that Ctr5 could serve to ensure stable assembly of the high-affinity copper-transport complex, because Ctr4 has residues at positions 236 (Phe) and 237 (Leu) in transmembrane domain three that hinder formation of a stable multimeric complex (Aller et al., 2004). Because Ctr5 was originally identified based on its ability to alleviate a block in Ctr4 sorting to the plasma membrane, it is quite possible that the Ctr5 protein is required for Ctr4 folding, hetero-oligomerization and exit from the secretory pathway. Further characterization of Ctr4 and Ctr5 will be required to elucidate how these two transmembrane proteins assemble as a multisubunit complex to migrate through the secretory pathway to the plasma membrane, where the complex can mediate copper uptake into the cell.

	ACKNOWLEDGEMENTS

 
We are grateful to Dr Maria M. O. Peña for critically reading the manuscript and for her constructive suggestions. We thank Benoit Pelletier for the plasmid pBPade6⁺. J. L. is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). This work was supported by the Canadian Institutes for Health Research (CIHR) Grant MOP-36450 to S. L. Infrastructure equipment essential for carrying out this investigation was obtained through the Canada Foundation for Innovation Grant NOF-3754 to S. L. S. L. is a New Investigator Scholar from the CIHR.

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Received 31 July 2005; revised 12 October 2005; accepted 14 October 2005.

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