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Keywords:

  • Atx1;
  • Ccc2;
  • complementation;
  • Cu(I);
  • domain–domain interactions

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In yeast, copper delivery to the trans-Golgi network involves interactions between the metallo-chaperone Atx1 and the N-terminus of Ccc2, the P-type ATPase responsible for copper transport across trans-Golgi network membranes. Disruption of the Atx1–Ccc2 route leads to cell growth arrest in a copper-and-iron-limited medium, a phenotype allowing complementation studies. Coexpression of Atx1 and Ccc2 mutants in an atx1Δccc2Δ strain allowed us to study in vivo Atx1–Ccc2 and intra-Ccc2 domain–domain interactions, leading to active copper transfer into the trans-Golgi network. The Ccc2 N-terminus encloses two copper-binding domains, M1 and M2. We show that in vivo Atx1–M1 or Atx1–M2 interactions activate Ccc2. M1 or M2, expressed in place of the metallo-chaperone Atx1, were not as efficient as Atx1 in delivering copper to the Ccc2 N-terminus. However, when the Ccc2 N-terminus was truncated, these independent metal-binding domains behaved like functional metallo-chaperones in delivering copper to another copper-binding site in Ccc2 whose identity is still unknown. Therefore, we provide evidence of a dual role for the Ccc2 N-terminus, namely to receive copper from Atx1 and to convey copper to another domain of Ccc2, thereby activating the ATPase. At variance with their prokaryotic homologues, Atx1 did not activate the Ccc2-derived ATPase lacking its N-terminus.


Abbreviations
MBD

metal-binding domain

TGN

trans-Golgi network

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Copper is an essential element for life because of its ability to switch between Cu(I) and Cu(II) in vivo. Yet, because of its redox properties, copper can also be toxic when present in excess. Accordingly, the intracellular concentration of this metal is tightly regulated by a cascade of cytosolic and membrane proteins that interplay to deliver copper to specific targets, namely the cytosolic superoxide dismutase, the mitochondrial cytochrome c oxidase and secreted enzymes maturing in the trans-Golgi network (TGN) [1]. In humans, the Menkes and Wilson proteins are copper ATPases embedded in the TGN membranes that play an essential role in copper delivery to the secretory pathway and in copper balance. These ATPases receive copper from the metallo-chaperone Atox1 and assure its further transport into the TGN where the metallic cation is inserted as a co-factor in various secreted enzymes [2]. Among them, ceruloplasmin, a multicopper ferroxidase secreted by hepatocytes, carries almost all of the copper circulating in the plasma. Two genetic disorders of copper metabolism, called Menkes and Wilson diseases, are caused by deficient copper ATPases and highlight the need for copper, as well as its toxicity [3,4].

The yeast Saccharomyces cerevisiae provides an excellent model with which to characterize copper delivery to the secretory pathway in further detail because the proteins involved in copper homeostasis are highly conserved from yeasts to humans. The first step occurs at the membrane level where a reductase converts extracellular Cu(II) into Cu(I); the latter then enters the cell via the high-affinity transporter Ctr1 [5] and is distributed to specific intracellular targets. Delivery to the secretory pathway is mediated by the metallo-chaperone Atx1, the homologue of Atox1, and by the copper ATPase Ccc2, homologous to the Menkes and Wilson proteins [6,7]. In the TGN lumen, copper binds to and activates the multicopper oxidase Fet3 [8], the homologue of the human ceruloplasmin. Fet3 is then secreted towards the plasma membrane as a complex with the permease Ftr1 [9]. This complex, in which Ftr1 transports Fe(II) across the plasma membrane and Fet3 transforms it into Fe(III), is responsible for high-affinity iron uptake in the absence of siderophores [5,10,11]. Therefore, copper-dependent interactions between Atx1 and Ccc2 and further copper transfer into the TGN lumen by Ccc2 are required to activate high-affinity iron uptake in yeast (Fig. 1A).

image

Figure 1.  Ccc2 function, enzymatic cycle and schematic representations. (A) Copper delivery to the secretory pathway in yeast. Black dots represent Cu(I), white dots represent Fe(II). Both Cu(I) and Fe(II) are produced by Fre1, a membrane reductase that is not represented here to simplify the scheme. Ctr1, high-affinity Cu(I) transporter at the plasma membrane; Atx1, cytosolic metallo-chaperone; Ccc2, copper ATPase at the TGN membrane; Ftr1, high-affinity Fe(II) transporter; Fet3, multicopper ferroxidase. The Fet3–Ftr1 complex is secreted to the plasma membrane. Under conditions where only high-affinity transporters are active, i.e. in copper- and iron-limited media, the physiological pathway comprises Atx1, which delivers Cu(I) to Ccc2 (plain arrow). However, when Ccc2 is overexpressed, an endocytosis-dependent pathway brings copper to Ccc2 independent of Atx1 (dotted arrow). (B) The enzymatic cycle of Ccc2. (1) The ATPase binds Cu(I) at its transport site, a step which induces a conformational change and (2) phosphorylation from ATP; (3) the energy is used to open the transport site on the other side of the membrane and to release Cu(I) into the TGN lumen; (4) the aspartyl-phosphate bound is hydrolyzed. (C) Wild-type Ccc2. A schematic representation of Ccc2 structure that comes from the calcium ATPase structure [40]. Empty circles represent known Cu(I)-binding sites. N-terminus includes M1 [white hexagon with the Cu(I)-binding sequence CASC] and M2 (grey hexagon with CGSC). The membrane domain comprises eight helices and the transport site (CPC) is on helix 6. The catalytic loop (cat), where phosphoryl transfer occurs from ATP to the phosphorylation site (Asp627 indicated by the star) when Cu(I) is bound to the transport site, is localized between helices 6 and 7. The actuator domain (act), between helices 4 and 5, participates in dephosphorylation of the catalytic loop. (D) Ccc2-like proteins bearing only one functional domain, either M1 or M2. (E) A Ccc2-like protein unable to bind copper at its N-terminus. In (D) and (E), all the constructs are named according to their N-terminus. Intact copper-binding motifs are denoted CXXC. SXXS refers to domains whose cysteines are changed to serines. Stripes recall their inability to bind Cu(I).

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Atx1 is a 73-amino-acid cytosolic protein featuring a βαββαβ ferredoxin-like fold with a metal-binding site located in its first loop. This site comprises the consensus sequence MXCXXC, whose two cysteines are involved in binding Cu(I) [12,13]. Ccc2 and the mammalian copper ATPases all possess a cytosolic N-terminus made of between two and six metal-binding domains (MBD) and each of these domains is similar to Atx1 in terms of structure, consensus sequence and copper-binding site [14]. The Ccc2 N-terminus (Met1–Ser258) is composed of two MBDs, designated M1 (Met1–Lys80) in tandem with M2 (Ser78–Gly151). Yeast two-hybrid experiments have shown that Atx1 interacts with each in the presence of copper [6,15,16]. In particular, it has been demonstrated in vitro that copper can be exchanged between Atx1 and the first MBD expressed separately [17].

Several aspects of copper delivery into the TGN have been studied. Under conditions where only high-affinity transporters can ensure uptake, i.e. in copper- and iron-limited media, deletion of ATX1 results in a deficient high-affinity iron uptake and this leads to growth arrest [15]. Consequently, atx1Δ deletion strains have been used as a tool to investigate whether homologous proteins would replace Atx1 and ensure copper transport into the TGN [18–22]. Deletion of the CCC2 gene also results in a deficient high-affinity iron uptake and leads to growth arrest in copper- and iron-limited media. Thus, ccc2Δ deletion strains have been used in complementation assays to assess the transport of copper inside the TGN by Ccc2 [8] and human homologues [23,24], or to investigate the role of various amino acids in these proteins [25–28]. Although these studies provide useful information about the functionality of the ATPase, they are all interpreted independently of the metallo-chaperone. Indeed, studying the Atx1–Ccc2 route in vivo remains challenging, because of the existence of an Atx1-independent pathway for copper delivery to the TGN [15]. This pathway was first discovered in atx1Δ, in which overexpression of Ccc2 allows growth in a copper- and iron-limited medium. The current hypothesis involves the plasma membrane transporter Ctr1 which would undergo endocytosis and deliver copper directly to Ccc2, bypassing the need for Atx1 (Fig. 1A).

We designed a sensitive complementation assay to study in vivo copper-dependent interactions between Atx1 or its homologues and Ccc2. The technical difficulty arising from activating Atx1-independent copper transfer to Ccc2 was first solved by expressing Ccc2 at a very low level in an atx1Δccc2Δ deletion strain. We show that the presence of one MBD on the Ccc2 N-terminus (M1 or M2) is necessary and sufficient to receive copper from Atx1 and to activate the ATPase, leading to copper transport into the TGN. The high sensitivity of our new complementation assay allowed us to determine that M1 and M2 expressed as independent cytosolic proteins were less efficient than Atx1 in transferring copper to the Ccc2 N-terminus. However, these independent MBDs were able to deliver copper directly to another copper-binding site in Ccc2, whose presence was revealed by truncation of the N-terminus. Our results suggest a dual role for the N-terminus of Ccc2, able to receive copper from Atx1, but also to transfer copper to another copper-binding site whose location in Ccc2 is discussed.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

A sensitive complementation assay to assess copper transfer from Atx1 to Ccc2 in vivo

The aim of this study was to investigate in detail copper delivery to the Ccc2 N-terminus by the metallo-chaperone Atx1 and its further transfer into the TGN lumen. As an alternative to the yeast two-hybrid system, we chose to coexpress Atx1 and Ccc2, the two partner proteins, in an atx1Δccc2Δ deletion strain and to assess growth restoration on a copper- and iron-limited medium, referred to as the selective medium (0.1–1 μm copper and 100 μm iron chelated by 1 mm ferrozine). Such low concentrations of copper and iron in the selective medium ensure that Ctr1 and the Fet3–Ftr1 complex are the only effective transporters for copper and iron uptake by the cells [5,8]. Bearing in mind that Fet3 is essential for cell growth and that its activity requires copper, the atx1Δccc2Δ deletion strain cannot grow unless Atx1 and Ccc2 are coexpressed and exchange copper, which then reaches Fet3 in the TGN (Fig. 1A, plan arrows). In this section, we report the design of a new complementation assay to dissect the role of Atx1, M1 and M2 in activating Ccc2 in vivo (the strains and plasmids needed for this assay are described in Table 1).

Table 1.   Yeast strains and plasmids.
YPH499Mata his3-Δ200 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 ade2-101 
atx1Δccc2ΔMata his3-Δ200 leu2-Δ1 lys2-801 ade2-101 atx1::ΤRP1 ccc2::URA3This study
BY4741Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 
BY4741ccc2ΔMata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ccc2ΔATCC
 Derived fromPlasmid namePromoterTerminator
MulticopypYEp181pY–PMA1ADC1
MonocopypRS313, pRS315pC–PMA1ADC1
Extra-low-copypRS313pX–LeakADC1
Plasmid nameExpressed proteinOriginRef.
Ccc2Wild-type Ccc2Saccharomyces cerevisiae[25]
M1ssM2Ccc2C13S,C16S in Ccc2This study
M1M2ssCcc2C91S,C94S in Ccc2This study
M1Ccc2del (E81–G151) from Ccc2This study
M1ssM2ssCcc2C13S,C16S,C91S,C94S in Ccc2This study
ΔMBDCcc2del (M1–G151)This study
Ccc2GFPGreen fluorescent protein tag in C-terThis study
ΔMBDCcc2GFPdel (M1–G151) green fluorescent protein tag in C-terThis study
Atx1Wild-type [22]
Atx1ssC15S,C18S in Atx1 [22]
Atx1HAWild-type HA tag in C-terThis study
Mbd1M1–K80 from Ccc2 [22]
Mbd1HAM1–K80 HA tag in C-terThis study
Mbd1ssC13S, C16S in Mbd1 [22]
Mbd1ssHAC13S, C16S, HA tag in C-terThis study
Mbd2S78–G151 from Ccc2 [22]
Mbd2HAS78–G151 HA tag in C-terThis study
Mbd3M1–G151 from Ccc2This study
CopZWild-typeBacillus subtilis[22]
NtkM1–A71 from CadAListeria monocytogenes[22]
MerPWild-typeCupriavidus metallidurans CH34[22]

Studying interactions between Atx1 and Ccc2 in vivo was challenging because of the presence of an Atx1-independent copper-delivery pathway to the TGN, activated by Ccc2 overexpression [15] (Fig. 1A, dotted arrows). Accordingly, expression of Ccc2 using a multicopy vector pY–Ccc2 (PMA1 promoter) restored the growth of atx1Δccc2Δ in the selective medium, even though Atx1 was absent (Fig. 2A, lane 2). We reasoned that lowering Ccc2 expression would impair the Atx1-independent pathway of copper delivery to the TGN. Thus, CCC2 was cloned in the monocopy plasmid pC, generating pC–Ccc2 (PMA1 promoter), but growth was still restored in the absence of Atx1 (Fig. 2A, lane 3). While trying to replace the PMA1 promoter with that of CCC2 [7] in pC–Ccc2, we obtained a plasmid denoted pX–Ccc2 with neither promoter. Although pX–Ccc2 was obtained through serendipitous discovery, it did not restore the growth of atx1Δccc2Δ in the selective medium (Fig. 2A, lane 4) unless Atx1 was coexpressed (Fig. 2A, lane 5). No complementation was obtained when the Atx1 copper-binding site was impaired as in Atx1ss (Fig. 2A, lane 6). Growth restoration of atx1Δccc2Δ in the selective medium using pC–Atx1 and pX–Ccc2 is therefore likely to reflect interactions between Atx1 and Ccc2. In all cases, growth was recovered by supplementation with 350 μm iron, because iron at high concentrations is imported into the cell by low-affinity transporters [8]. Immunodetection of Ccc2 showed that pX–Ccc2 provides a very low expression of Ccc2, weaker than the expression level detected in YPH499, the wild-type strain (Fig. 2B).

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Figure 2.  Design of a sensitive complementation system to study Atx1–Ccc2 interactions in vivo. (A) The growth of atx1Δccc2Δ cotransformed with indicated plasmids was assessed on the selective medium (1 μm copper, 100 μm iron and 1 mm ferrozine) or the iron-supplemented medium (1 μm copper, 350 μm iron and 1 mm ferrozine). Plasmids are indicated on the left, pC– stands for empty plasmids, required to provide accurate selection markers. (B) Expression of Ccc2 using the pX vector. Immunodetection of endogenous Ccc2 in the wild-type strain YPH499 (1) and immunodetection of Ccc2 provided by the pX– vector in the presence of Atx1 (pC–Atx1) in atx1Δccc2Δ (2). The proteins are indicated by *. (C) The growth of atx1Δccc2Δ cotransformed with indicated plasmids (and empty plasmids when required) was assessed on the selective (100 μm) and iron-supplemented (350 μm) media.

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Two-hybrid experiments [6,16] suggested that Atx1 interacts with both domains of the Ccc2 N-terminus (M1 and M2). Our new complementation assay using pX was further assessed by studying copper transfer from Atx1 to each MBD. Accordingly, we chose to express Atx1 together with Ccc2 proteins bearing only one intact MBD. We first generated pX–M1ssM2Ccc2 in which the copper-binding cysteines of M1 are changed to serines (i.e. M1 cannot bind copper) (Fig 1D). Coexpression of Atx1 with M1ssM2Ccc2 restored the growth of atx1Δccc2Δ in the selective medium, suggesting that copper transfer occurred in vivo between Atx1 and M2 (Fig. 2C, lane 3). Growth restoration was dramatically reduced when Atx1 was not coexpressed (Fig. 2C, lane 4), or when Atx1ss was coexpressed (data not shown). To investigate whether copper transfer would also occur between Atx1 and M1 in our system, we tried to generate M1M2ssCcc2, but this protein was not expressed using either pX–M1M2ssCcc2 or pC–M1M2ssCcc2. Alternatively, another construct denoted M1Ccc2 was created in which M2 was deleted and replaced by M1 (Fig. 1D). Coexpression of Atx1 with M1Ccc2 restored the growth of atx1Δccc2Δ in the selective medium, suggesting that copper transport occurs between Atx1 and M1 (Fig. 2C, lane 5).

These results are in agreement with the conclusions drawn from two-hybrid experiments [6,16] and in addition show that Atx1–M1 or Atx1–M2 copper-dependent interactions activate the ATPase in vivo, leading to copper transfer in the TGN lumen and growth restoration in the selective medium. Our findings also agree with those obtained with both human ATPases, whose fifth or sixth MBD (of six) is sufficient for normal transport activity of the protein in Chinese hamster ovary cells [29,30].

Atx1 homologues and copper transfer to the Ccc2 N-terminus

In a previous study, some structural homologues of Atx1 (namely MerP, a mercury-binding protein from Cupriavidus metallidurans CH34, Ntk, the MBD of the cadmium ATPase from Listeria monocytogenes, and CopZ, the metallo-chaperone from Bacillus subtilis), were found to be able to deliver copper to endogenous Ccc2 in an atx1Δ strain [22]. This study also showed that M1 and M2, expressed as independent proteins denoted Mbd1 and Mbd2, could also play the role of Atx1. Given the higher sensitivity of our new expression system, we reasoned that expressing these Atx1 homologues would allow us to compare their ability to transfer copper to Ccc2. For this purpose, Mbd1, Mbd2, Mbd3 (M1 in tandem with M2 expressed as an independent protein), MerP, CopZ and Ntk were coexpressed in atx1Δccc2Δ with Ccc2 and growth was assessed in the selective medium (Fig. 3A). On the whole, Mbd1, Mbd2 and Mbd3 were less efficient than the other homologues in transferring copper to Ccc2, a feature that was not observed in our previous study [22]. Coexpression of Mbd1 or Mbd3 with Ccc2 showed a quite similar growth, in agreement with structural data obtained in vitro for the whole N-terminus of Ccc2 showing unfolded M2 [31]. However, coexpression of Ccc2 and Mbd2 on its own was more efficient than coexpression with Mbd1, however, it still did not reach the growth levels obtained by coexpression with Atx1, CopZ, Ntk or MerP.

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Figure 3.  Evaluation of the Atx1 analogues efficiency in transferring copper to Ccc2 N-terminus. The growth of atx1Δccc2Δ cotransformed with indicated plasmids was assessed on the selective medium. Proteins used as potential metallo-chaperones are indicated on the left side. (A) The potential metallo-chaperones are expressed together with Ccc2; (B) Ccc2 is replaced by M1ssM2Ccc2; (C) Ccc2 is replaced by M1Ccc2.

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As we replaced Ccc2 by M1ssM2Ccc2, the growth pattern was similar (Fig. 3B). Interestingly, when M1Ccc2 was expressed in place of Ccc2 neither Mbd1 nor Mbd2 seemed efficient in restoring copper transfer (Fig. 3C). Taken together, these experiments suggest that interactions between the independent MBDs and one or the other MBD tethered to the membrane protein are poorly efficient or even not efficient in restoring copper transfer. Recalling that copper transfer from Atx1 to M1 or M2 is thought to occur via a copper-mediated interaction [6,32], one possible explanation is that M1 and M2 do not form homo- or hetero-dimers in the presence of copper. This may be a special feature of copper ATPase MBDs, as opposed to the metallo-chaperones, because Atox1, Atx1 and CopZ are known to form dimers in the presence of copper [33–35]. Such an inability to form a dimer would be beneficial to wild-type Ccc2, avoiding a dead-end heterodimer between M1 and M2 and favouring their interaction with Atx1 to collect copper. These results prompted us to investigate further the role of the N-terminus in the overall function of Ccc2. In particular, we investigated whether Mbd1 or Mbd2 could deliver copper to some other site in Ccc2, apart from the N-terminus.

Is the N-terminus dispensable in vivo?

In vitro studies of several ATPases have shown that their N-terminus modulates their activity [36,37]. The current hypothesis involves domain–domain interactions between the N-terminus and other domains of the ATPase. Indeed, in vitro studies of the purified N-terminus and catalytic loop of the Wilson ATPase have shown that these two domains interact in the absence of copper and that their interaction is diminished by copper binding to the N-terminus [38]. Such a domain–domain interaction has been proposed to hinder access to the membrane transport site and therefore to decrease the apparent affinity of the Wilson ATPase for copper [39]. Similar conclusions arose from the study of CadA, a cadmium ATPase expressed in Sf9 cells [36,40]. It is currently thought that these inhibitory domain–domain interactions are relieved by binding of the metal to the N-terminus of the ATPase. To further study the role of the Ccc2 N-terminus in the overall function of the ATPase in vivo, we engineered ΔMBDCcc2 (Fig. 1E), a protein in which the N-terminus is truncated from both M1 and M2. As shown by the complementation assays in Fig. 4A, coexpression of ΔMBDCcc2 with Atx1 did not restore the growth of atx1Δccc2Δ in the selective medium. Using green fluorescent protein fusions, the correct intracellular location of ΔMBDCcc2GFP was demonstrated (Fig. 4B). Therefore, mislocation could not explain this nongrowth phenotype and the enzymatic activity of ΔMBDCcc2 had to be verified.

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Figure 4.  ΔMBDCcc2 is active in vitro but does not restore copper transport to the TGN. (A) The growth of atx1Δccc2Δ cotransformed with the indicated plasmids was assessed on the selective (100 μm) and iron-supplemented (350 μm) media. (B) Localization of green fluorescent protein (GFP)-tagged proteins. BY4741ccc2Δ cells transformed with pC–Ccc2GFP or pC–ΔMBDCcc2GFP were grown until the exponential phase in liquid cultures. Ccc2GFP is localized partly at the vacuole membranes, as originally observed [7]. A similar pattern was observed with ΔMBDCcc2. The bar indicates 5 μm. (C) Phosphorylation assay. Ccc2, its non-phosphorylatable mutant Asp627Ala and ΔMBDCcc2 were expressed in Sf9 cells. Membrane preparations were incubated in ice-cold buffer (20 mm bis-Tris propane pH 6.0, 200 mm KCl, 5 mm MgCl2), phosphorylated with 5 μm radioactive ATP (1500 cpm·pmol−1) and samples were taken at different times, as indicated. All phosphoenzyme levels were compared with the value measured with the wild-type Ccc2 at 1 min. (▮) Ccc2, (◆) Asp627Ala, (□) ΔMBDCcc2.

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Copper ATPases such as Ccc2 and its human homologues are P-type ATPases, whose enzymatic cycle has been well described (Fig. 1B) [25,41,42]. As for all P-type ATPases, the energy for the active transport of copper is delivered by phosphoryl transfer from ATP to the aspartate belonging to the consensus sequence DKTGT in the catalytic loop. A tight coupling between this chemical reaction and ion transport across the protein is ensured by phosphoryl transfer requiring copper to be bound to the so-called transport site [43]. Once phosphorylated, the ATPase undergoes a conformational change and copper can dissociate towards the TGN lumen. The aspartyl-phosphate is then hydrolysed and the ATPase is ready for a new cycle. According to the known structures of other P-type ATPases, the transport site is located among the transmembrane helices, as indicated in Fig. 1C [44]. In the case of Ccc2, copper needs to bind to the CPC motif located at the sixth helix to allow the phosphorylation reaction, and thereby the release of copper in the TGN lumen [25]. When copper cannot bind to the transport site, the ATPase cannot be phosphorylated by ATP and is considered inactive.

ΔMBDCcc2, Ccc2 and its negative control, the non-phosphorylatable mutant Asp627Ala, were overproduced in Sf9 cells [25]. Membrane fractions were used to measure phosphoenzyme formation from radioactive ATP in the presence of contaminating copper, an assay which also evaluates whether the transport site has bound copper (Fig. 1B). The membrane fractions were mixed with [32P]ATP[γP], the reaction was acid-quenched at different times, samples were loaded onto acidic gels and the radioactivity incorporated at 110 kDa (Ccc2) or 94 kDa (ΔMBDCcc2) was evaluated. Ccc2 displayed a strong and transient signal, similar to the one obtained for ΔMBDCcc2, in comparison with the background, evaluated with Asp627Ala (Fig. 4C). In conclusion, ΔMBDCcc2 was well localized in the cell and active in vitro but could not deliver copper to the TGN because the N-terminus was missing and Atx1–MBD copper-dependent interactions could no longer occur to activate the ATPase.

We propose that, as described for the Wilson protein, the N-terminus interacts with another domain of Ccc2 (the catalytic loop for example) and this interaction inhibits the ATPase activity. Truncation of the N-terminus alleviated these interactions in ΔMBDCcc2, thereby facilitating the access of copper at the transport site in vitro, as shown previously for CadA [36,40]. Consequently, because Cu(I) is not available in the cytosol [45], a suitable copper carrier may be necessary to deliver copper to ΔMBDCcc2 in vivo. This is why we investigated whether Mbd1 or Mbd2 could directly deliver copper to ΔMBDCcc2.

A role for M1 and M2 in delivering copper to another binding site in Ccc2

In the first section of this study, we showed that Mbd1 and Mbd2 (M1 and M2 expressed as independent proteins) were barely able to deliver copper to the N-terminus. To evaluate the possibility that a native N-terminal MBD delivers copper to a domain other than the N-terminus, we coproduced Mbd1 or Mbd2 as independent proteins together with ΔMBDCcc2 in atx1Δccc2Δ. Mbd1ss was also generated where the cysteines from the copper-binding site have been changed to serines (i.e. Mbd1ss cannot bind copper). Unlike Atx1 (Fig. 5A, lane 2), Mbd1 and Mbd2 coexpressed with ΔMBDCcc2 restored the growth of atx1Δccc2Δ in the selective medium (Fig. 5A, lanes 4,5). No growth restoration was observed with Mbd1ss, showing that copper binding to Mbd1 or Mbd2 is a necessary step to allow copper translocation into the TGN by ΔMBDCcc2 (Fig. 5A, lane 3). In addition, no growth restoration was obtained when CopZ or MerP was coexpressed with ΔMBDCcc2 (data not show). Interestingly, CopZ has an electrostatic potential surface similar to that of Mbd1 and Mbd2 [22], but was still unable to transfer copper to ΔMBDCcc2, suggesting that electrostatic interactions are not the driving force for copper transfer to ΔMBDCcc2. HA-tagged Mbd1, Mbd1ss, Mbd2 and Atx1 were also coexpressed with ΔMBDCcc2 to evidence their expression by immunodetection (Fig. 5B). Thus, our data disclose special properties of Mbd1 and Mbd2 which enable both to transfer copper to ΔMBDCcc2, although this transfer does not occur with Atx1.

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Figure 5.  M1 and M2 domains produced as independent proteins (Mbd1 and Mbd2) are able to deliver copper to ΔMBDCcc2. (A) The growth of atx1Δccc2Δ cotransformed with indicated plasmids was assessed on the selective (100 μm) and iron-supplemented (350 μm) media. (B) Immunodetection of HA-tagged proteins expressed in atx1Δccc2Δ using pY–Atx1HA, pY–Mbd1HA, pY–Mbd2HA or pY–Mbd1ssHA together with pC–ΔMBDCcc2.

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Therefore, we can conclude that both M1 and M2, when expressed as independent cytosolic proteins, are able to bind copper in the cytosol and transfer this metal to a copper-binding site in Ccc2, out of the N-terminus. Although the site that receives copper from Mbd1 or Mbd2 has not yet been identified in ΔMBDCcc2, we can assume that when tethered to Ccc2, M1 and M2 deliver copper to the same site. The identity of this copper-binding site remains unknown but one good candidate is the membrane transport site, which comprises a CPC motif in the sixth helix, whose cysteines are essential for ATPase activity [25]. Therefore, unless another copper-binding site is disclosed in Ccc2, we propose that the copper ion bound to either N-terminal MBD is transferred directly to the membrane transport site, thereby activating the ATPase. The possibility of a still unidentified protein participating in copper transfer between the MBDs and the membrane transport site cannot be excluded here. However, activation of the Wilson ATPase by Atox1 in vitro suggests that no other protein is necessary to transport copper [46]. To conclude, our results suggest that the N-terminus of Ccc2 is not only involved in receiving copper from Atx1, but also plays a crucial role in the overall function of the ATPase by delivering copper to another site belonging to the rest of the protein. It is an important link in the cascade that delivers copper to the ATPase transport site, immediately before the active transport step occurs.

A model for the Atx1–Ccc2 route to the TGN under iron- and copper-limited conditions

Our study, in synergy with data gathered from the literature, allows us to propose a mechanism regarding copper transport to the Golgi lumen, enhancing the crucial role of Atx1 and the Ccc2 N-terminus. This model is described in Fig. 6 and shows step-by-step copper transport to the Golgi.

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Figure 6.  Model for the copper route to the TGN. (A) Wild-type Ccc2: (1) Atx1 can deliver Cu(I) to M1 or M2; (2) M2 has bound Cu(I); (3) Cu(I) at the transport site (CPC) induces phosphorylation and transport into the TGN. (B) One MBD is necessary and sufficient to receive copper from Atx1 and to activate the ATPase. In this representation, Atx1 delivers Cu(I) to the M2 domain of M1ssM2Ccc2. (C) ΔMBDCcc2 receives Cu(I) from Mbd1 (or Mbd2) (1) and transports Cu(I) into the TGN (2).

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First, we hypothesized the presence of inhibitory domain–domain interactions involving the Ccc2 N-terminus, as represented in Fig. 6A1, and similar to those demonstrated for the Wilson ATPase in vitro [38,39]. These interactions were functionally evidenced by the Cys to Ser mutations in all MBDs. The -SH to -OH substitutions made the N-terminus unable to bind copper, but still able to interact with the catalytic loop, leading to a conformation that was inactive. Recent structural data from the Archaeoglobus fulgidus copper ATPase are in agreement with such interactions [47].

To alleviate these inhibitory domain–domain interactions in wild-type Ccc2, copper transfer from Atx1 or any other efficient metallo-chaperones to M1 or M2 domains of Ccc2 is essential (Fig. 6A1 and A2). We demonstrate here that one, and only one, functional copper-binding site on the Ccc2 N-terminus is required for efficient copper transfer from Atx1 to Ccc2 (Fig. 6B where M1 is impaired and unable to bind copper). Another way to disrupt these inhibitory domain–domain interactions in Ccc2 is to delete the N-terminus, as in ΔMBDCcc2. In vitro, ΔMBDCcc2 is active and phosphorylatable by ATP in the presence of copper. Copper binding at the membrane transport site CPC (i.e. distinct from the N-terminus copper-binding sites) induces phosphoryl transfer from ATP to Asp627 (Fig. 1B) (for a description of the Ca2+-ATPase transport cycle based on recent structural data, see Olesen et al. [44]). However, in vivo, Cu(I) is not freely available [45] and ΔMBDCcc2 did not restore copper homeostasis because the N-terminus was not present to receive copper from Atx1 (Fig. 5). The suitable copper carriers that were found here are Mbd1 and Mbd2, the two domains of the N-terminus expressed as independent proteins, which could directly deliver copper to another binding site in ΔMBDCcc2 (Fig. 6C1 and C2).

We conclude that the copper cargo, initially delivered by Atx1 and bound to the Ccc2 N-terminus, is next transferred to another binding site in the protein through domain–domain interactions. Interestingly, a recent report on in vitro copper transfer from the metallo-chaperone CopZ to the solubilized copper ATPase CopA from A. fulgidus reaches the opposite conclusion [48]. CopA possesses one MBD in its N-terminus and another in its C-terminus and although both MBDs are able to receive copper from CopZ this does not activate the ATPase. Activation is obtained by direct delivery of copper by CopZ to CopA transport site in the membrane.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The complementation studies reported here have led us to propose a model for in vivo copper delivery to the secretory pathway in S. cerevisiae which takes into account in vitro studies performed on the human Wilson ATPase [38,39,46]. Our model depicts protein–protein and intraprotein domain–domain interactions leading to copper transfer from Atx1 to the core of the copper ATPase. These interactions ensure that no release of free copper occurs in the cytosol during the transfer of the ion to the secretory pathway. The assay designed here should be useful for designing a functional Atox1–Menkes ATPase or Atox1–Wilson ATPase pathway for copper delivery into the TGN in yeast. This could enable further investigation of the effect of mutations in Atox1 or in the Menkes or Wilson ATPase N-terminus that may impair copper transfer and result in disorders of copper homeostasis.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Yeast strains

Genetic modifications were performed on the YPH499 strain of S. cerevisiae (a gift from Pierre Thuriaux, CEA, Gif/Yvette, France). The chromosomal ATX1 gene was disrupted in YPH499 using a deletion cassette bearing the TRP1 gene as a selection marker generating atx1Δ [22]. The chromosomal CCC2 gene was then disrupted in atx1Δ using a deletion cassette inserting the URA3 gene into the CCC2 coding sequence [25]. Yeast transformants were selected on Drop Out medium without uracil or tryptophan and their genomic DNA was prepared according to Zhang et al. [49]. PCR analysis confirmed the correct insertion of deletion cassettes in the yeast genome. The double mutant strain was designated atx1Δccc2Δ.

Expression constructs

Soluble proteins

Genomic DNA was used to amplify the ATX1 gene. The metal-binding domains of Ccc2, Mbd1 (Met1–Lys80), Mbd2 (Ser78–Gly151) and Mbd3 (Met1–Gly151) were generated by PCR amplification of the corresponding segments of CCC2 cDNA [25]. Mbd1ss (i.e. Mbd1 bearing C13S and C16S mutations), was generated by PCR starting from Mbd1. For expression in atx1Δccc2Δ, the coding sequences were inserted into mono- or multicopy plasmids containing the PMA1 promoter and the ADC1 terminator and all bearing LEU2 as selection marker (Table 1). The monocopy plasmid (pC-) was derived from pRS315 [50]; the multicopy plasmid (pY-), from pYep181 [51]. Site-directed mutagenesis was used to fuse the HA-coding sequence at the end of Atx1, Mbd1, Mbd1ss and Mbd2 coding sequences in the multicopy plasmid pY (Table 1).

Expression constructs for mutants of Ccc2

Plasmids encoding the modified Ccc2 proteins described in Fig. 1 were generated with the QuickChange site-mutagenesis kit (Stratagene, Agilent Technologies, Massy, France) using pSP72CCC2 as the template [25]. In all cases, the presence of designed mutations and the absence of fortuitous mutations were verified by automated DNA sequencing. CCC2 was first cloned in a multicopy plasmid (pY) and in a monocopy plasmid (pC) under the control of the PMA1 promoter and the ADC1 terminator and all bearing HIS3 as selection marker (Table 1). The monocopy plasmid was derived from pRS313 [50], the multicopy plasmid from pYep181 in which the selection marker was changed to HIS3 [51]. In an attempt to clone the CCC2 promoter in place of the PMA1 promoter in pC–Ccc2, a fortuitous ligation occurred between SacII and StuI sites generating a plasmid with neither CCC2 nor PMA1 promoters. This plasmid was called pX–Ccc2, where pX stands for extra-low-copy expression plasmid, and used for extra-low expression of other constructs (Table 1). When the enzymatic activity of ΔMBDCcc2 protein had to be evaluated, the mutant was cloned in the pFastBac plasmid for expression in Sf9 cells [25]. When the location of the copper ATPase had to be verified, site-directed mutagenesis was used to fuse the green fluorescent protein-coding sequence at the end of Ccc2 and ΔMBDCcc2 in the monocopy plasmid pC.

Expression in yeast and complementation assay

atx1Δccc2Δ was co-transformed with one plasmid expressing Atx1 or an Atx1-like protein and another expressing wild-type or modified Ccc2. In some experiments, empty plasmids with the HIS3 or the LEU2 markers were used. Transformants were selected on DO–Leu–His–Trp–Ura plates. Randomly selected clones were grown for 3 days at 30 °C in copper- and iron-limited medium to assess growth restoration by expression of the proteins of interest (105 and 104 cells per drop) [52]. The medium designated selective contained 1.7 g·L−1 Yeast Nitrogen Base without copper or iron (Qbiogene, MP Biochemicals, Illkirch, France), 5 g·L−1 ammonium sulfate, 20 g·L−1 glucose, 0.6 g·L−1 CSM–Leu–His–Trp–Ura (Qbiogene), 50 mm Mes/NaOH (pH 6.1), 1 μm CuSO4, 100 μm NH4(FeSO4)2.6H2O and 1 mm ferrozine. Higher concentrations of iron in this medium [350 μm NH4(FeSO4)2.6H2O] allow yeast cells with an Atx1–Ccc2-deficient pathway to incorporate iron and therefore to grow. The iron-supplemented medium was systematically used as a control after each transformation of atx1Δccc2Δ with modified Atx1 and/or Ccc2 proteins. For each complementation assay, we used three different clones obtained from at least two different transformations. Plates were photographed after 3 days of growth at 30 °C.

Yeast proteins extracts: expression level analysis

Cells were spun down for 10 min at 1000 g and 4 °C and washed twice with ice-cold water. They were then suspended in buffer A (50 mm Tris/HCl, pH 8.0, 300 mm sorbitol, 2 mm EDTA) supplemented with antiproteases [one tablet of complete EDTA-free protease inhibitor (Roche Diagnostics, Meylan, France) and 1 mm phenylmethanesulfonyl fluoride]. After disruption with glass beads, the lysate was centrifuged for 10 min at 1000 g to pellet cell debris and nuclei. The resulting supernatant was spun down for 60 min at 100 000 g and 4 °C and either the pellet was suspended in buffer A for membrane protein determination or the supernatant was concentrated using a 5000 Da molecular mass cut-off concentrator (Vivaspin, dominique Dutscher, Brumath, France) for HA-tagged metallo-chaperone detection. Protein concentration was determined using DC protein assay (Bio-Rad, Marnes-la-Coquette, France). For Ccc2 and Ccc2-like proteins, 30 μg of total protein was measured and loaded on SDS/PAGE gels prior to western blotting. Proteins were immunodetected using the BM Chemiluminescence Western Blotting kit (Roche Diagnostics), Ccc2 with the polyclonal anti-Ccc2GB [25] and the metallo-chaperones with the anti-HA-peroxidase system (Roche Diagnostics).

Expression in Sf9 cells, membrane fraction preparation, phosphorylation from ATP and isotopic dilution

All these procedures were performed as described earlier [25].

Fluorescence microscopy

BY4741ccc2Δ cells were transformed with pC–Ccc2GFP or pC–ΔMBDCcc2GFP and grown in minimum medium without Leu at 30 °C with agitation for 2 h. Cells were collected and imaged using a 100 × oil immersion objective on a fluorescence microscope (Axiovert 200M; Carl Zeiss, Le Pecq, France) and the appropriate filters for GFP fluorescence and Nomarski imaging.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We would like gratefully to thank Patrice Catty and Florent Guillain for their participation in fruitful discussions and Naima Belgareh-Touze for her advice on performing fluorescence microscopy with yeast. Funding of this work was provided in part by the Programme de Toxicologie Nucléaire Environnementale. IM received support from a fellowship from the Programme de Toxicologie Nucléaire du CEA.

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  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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