Maintenance of copper homeostasis is essential for cell survival. Consequently sophisticated mechanisms have evolved for copper acquisition, distribution and excretion. In mammalian cells, copper is acquired by the copper transport protein Ctr1 and distributed to target proteins via dedicated copper metallochaperones such as CCS, Atox1 and Cox17 [
7,
8]. To avoid the potentially toxic effects of copper, Cu-ATPase proteins mediate the excretion of excess copper [
9] and a
P. falciparum Cu-ATPase ortholog has been identified [
5]. One
P. falciparum copper source appears to be from the ingestion of host erythrocyte Cu/Zn SOD [
5]. A screen of the PlasmoDB database, performed in the present study, identified six
P. falciparum copper-requiring protein orthologs and a candidate copper transport protein. Native expression of the putative copper transporter was confirmed by Western blot and the protein’s subcellular location identified by immunofluorescence microscopy. A recombinant form of the transporter's amino terminal domain was shown to bind copper
in vitro and within
E. coli host cells, supporting the possibility that the full-length protein functions to acquire copper.
Identification of a putative P. falciparum copper transport protein
The identification of six
P. falciparum copper-requiring protein orthologs suggested an important role for copper in
P. falciparum metabolism. Treating
P. falciparum parasites with the intracellular copper chelator, neocuproine, inhibited ring-to-trophozoite transition
in vitro[
5], highlighting the importance of copper for the parasite. During the course of its asexual cycle,
P. falciparum appears to digest host erythrocyte Cu/Zn SOD to release copper [
5]. The presence of a membrane associated copper binding protein with copper transport protein characteristics in plasmodia suggests a mechanism for the transport of copper similar to that played by the Ctr1 copper transport protein in yeast and mammalian cells [
7,
8]. Candidate copper transporter sequences were identified for eight species of the
Plasmodium parasite and each sequence was shown to contain the essential and largely definitive Mx
3M and Gx
3G motifs (Figure
1B) [
30,
45]. The predicted amino terminal domain of each putative transporter was found to contain one or more methionine motifs (MxM or MxxM). Although not limited to copper transporters, these motifs are important for Ctr1 protein function [
30].
The presence of three putative membrane-spanning regions is considered definitive for copper transport proteins [
33] and topological analysis of the putative
P. falciparum copper transporter identified three such domains. Since three transmembrane domains cannot form a functional channel or pore for ion transport [
10], a homotrimeric complex of copper transport protein monomers is formed for the characterized mammalian and yeast copper transport proteins [
33]. The
P. falciparum copper transport protein may form a similar complex since the first transmembrane domain of the copper transport protein family serves as an adaptor allowing evolutionarily distant copper transporters to adopt a similar overall structure [
46]. An immunoblot of yeast and mammalian copper transport proteins identified monomeric and dimeric species of the trimeric complex [
37]. Similar monomeric and dimeric species were detected in an immunoblot of the putative
P. falciparum copper transport protein in this study.
Recombinant expression and copper binding to MBP-Pf Ctr369Nt-S
Signal peptides, transmembrane domains, rare codons, introns and genome AT-richness affect recombinant expression of
P. falciparum proteins [
34]. The amino terminal signal peptide of the putative
P. falciparum copper transporter was, therefore, excluded from the expression construct, resulting in periplasmic expression. The purified MBP-
Pf Ctr369Nt
-S recombinant protein bound reduced copper
in vitro and within a cellular environment. A second copper transport protein, MBP-
Pf Ctr211Nt
-S, from the PF14_0211 gene was recombinantly expressed and isolated. Expression of the native
P. falciparum protein encoded by this gene has not been ascertained at this stage.
In mammalian and yeast cells, the reduced cuprous ion (Cu
+) is favoured for transfer and transport [
30], since Cu
+ is more exchange labile than Cu
2+[
36]. Cu
+ is highly reactive in an oxidizing environment and methionine motifs in the extracellular amino terminal domain of the copper transporter family are thought to sequester copper prior to its transport across the lipid bilayer [
30]. This was supported by a study demonstrating that methionine motif peptides inhibited copper-catalysed ascorbate oxidation through Cu
+ chelation [
36]. MBP-
Pf Ctr369Nt
-S inhibited copper-catalysed ascorbate oxidation through copper chelation. Taken together with the data showing copper binding to MBP-
Pf Ctr369Nt
-S, it appeared that the amino terminal domain of the putative
P. falciparum copper transporter preferably coordinates the Cu
+ ion. Copper coordination was presumably a result of the methionine motif present in MBP-
Pf Ctr369Nt
-S.
The methionine motifs contained in the amino terminal domains of yeast and human copper transport proteins are essential for copper binding under copper limiting conditions [
30]. The yeast copper transport protein contains eight methionine motifs, but only the last methionine of the eighth motif was shown by Puig
et al.[
30] to be essential for copper binding capability. The position of this methionine is conserved between copper transport proteins and is 20 amino acid residues N-terminal to the transporter’s first transmembrane domain [
30]. Interestingly, the last methionine of the methionine motif in the amino terminal domain of the putative
P. falciparum copper transport protein is located in a similar position. However, the involvement of other amino acid residues, like cysteine and histidine residues that bind metal ions [
47,
48], and are present in this protein cannot be excluded. Identification of the specific residues coordinating copper will be explored in future experiments.
MBP-Pf Ctr369Nt-S recombinant protein likely interacts with E. coli copper binding proteins
An interesting consequence of expressing MBP-
Pf Ctr369Nt
-S in the presence of copper was a significant increase in recombinant protein yield. In the presence of excess copper it is a possibility that the copper bound to and stabilized the recombinant protein [
48]. The cytoplasm and periplasm of
E. coli cells do, however, have different copper requirements since almost all bacterial copper proteins, such as multi-copper oxidases, amine oxidases and lysine oxidases are found in the periplasm or excreted extracellularly [
49]. Copper availability in
E. coli is thought to be regulated by the actions of the DNA-binding metal sensor CueR, which controls the expression of genes encoding proteins involved in metal homeostasis [
50]. A copper-requiring protein is thought to gain access to copper only if the protein’s affinity for the metal ion is greater than the buffered cellular concentration of copper [
51]. Interaction of a recombinant
P. falciparum protein with native
E. coli proteins has been demonstrated for
Pf Hsp70 [
52], supporting the possibility that MBP-
Pf Ctr369Nt
-S interacts with native
E. coli copper proteins for copper loading. These interactions could, in turn, influence the availability of copper for native
E. coli copper-requiring proteins resulting in tightly regulated MBP-
Pf Ctr369Nt
-S expression under standard growth conditions. The addition of excess extracellular copper appears to have alleviated this growth stress producing the increase in the expression of MBP-
Pf Ctr369Nt
-S observed here.
The putative P. falciparum copper transport protein shows stage-specific localization
During early stages of asexual development, the putative
P. falciparum copper transporter appears to be targeted to the plasma membrane of the infected erythrocyte. As the parasite matures through its asexual cycle the protein was detected on a parasite membrane. This may represent a turnover of host cell membrane associated malarial proteins, followed by trafficking of the copper transporter to a parasite membrane. The precise localization of the copper transporter, when associated with the parasite, was not elucidated, due to limits to the fluorescence detection methods. The parasitophorous vacuolar membrane contains a “promiscuous” pore that permits the passage of solutes, nutrients and macromolecules [
53,
54] and thus it is unlikely for there to be the need for a separate copper transporter. The parasite plasma membrane contains a variety of selective transporters (Reviewed by Martin
et al.[
4]). The copper transporter is thus suggested to be associated with the parasite plasma membrane.
Pf CuP-ATPase was similarly localized to a parasite plasma membrane and the surface of the infected erythrocyte. However, unlike the putative copper transport protein,
Pf CuP-ATPase was associated with both the parasite plasma membrane and the erythrocyte membrane at the same time in trophozoites and schizonts, whilst no expression was detected in rings [
5]. These authors suggested that dual localization of
Pf CuP-ATPase represents a novel mechanism by which the parasite reduces copper toxicity through copper efflux [
5]. Reasons for the different localities of the copper transport protein are less apparent. There is the possibility that during ring stages, extracellular copper is required until the parasite digests endocytosed host cell Cu/Zn SOD and translocates the copper transporter protein to a parasite membrane.
The concentration of copper in normal red blood cells has been reported as18 μM and copper is imported into red cells by a Cl
--HCO
3- anion exchanger [
55]. Inside the red cell most of the copper is associated with Cu/Zn SOD and a 30 and a 40 kDa red cell protein [
56]. Rasoloson
et al.[
5] reported a lower red cell copper concentration at 10 μM and further suggested that copper concentrations in parasitized cells was lower than uninfected cells. Copper in isolated parasites was lower than in red cells at 2 μM in rings and between 6 and 6.6 μM in trophozoites (depending on the isolation method). This data disagrees with recent data indicating that malaria infected red cell copper concentration was 30 μM, and double that of uninfected red cells in the same experiment [
57]. The different values for copper concentrations in infected red cells may be due to the different measuring methods employed and needs to be reconciled [
5,
57]. Interestingly as the parasite develops from trophozoites to schizonts, the concentration of ingested Cu/Zn SOD decreases [
5] and this coincides with data presented here showing the appearance of the putative copper transporter on parasite membranes, the highest level of expression of the protein, and mRNA levels described by Le Roch
et al.[
38]. Thus as the source of copper in the form of Cu/Zn SOD decreases in the parasite [
5], the parasite compensates with an increase in expression of the putative copper transport protein. Given the potential toxicity of copper and the need for the regulation of copper homeostasis, it is likely that the parasite employs a copper transport protein alongside the
Pf CuP-ATPase copper export protein described by Rasoloson
et al.[
5]. It is suggested that, given the presence and membrane location of a copper binding protein with copper transporting motifs, and the increase in copper concentration in malaria infected red cells [
57], that the protein described here has a role in copper transport. Data to support a transport role for the copper binding protein is being pursued.
Treatment of
P. falciparum parasites with neocuproine inhibited ring-to-trophozoite transition [
5], implicating copper metabolism as a potential target for novel anti-malarial drug development. Copper, when the concentration is above a critical level, has been shown to be toxic to the parasite [
58]. However, from
in silico findings it seems unlikely that
P. falciparum contains unique copper-dependent metabolic pathways. Copper-binding protein motifs also appear to be conserved from prokaryotes to eukaryotes [
59], suggesting that designing a compound specific for
P. falciparum copper-dependent proteins could prove difficult. An alternative approach would be to deliver anti-malarial compounds to the parasite by exploiting transport mechanisms, as suggested for the
P. falciparum new permeation pathway and choline carrier [
60]. The human copper transport protein has been shown to transport the platinum containing anti-cancer drugs cisplatin and oxaliplatin [
61]. Cisplatin also exhibits anti-malarial activity through DNA damage [
62,
63]. Given the structural similarities between the putative
P. falciparum copper transport protein and the human copper transporter, it is possible that cisplatin is delivered to the parasite by the putative copper transport protein. This transport mechanism could perhaps be exploited for the delivery of novel platinum-based anti-malarial compounds.
Plasmodium falciparum actively remodels the erythrocyte during infection, leading to an increase in the permeability of the host cell membrane to low molecular weight solutes [
64]. This increase is mediated by ‘new permeability pathways’ that have also been shown to greatly facilitate the uptake of the antibiotics fosmidomycin and its derivative FR900098 [
62]. Parasitized erythrocytes also show a significant increase in the uptake of a copper-neocuproine complex when compared to uninfected erythrocytes [
65]. Association of the putative
P. falciparum copper transport protein with the erythrocyte membrane, during early asexual development, therefore makes it tempting to speculate that the copper transport protein’s mechanism accounts for the increased rate of copper-neocuproine complex uptake by parasitized erythrocytes. Complexation of the anti-malarial buparvaquone to copper(II) significantly enhanced its anti-malarial activity [
66]. This was proposed to be a consequence of improved compound internalisation, which may be related to the transport mechanism of the putative copper transport protein. Localization of the
P. falciparum copper transport protein to the erythrocyte and parasite membranes in late ring and early trophozoite stages may explain the increased susceptibility of these stages to cisplatin [
67].
The putative copper transport protein sequence lacks an identifiable export motif
Plasmodium falciparum protein export beyond the parasitophorous vacuole membrane was suggested to be mediated by the PEXEL/HT motif, but this motif was later shown not to be the sole determinant of protein export. Over 300 PEXEL/HT proteins have been predicted, whereas only a few PEXEL negative export proteins (PNEPs) have been identified and a common PNEP motif is yet to be identified [
68]. One important feature of PNEP proteins is the presence of a transmembrane domain, since removal of this domain from the PNEPs MAHRP1, SBP1 and REX2 inhibited protein export [
43,
44]. Similarly, the transmembrane domain of SURFIN
4.2 was essential for protein trafficking to the infected erythrocyte and Maurer’s clefts [
69].
Analysis of the PNEPs REX2 [
43] and SBP1 [
44] identified partial PEXEL/HT motifs (LAE and LAD, respectively) as being essential for protein export. Analysis of the PF14_0369 sequence identified an identical LAD motif within the first 20 amino acids of its amino terminal domain, a similar position to the LAD motif in SBP1. Mutational analysis of the related LAE motif in REX2 established that the glutamate residue was essential for export [
43]. This suggests the related aspartate residue in the LAD motif of PF14_0369 may be important for export. Cleavage of the signal peptide in the PF14_0369 sequence would generate a new amino terminus similar to that generated following cleavage of the PEXEL motif in PEXEL/HT proteins. PEXEL motif cleavage generates an xD/E/Q amino terminus, with the presence of D/E/Q being essential for protein export [
70]. Another potential export signal in SBP1 is located N-terminal of its transmembrane domain, between amino acids 180 and 190 [
44]. A comparison of this short signal sequence (
N EY
E VE
S) with the PF14_0369 sequence identified a similar sequence upstream of the first predicted transmembrane domain (
N KW
E TK
S), thereby implicating this sequence as a potential contributor to successful protein export. The presence of motifs in PF14_0369 similar to those important for PNEP export suggests that these motifs play a role in the export of the putative copper transporter.
The amino terminal domain of the
P. falciparum copper binding protein described here binds copper
in vitro and in an
in vivo expression system. The protein has copper transport motifs and has been shown to be expressed by malaria parasites and locate to two different membranes (the erythrocyte and the parasite membrane) as the parasite develops within the infected red cell. This evidence implicates a copper transport role for the protein in malaria infected erythrocytes and this implication is being explored. Whether the protein imports copper or has a copper export role alongside the Cu-ATPase protein described by others [
5], remains to be elucidated.