CHAPTER 6 General discussion
6.6 Proposed mechanism for Plasmodium parasite copper acquisition
The presence of important functional copper transport protein motifs in the putative Plasmodium copper transporters (Figure 3.1 and Figure 3.2) suggests they are membrane bound and play a similar role to yeast and mammalian copper transporters. Association of a putative murine malaria parasite copper transporter with what appears to be a parasite membrane (Figure 4.8) supports this possibility. Furthermore, it was established that a recombinant form of the amino terminal domain of the Plasmodium parasite's copper transporter could bind copper (Figure 4.20 and Figure 4.21), which has similarly been observed for the human copper transporter ortholog (De Feo et al., 2009; Jiang et al., 2005; Larson et al., 2010). However, it is still necessary to confirm the location, orientation and direction of transport for the Plasmodium copper transporter. Establishing protein location, in particular, will assist in elucidating protein function since it is predicted that each of the two putative P. falciparum copper transporters are targeted to different organelles (Figure 6.1). Considering the similarities identified between the Plasmodium transporters and other characterised copper transporters, two potential mechanisms for parasite copper acquisition are proposed (Figure 6.1).
Figure 6.1 Proposed mechanism for P. falciparum copper acquisition
Following invasion of the red blood cell (RBC), the Plasmodium parasite is encapsulated within the parasitophorous vacuole membrane (PVM). Two putative copper transporters are predicted to be transcribed by P. falciparum, PF14_0211 and PF14_0369. PF14_0211 is predicted to be in a parasite membrane (A), whilst PF14_0369 is predicted to be in an apicoplast membrane (B). The Cox17 metallochaperone is predicted to load copper into cytochrome-c oxidase, which is thought to be associated with the mitochondrion (van Dooren et al., 2006). Unknown transport or transfer steps are marked with a question mark (?).
A number of Plasmodium parasite transport proteins are targeted to the surface of the parasite, whilst others are localised to the membranes of the parasite apicoplast, mitochondrion, digestive vacuole and organelles of the secretory pathway. The likely cellular destination(s) of a given transporter can be inferred by signals present in its protein sequence and/or by its close similarity to a transport protein of known cellular location (Martin et al., 2005; Martin et al., 2009a). Analysis of the two putative P. falciparum copper transporters identified a signal peptide in both proteins, whilst an apicoplast transit peptide was also identified in PF14_0369. This suggests PF14_0211 is targeted to the parasite plasma membrane, whereas PF14_0369 is targeted to the apicoplast (Figure 6.1). Although targeting of PF14_0211 to the parasite membrane seems most likely, it cannot be excluded that PF14_0211 might also be targeted to the parasitophorous vacuole membrane. This is a possibility since the signal/s for targeting membrane proteins to the parasitophorous vacuole membrane remain unknown (Martin et al.,
2009a). Should PF14_0211 be localised to the vacuolar membrane, it would presumably transport copper from the erythrocyte cytosol into the parasitophorous vacuole for the parasite (Figure 6.1). However, it is currently believed that only high-capacity, low-selectivity channels are located in the vacuolar membrane to render it freely permeable to low-molecular-weight solutes (Desai et al., 1993; Martin et al., 2009a). Since these solutes could include copper, it is possible that PF14_0211 location in the vacuolar membrane is unnecessary, suggesting this transporter could be exclusively in the parasite plasma membrane.
The P. falciparum apicoplast is a plastid bound by four membranes that is homologous and conceptually similar to the plant chloroplast, which was derived from a modified cyanobacterium in a eukaryotic host cell (Lim and McFadden, 2010; Ralph et al., 2004). The apicoplast is essential for parasite survival (Goodman et al., 2007; Vaughan et al., 2009) and contains several plastid-derived biochemical pathways (Lim and McFadden, 2010). A striking feature of the apicoplast is its close association with the parasite mitochondrion (Bannister et al., 2000; van Dooren et al., 2006). This association is thought to be conducive to substrate exchange (Ralph et al., 2004), but the majority of the membrane-bound transporters required for such processes remain to be identified (Lim and McFadden, 2010; van Dooren et al., 2006).
In the present study it was proposed that one of the P. falciparum copper transporters (PF14_0369) is located in an apicoplast membrane (Figure 6.1). Reasons for this association remain elusive, particularly since the membrane orientation and direction of transport remain unknown. However, the possibility exists that the parasite apicoplast acts as a site for copper storage, similar to the Chlamydomonas chloroplast (Merchant et al., 2006). This therefore implies that, depending on its orientation, the PF14_0369 copper transporter could either function to redistribute stored copper for intracellular use or alternatively acquire surplus cellular copper for storage. However, it is perhaps more likely that PF14_0369 functions in copper redistribution since in the Chlamydomonas chloroplast, for example, copper is acquired through the action of two independent P-type ATPases whilst copper transporter-type proteins are thought to mobilise stored copper (Merchant et al., 2006; Shcolnick and Keren, 2006).
Considering the close association between the apicoplast and mitochondrion it is plausible that, if copper were stored in the apicoplast, PF14_0369 could provide a more direct route of copper delivery to Cox17 for subsequent insertion into cytochrome-c oxidase.
Regardless of their specific loctions, it is presumed that both PF14_0211 and PF14_0369 would transport copper across the membrane via a passive mechanism, similar to that suggested for the human copper transporter (Lee et al., 2002). Another similarity likely to exist between the human and putative Plasmodium copper transporters, is that copper is transported
across the membrane in its reduced, cuprous form. This probability was based on the data obtained from copper binding studies with MBP-PfCtr211NtTD and MBP-PfCtr369NtTD (Figure 4.21). Once copper is transported across the relevant parasite membrane(s) and into the cytosol, it is presumably distributed to target proteins by intracellular metallochaperones.
Although the majority of these target proteins and metallochaperones remain to be identified, a putative Plasmodium parasite Cox17 metallochaperone ortholog was identified in the present study and shown to bind copper (Figure 5.9). Supporting a parasite requirement for this metallochaperone was the in silico identification of parasite cytochrome-c oxidase complex orthologs. In yeast and mammalian cells, Cox17 is required for copper delivery to cytochrome-c oxidase for its activation in the mitochondrion (Kim et al., 2008; Lutsenko, 2010). However, in yeast and mammalian cells, the mechanism by which copper is delivered to the mitochondrion still requires further elucidation (Lutsenko, 2010). This therefore limits the similarities that can be drawn between the mammalian and corresponding parasite mechanisms, apart from the fact that Cox17 is likely to be required for copper delivery to the cytochrome-c oxidase complex (Figure 6.1).