Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of cation/H+ antiporter (PbCAX)

Protein (function)
Ca2+/H+ exchanger (PfCAX, also termed the Ca2+/H+ antiporter, PfCHA) has been identidied in P. falciparum. PfCAX and other apicomplexan orthologues belong to the Ca2+/cation antiporter (CaCA) superfamily CAX genes are classified into 3 subfamilies. Type II CAXs are found in fungi, Dictyostelium, and lower vertebrates and Type III CAXs are found in bacteria, while Type I CAXs include bacterial, fungal, plant and protozoan CAXs. Type I CAXs are divided into 8 subgroups (A to H) and protozoa are classified into the Type I-C phylogenetic group. Functional characterisation of CAX proteins (mainly from plants and fungi) has shown that these H+ coupled exchangers all transport Ca2+, with some being highly specific for Ca2+, whilst others mediate the transport of a broad range of additional divalent cations or, in some cases, transport additional monovalent cations. Their primary role in plants and fungi is to enable tolerance to high extracellular Ca2+concentrations, by internal sequestration of Ca2+into acidic organelles when cytosolic levels rise.

The phenotype analyses of P. berghei mutants lacking PbCAX (RMgm-846, RMgm-847) indicate that PbCAX has no essential role in asexual blood stages. Phenotype analyses indicate that PbCAX is involved in development of the female gamete/zygote into ookinetes. Mutant parasites failed to develop further from ‘round’ form zygotes into mature ookinetes.

Phenotype
The phenotype analyses of P. berghei mutants lacking PbCAX indicate that PbCAX has no essential role in asexual blood stages. Phenotype analyses indicate that PbCAX is involved in development of the female gamete/zygote into ookinetes. Mutant parasites failed to develop further from ‘round’ form zygotes into mature ookinetes.

Analysis of the transgenic Pbcax-gfp parasites showed the following: Only very low level, diffuse fluorescence signals in asexual blood stages, with stronger parasite-associated GFP signals observed only on rare occasions. Stronger GFP signal was observed in female but not male gametocytes and the same was true for gametes. Thus, the asexual parasites with stronger GFP signal may have been immature female gametocyte stages. Good GFP signal was also observed in zygotes, ookinetes and oocysts. Little GFP-signal co-localised with Mito-Tracker, used as a marker for parasite mitochondrion.

Additional information
 Analysis of P. falciparum PfCAX has demonstrated Ca2+/H+ exchange activity, an ability to exchange a limited range of other divalent cations, and a high transport capacity but low affinity (Km value of ,2 mM) for Ca2+, when expressed in Xenopus oocytes. In vivo studies characterising PfCAX are consistent with an atypical localisation to the inner mitochondrial membrane, and an atypical function, where the protein provides a pathway for removal of Ca2+ from this organelle back into the parasite cytosol.

The studies on P. berghei PbCAX provided no evidence for a location or role in mitochondria.

PfCAX has a predicted mitochondrial targeting sequence at residues 11–18 (YVRRTISQ), consistent with mitochondrial localisation, and this is conserved throughout the apicomplexan CAXs. Interestingly, this protein sequence has been identified in phosphoproteomic studies, using preparations derived from mature trophozoite-infected erythrocytes, and two of these residues, T15 and S17, are putative phospho-acceptor sites (with ascores of 1000, suggesting the annotations have a high degree of confidence). Phosphorylation of the S17 residue of PbCAX has also been reported, in a similar study, using ookinete preparations (available on GeneDB). The homologous protein region of TgCAX was also identified in tachyzoite preparations, although it contained no phosphorylated sites. However, neighbouring residues at positions S26, S27 and T46 were identified as being phosphorylated, albeit with lower ascores of 19, 13 and 6, respectively. Previous work has demonstrated that phosphorylation of the mitochondrial signal sequence of 29,39-cyclic nucleotide-39-phosphodiesterase 2 (CNP2) alters its localisation so that it is retained in the cytoplasm and its possible a similar mechanism could alter the location of apicomplexan CAXs. However, the localisation evidence presented for PbCAX provides little evidence for mitochondrial function of CAXs in apicomplexan parasites.

Other mutants
P. berghei mutants lacking PbCAX (RMgm-846, RMgm-847)