Membrane transporters in the relict plastid of malaria parasites

Abstract

Malaria parasites contain a nonphotosynthetic plastid homologous to chloroplasts of plants. The parasite plastid synthesizes fatty acids, heme, iron sulfur clusters and isoprenoid precursors and is indispensable, making it an attractive target for antiparasite drugs. How parasite plastid biosynthetic pathways are fuelled in the absence of photosynthetic capture of energy and carbon was not clear. Here, we describe a pair of parasite transporter proteins, PfiTPT and PfoTPT, that are homologues of plant chloroplast innermost membrane transporters responsible for moving phosphorylated C3, C5, and C6 compounds across the plant chloroplast envelope. PfiTPT is shown to be localized in the innermost membrane of the parasite plastid courtesy of a cleavable N-terminal targeting sequence. PfoTPT lacks such a targeting sequence, but is shown to localize in the outermost parasite plastid membrane with its termini projecting into the cytosol. We have identified these membrane proteins in the parasite plastid and determined membrane orientation for PfoTPT. PfiTPT and PfoTPT are proposed to act in tandem to transport phosphorylated C3 compounds from the parasite cytosol into the plastid. Thus, the transporters could shunt glycolytic derivatives of glucose scavenged from the host into the plastid providing carbon, reducing equivalents and ATP to power the organelle.

Fig. 1.

Schematics of the PfiTPT and PfoTPT proteins showing 10 transmembrane domains, two putative substrate binding sites, and a 140-aa leader comprising a signal peptide (SP) and a transit peptide (TP) in PfiTPT. Motifs selected from PfiTPT and PfoTPT for synthetic peptides used to generate antibodies are shown in dashed red lines. The N-terminal processing site for PfiTPT is marked by an arrow. Predicted molecular masses are shown.

Fig. 2.

Immunolocalization of PfiTPT and PfoTPT to the apicoplast membranes. (A) Anti-PfiTPT (red) colocalizes with apicoplast-targeted GPF (green) in parasites expressing PfACP(leader)-GFP (18). PfiTPT is in the membrane fraction (M) not the soluble protein fraction (S) and has a molecular mass of 43 kDa (B) Anti-PfoTPT (red) colocalizes with apicoplast-targeted GFP (green). PfoTPT is in the membrane fraction (M) not the soluble protein fraction (S) and has a molecular mass of 34 kDa. (C) Anti-hemagglutinin (green) colocalizes with apicoplast-located protein PfACP (red) in parasites transfected with a C-terminal tagged copy of PfiTPT (iTPT-HA). PfiTPT-HA is in the membrane fraction (M) not the soluble protein fraction (S) and has a molecular mass of 49 kDa. (D) Anti-hemagglutinin (green) colocalizes with apicoplast-located protein PfACP (red) in parasites transfected with a C-terminal tagged copy of PfoTPT (oTPT-HA). PfoTPT-HA is in the membrane fraction (M) not the soluble protein fraction (S) and has a molecular mass of 38 kDa. (E) Anti-hemagglutinin (green) colocalizes with apicoplast-located protein PfACP (red) in parasites transfected with an N-terminal tagged copy of PfoTPT (HA-oTPT). HA-PfoTPT is in the membrane fraction (M) not the soluble protein fraction (S) and has a molecular mass of 40 kDa. (F) Fusion protein comprising the N terminus of PfiTPT and yellow fluorescent protein (green) colocalizes with apicoplast-located protein PfACP (red) in parasites transfected PfiTPT(l)-YFP. Mature YFP (27 kDa) and a precursor bearing the N-terminal transit peptide (39 kDa) are detected by Western blotting.

Fig. 3.

Pulse–chase analysis of PfiTPT and PfoTPT processing. (A) A 57-kDa PfiTPT precursor (arrowhead) is depleted during a 2-h chase, whereas a 43-kDa mature form of PfiTPT (arrow) increases in abundance. The 43-kDa immunoprecipitated protein was N-terminally sequenced and lacks the first 140 aa encoded by the PfiTPT gene. (B) The 34-kDa PfoTPT protein does not undergo any size change during a 2-h chase, suggesting no processing.

Fig. 4.

Immunolocalization of PfoTPT to the outside of free, intact apicoplasts. (A) Z-stack of immunofluorescent images (–8) showing localization of anti-PfoTPT (red) around apicoplast-targeted GPF (green) in parasites expressing PfACP(leader)-GFP (18). (B) Electron micrograph of intact apicoplast labeled with anti-PfoTPT antibodies and 20-nm colloidal gold. (C) Tannic acid fixation showing four bounding membranes around isolated apicoplast labeled with anti-PfoTPT antibodies and 10-nm colloidal gold. (Scale bars, 200 nm.)

Fig. 5.

Proteolysis of free, intact apicoplasts demonstrating exposure of the N and C-termini of PfoTPT on the outside of apicoplasts. (A) In free, intact apicoplasts from wild-type parasites (3D7), thermolysin degrades PfoTPT but not the innermost membrane protein PfiTPT or the apicoplast stromal protein ACP. (B) Thermolysin digestion of free, intact apicoplasts from parasites transfected with a C-terminally tagged copy of PfiTPT (iTPT-HA) shows that PfiTPT is protected whereas PfoTPT is exposed. (C) Thermolysin digestion of free, intact apicoplasts from parasites transfected with a C-terminally tagged copy of PfoTPT (oTPT-HA) shows that the innermost membrane protein PfiTPT and the apicoplast stromal protein ACP are protected, whereas oTPT-HA is exposed on the apicoplast surface. (D) Thermolysin digestion of free, intact apicoplasts from parasites transfected with an N-terminally tagged copy of PfoTPT (HA-oTPT) shows that the innermost membrane protein PfiTPT and the apicoplast stromal protein ACP are protected whereas HA-oTPT is exposed on the apicoplast surface.

Fig. 6.

Model for the evolution of plastid transporters. Both importers and exporters are targeted to the innermost membrane of photosynthetic primary plastids (such as those of plants) courtesy of removable transit peptides (TP) (31). During establishment of a secondary plastid, such as that of the malaria parasite, the transporter genes (complete with transit peptide) are relocated from the endosymbiont nucleus to the host nucleus and must acquire a signal peptide (SP) in order for the protein to traffic through the outermost membrane of the secondary plastid and into its innermost membrane (e.g., PfiTPT) (39). However, loss of the transit peptide and no acquisition of an N-terminal signal peptide for a copy of iTPT (exp) results in targeting into the endomembrane (ER) courtesy of an internal signal peptide that also does duty as the first transmembrane domain. ER-derived vesicles then fuse with the outermost apicoplast membrane delivering oTPT to the outermost secondary plastid membrane. Because PfoTPT now enters the membrane in reverse orientation, it has been converted from an exporter to an importer.