A newly discovered protein export machine in malaria parasites

Abstract

Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is central to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here we identify in Plasmodium falciparum a translocon of exported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered, and comprises heat shock protein 101 (HSP101; a ClpA/B-like ATPase from the AAA+ superfamily, of a type commonly associated with protein translocons), a novel protein termed PTEX150 and a known parasite protein, exported protein 2 (EXP2). EXP2 is the potential channel, as it is the membrane-associated component of the core PTEX complex. Two other proteins, a new protein PTEX88 and thioredoxin 2 (TRX2), were also identified as PTEX components. As a common portal for numerous crucial processes, this translocon offers a new avenue for therapeutic intervention.

Fig. 1. HSP101 and PTEX150 co-localise and have dual apical merozoite and PVM localisation

Western blot analysis of parasite proteins extracted from: a, parental 3D7 and transgenic 3D7-150HA parasites; b, 3D7 and transgenic 3D7- 101HA parasites; c, 3D7-150HA parasites harvested at mixed schizont/ring (S/R), ring (R), early trophozoite (ET), late trophozoite (LT) and early schizont (ES) stages or from mature magnet purified schizont (S) stages, using the indicated antibodies. AMA1 represents a marker for a protein expressed only at the late-stages of parasite development. Molecular weight standards (kDa) are shown on the left; d, Double labelling IFA on fixed 3D7-150HA and 3D7-101HA ring- and merozoite-stage parasites using the antibodies as indicated. The arrowheads indicate the apical end of the merozoite.

Fig. 2. Isolation of a five-member PTEX complex

Coomassie-stained SDS-PAGE gels of material eluted from immune-precipitations performed using HA antibodies on parasite lysates from: a, 3D7 and 3D7-150HA or b, 3D7 and 3D7-101HA. The total number of peptides present in all excised bands for those parasite-specific proteins that were the top peptide hit in the visible bands unique to pull-downs using transgenic parasites (labelled 1-5 t are indicated, as is the peptide abundance of Pfcdc48 and all other parasite and host cell proteins identified; c, Immunoblot analysis of immune-precipitations performed on 3D7 parental and transgenic HA-tagged parasite lysates using antibody combinations for immuneprecipitation (IP Ab) and Western blot (WB Ab) as indicated; d, Double labelling IFA on fixed 3D7-150HA ring-stage parasites using a mixture of antibodies specific for PTEX150 and EXP2; e, Schematic of the five members of the PTEX complex showing that each possesses a ER signal sequence and other distinguishing features such as regions that are highly conserved across the genus.

Fig. 3. The PTEX complex interacts with PEXEL proteins

a, Upper panel - GFP fluorescence of non-exported (K-GFP) versus exported (K+GFP) reporters in live transgenic parasites. The arrows highlight the differences in fluorescence at the parasitophorous vacuole, with the exported protein displaying a `necklace of beads' appearance. Lower panel - Western-blot analysis of immune-precipitations with HSP101 antibodies using transgenic parasites, which express various GFP reporter proteins (See Fig. S5). Despite equivalent levels of interaction of PTEX150 with HSP101 in all transgenic parasite lysates (bottom panels), greater amounts of the exported K+GFP protein (asterisks) is pulled down in comparison to any of the non-exported reporters proteins. b, Immune-precipitations in the K+GFP transgenic line using antibodies against 3 exported proteins (shown in red) probed in Western blot with antibodies against PTEX components. No antibody, pre-immune and irrelevant (anti-AMA1) IgG were included as negative controls.

Fig. 4. Model for PTEX function

a, Differential solubilisation of schizont-stage parasites to analyse membrane association. Parasites were first solubilized in PBS into supernatant (S) and pellet (P) fractions and analysed by Western blotting. The PBS insoluble pellet was further solubilized in Na₂CO₃ and similarly analysed. The P83 fragment of MSP1 is included as a control for solubilisation of a peripherally-associated membrane protein; b, We propose the following model for PEXEL-protein export. Once deposited into the vaculoar space, proteins destined for export are recognised by some member(s) of the PTEX complex and deposited into the N-terminal domain of HSP101 where they are unfolded. These proteins are fed through the central channel of HSP101 and ultimately through a membrane-associated channel, predicted here to be EXP2. On the cytosolic face, host (eg, human HSP70) and/or exported parasite proteins may assist in energising translocation and/or in re-folding proteins.