3.A.26 The Plasmodium Translocon of Exported Proteins (PTEX) Family
Protein export is central for the survival and virulence of intracellular Plasmodium falciparum blood stage parasites. To reach the host cell, exported proteins cross the parasite plasma membrane (PPM) and the parasite-enclosing parasitophorous vacuole membrane (PVM), a process that requires unfolding, suggestive of protein translocation. Components of a proposed translocon at the PVM termed PTEX are essential in this phase of export, but questions have been raised about its proposed membrane pore component EXP2 for which little functional data are available in P. falciparum. It is also unclear how PTEX mediates trafficking of both soluble and transmembrane proteins. Taking advantage of conditionally foldable domains, Mesén-Ramírez et al. 2016 dissected the translocation events in the parasite periphery, showing that two successive translocation steps are needed for the export of transmembrane proteins, one at the PPM and one at the PVM. Depending on the length of the C-terminus of the exported substrate, these steps occur by transient interaction of the PPM and PVM translocon, similar to the situation for protein transport across mitochondrial membranes. Constructs of exported proteins remained arrested in the process of being translocated across the PVM. This clogged the translocation pore, prevented the export of all types of exported proteins and, as a result, inhibited parasite growth. The substrates stuck in translocation were found in a complex with the proposed PTEX membrane pore component EXP2, suggesting a role of this protein in translocation. These data provided evidence that EXP2 is part of a translocating entity, suggesting that PTEX has translocation activity and provide a mechanistic framework for the transport of soluble as well as transmembrane proteins from the parasite boundary into the host cell (Mesén-Ramírez et al. 2016).
Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. P. falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases, ClpB1 and ClpB2, involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of the parasite-derived protein secretion complex, PTEX. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains - a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. AhYoung et al. 2015 reported and compared the first crystal structures of the N terminal domains of ClpB1 and ClpB2 and analyzed their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 showed that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX complex.
Plasmodium falciparum requires the Plasmodium translocon of exported proteins (PTEX) to proliferate in human red blood cells. During the blood stages of malaria, several hundred parasite-encoded proteins are exported from the parasite into the cytosol of red blood cells. PTEX is the translocon for protein export and comprises 5 proteins: EXP2, PTEX150, PTEX88, Hsp101 and TRX2 (Hakamada et al. 2016). Among them, EXP2 constitutes the transmembrane pore, whereas the other components play roles in substrate protein unfolding or providing a driving force. Hakamada et al. 2016 expressed and characterized the membrane-associated component EXP2 (one (or possibly two) N-terminal α-helical TMSs plus 6 - 8 transmembrane β strands. EXP2 formed pores in bilayer lipid membranes with an inner pore diameter of about 3.5 nm based on electron microscopy images and channel currents. The pore comprises approximately 10-12 EXP2 subunits (Hakamada et al. 2016). The PTEX translocon is believed to be responsible for the membrane insertion of the RhopH complex (TC# 1.A.91.1.1) into the erythrocyte membrane (Schureck et al. 2021).
The Plasmodium translocon exports proteins (PTEX) into human red blood cells. During the blood stages of malaria, several hundred parasite-encoded proteins are exported from the parasite into the cytosol of red blood cells. PTEX is the translocon for protein export and comprises 5 proteins with EXP2 constituting the transmembrane pore, whereas the other components may play roles in unfolding the luggage proteins or providing the driving force. Hakamada et al. 2017 expressed and characterized EXP2. Expression of EXP2 is lethal to E. coli. EXP2 forms pores in bilayer lipid membranes, with the inner diameter estimated to be approximately 3.5 nm based on electron microscopy images and channel currents. From this size and the molecular mass as determined by size exclusion chromatography and blue native polyacrylamide gel electrophoresis, it was determined that the pore comprises approximately 10-12 EXP2 subunit (Hakamada et al. 2017).
Plasmodium exported proteins must cross the parasite plasma membrane (PPM) and the parasitophorous vacuolar membrane (PVM), encasing the parasite, to access the host cell. Crossing the PVM requires protein unfolding and passage through PTEX. Matthews et al. 2019 provided direct evidence that heat shock protein 101 (HSP101) unfolds proteins for translocation across the PVM by creating transgenic Plasmodium parasites in which the unfoldase and translocation functions of HSP101 have become uncoupled. While these parasites can export native proteins, they are unable to translocate soluble, tightly folded reporter proteins bearing the Plasmodium export element (PEXEL) across the PVM into host erythrocytes. In contrast, an identical PEXEL reporter protein harboring a transmembrane domain could be exported, suggesting that a prior unfolding step occurs at the PPM. These results demonstrate that the export of parasite proteins is dependent on how these proteins are presented to the secretory pathway before they reach PTEX as well as their folded status. Accordingly, only tightly folded soluble proteins secreted into the vacuolar space, and not proteins containing transmembrane domains or the majority of erythrocyte-stage exported proteins, have an absolute requirement for the full unfoldase activity of HSP101 to be exported.
The Plasmodium parasites that cause malaria export hundreds of proteins into their host red blood cell (RBC). These exported proteins drastically alter the structural and functional properties of the RBC and play critical roles in parasite virulence and survival. To access the RBC cytoplasm, parasite proteins must pass through the Plasmodium translocon of exported proteins (PTEX) located at the membrane interfacing the parasite and host cell. HSP101 serves to unfold protein cargo requiring translocation.Moreover, addition of a transmembrane domain to soluble cargo influences its ability to be translocated by parasites in which the HSP101 motor and unfolding activities have become uncoupled. Thus, proteins with transmembrane domains use an alternative unfolding pathway prior to PTEX to facilitate export (Matthews et al. 2019).