Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity

Abstract

Protein export is central for the survival and virulence of intracellular P. 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 translocation activity has not been shown for the complex and questions have been raised about its proposed membrane pore component EXP2 for which no functional data is available in P. falciparum. It is also unclear how PTEX mediates trafficking of both, soluble as well as transmembrane proteins. Taking advantage of conditionally foldable domains, we here 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. Our data provide evidence that, 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 the mitochondrial membranes. Remarkably, we obtained constructs of exported proteins that 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 for the first time provide evidence for EXP2 to be 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.

An exported protein-interacting complex involved in the trafficking of virulence determinants in Plasmodium-infected erythrocytes

“…Here, we provide evidence of a close interaction between Pf EMP1 and PTEX, and corroborate suggestions that PEXEL and PEXEL-negative protein trafficking pathways converge in the PV10112241. In addition, our semiquantitative analysis of Pf EMP1B IPs indicated that Pf EMP1B is most strongly associated with HSP101.…”

“…Indeed, the majority of exported proteins pulled down with EPIC have peak expression in early- to mid-stage trophozoites (when IPs were performed) consistent with active transport of these proteins in the PV. This concept of facilitated sorting in the PV is consistent with the inability of position-5 PEXEL mutants to be exported into the host RBC despite successful entry into the PV45464748, and provides an explanation for the convergence of the PEXEL and PEXEL-negative trafficking pathways in the PV10112241.…”

Spatial organization of protein export in malaria parasite blood stages

“…To probe the function of the PTEX complex, conditionally trappable exported cargo molecules have been expressed in parasites and the effects of conditionally trapping and releasing of the cargo has been assessed . Immunoprecipitation (IP) assays revealed that the trapped cargo associates strongly with PTEX component, EXP2 but not HSP101 .…”

“…To probe the function of the PTEX complex, conditionally trappable exported cargo molecules have been expressed in parasites and the effects of conditionally trapping and releasing of the cargo has been assessed . Immunoprecipitation (IP) assays revealed that the trapped cargo associates strongly with PTEX component, EXP2 but not HSP101 . Given that previous studies have shown exported cargo can be co‐precipitated with each of the core PTEX components, we decided to employ a different trappable cargo approach to glean further insights into the process of protein export at the PVM.…”

Plasmodium species: master renovators of their host cells

“…Although protein substrates that were stuck in the process of translocation across the PVM were present in a complex with EXP2 (REF. 65), functional evidence that EXP2 acts as the PTEX pore is still lacking. Interestingly, all apicomplexans that reside in a parasitophorous vacuole encode an orthologue of EXP2…”