Malaria parasite translocon structure and mechanism of effector export

Abstract

The putative Plasmodium translocon of exported proteins (PTEX) is essential for transport of malarial effector proteins across a parasite-encasing vacuolar membrane into host erythrocytes, but the mechanism of this process remains unknown. Here we show that PTEX is a bona fide translocon by determining structures of the PTEX core complex at near-atomic resolution using cryo-electron microscopy. We isolated the endogenous PTEX core complex containing EXP2, PTEX150 and HSP101 from Plasmodium falciparum in the 'engaged' and 'resetting' states of endogenous cargo translocation using epitope tags inserted using the CRISPR-Cas9 system. In the structures, EXP2 and PTEX150 interdigitate to form a static, funnel-shaped pseudo-seven-fold-symmetric protein-conducting channel spanning the vacuolar membrane. The spiral-shaped AAA+ HSP101 hexamer is tethered above this funnel, and undergoes pronounced compaction that allows three of six tyrosine-bearing pore loops lining the HSP101 channel to dissociate from the cargo, resetting the translocon for the next threading cycle. Our work reveals the mechanism of P. falciparum effector export, and will inform structure-based design of drugs targeting this unique translocon.

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

“…To query EXP1 function in PV biology, we employed a dual aptamer TDA strategy using a linear plasmid system to replace the endogenous exp1 coding sequence in an NF54 attB parasite line bearing a 3xFLAG tag on the endogenous hsp101 gene (Garten et al, ; Ho et al, ). This was accomplished by LbCpf1 editing to introduce an aptamer just upstream of the start codon and a 10X aptamer array just downstream of the stop codon (Figure 2a and Figure S3A,B).…”

“…Similar loop‐like structures containing EXP2 but not other PTEX components have been observed to form in parasites expressing conditionally unfoldable exported cargo, suggesting they may represent a generalised response to perturbations in the PV (Charnaud, Jonsdottir, et al, ). EXP2 forms a dual functional pore in the PVM that is required for small molecule transport and effector protein translocation (Garten et al, ; Ho et al, ). Remarkably, the dramatic alteration in EXP2 distribution following EXP1 knockdown did not reduce protein export.…”

Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism

“…In this case, each of the six subunits has a loop analogous to the two-helix finger of SecA, which pushes the polypeptide chain through the central pore (Hinnerwisch et al, 2005;Martin et al, 2008;Han et al, 2017;Puchades et al, 2017;Ho et al, 2018). In this case, each of the six subunits has a loop analogous to the two-helix finger of SecA, which pushes the polypeptide chain through the central pore (Hinnerwisch et al, 2005;Martin et al, 2008;Han et al, 2017;Puchades et al, 2017;Ho et al, 2018).…”

“…In this case, each of the six subunits has a loop analogous to the two-helix finger of SecA, which pushes the polypeptide chain through the central pore (Hinnerwisch et al, 2005;Martin et al, 2008;Han et al, 2017;Puchades et al, 2017;Ho et al, 2018). In fact, recent structures of the Plasmodium translocon of exported proteins (PTEX) suggest an analogous model for the Hsp101 ATPase (Ho et al, 2018). In fact, recent structures of the Plasmodium translocon of exported proteins (PTEX) suggest an analogous model for the Hsp101 ATPase (Ho et al, 2018).…”

Illuminating how malaria parasites export proteins into host erythrocytes

“…Irrespective of how the diverse types of cargo cross the PPM, the trafficking pathways converge at the PVM (Beck, Muralidharan, Oksman, & Goldberg, ; Elsworth, Crabb, & Gilson, ; Mesen‐Ramirez et al, ). Crossing the PVM requires proteins to be in an unfolded state (Gehde et al, ), an energy force (Ansorge, Benting, Bhakdi, & Lingelbach, ), and a portal through the PVM, the latter of which is created by the assembly of an ~1.6 mega dalton proteinaceous complex termed PTEX (de Koning‐Ward et al, ; Bullen et al, ; Ho et al, ).…”