PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins.

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric Plasmodium export element (PEXEL)/host-targeting motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here, we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood stage. While the N termini of exported proteins containing the PEXEL and immediately downstream ~10 residues are sufficient to mediate translocation into the RBC, the equivalent UIS2 N terminus does not promote the export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position, which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RIL↓DE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N terminus mediates export, not PEXEL processing per se. Prompted by this observation, we further show that PEXEL sequences in the N termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export.IMPORTANCEHost erythrocyte remodeling by malaria parasite-exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum, most exported proteins undergo proteolytic maturation via recognition of the pentameric Plasmodium export element (PEXEL)/host-targeting motif by the aspartic protease Plasmepsin V, which exposes a mature N terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate that PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing, reinforcing that features of the mature N terminus, and not PEXEL cleavage, identify cargo for export. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

Nuclear pore complexes undergo Nup221 exchange during blood stage asexual replication of Plasmodium parasites.

Plasmodium parasites, which are the causative agents of malaria, undergo closed mitosis without breakdown of the nuclear envelope. Unlike the closed mitosis in yeast, P. berghei parasites undergo multiple rounds of asynchronous nuclear divisions in a shared cytoplasm result in a multinucleated (8-24) organism prior to formation of daughter cells within an infected red blood cell. During this replication process, intact nuclear pore complexes (NPCs) and their component nucleoporins are likely to play critical roles in parasite growth, facilitating selective bi-directional nucleocytoplasmic transport and genome organization. Here we utilize ultrastructure expansion microscopy (U-ExM) to investigate P. berghei Nup138, Nup221, and Nup313 at the single nucleus level throughout the 24 hour blood-stage replication cycle. Our findings reveal that these Nups are evenly distributed around the nuclei and organized in a rosette structure previously undescribed around the centriolar plaque, which is responsible for intranuclear microtubule nucleation during mitosis. We also detect an increased number of NPCs compared with previously reported, highlighting the power of U-ExM. By adapting the recombination-induced tag exchange (RITE) system to P. berghei, we provide evidence of NPC maintenance, demonstrating Nup221 turnover during parasite asexual replication. Our data shed light on the distribution of NPCs and their homeostasis during the blood-stage replication of P. berghei parasites. Further studies into the nuclear surface of these parasites will allow for a better understanding of parasites nuclear mechanics and organization.

Knock-sideways by inducible ER retrieval enables a unique approach for studying Plasmodium-secreted proteins.

Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting Plasmodium-secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple Plasmodium species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.

PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins.

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric P lasmodium ex port el ement (PEXEL)/Host Targeting Motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N-terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood-stage. While the N-termini of exported proteins containing the PEXEL and immediately downstream ∼10 residues is sufficient to mediate translocation into the RBC, the equivalent UIS2 N-terminus does not promote export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RILτDE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N-terminus mediates export, not PEXEL processing per se . Prompted by this observation, we further show that PEXEL sequences in the N-termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export. Importance: Host erythrocyte remodeling by malaria parasite exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum , most exported proteins undergo proteolytic maturation via recognition of the pentameric P lasmodium ex port el ement (PEXEL)/Host Targeting motif by the aspartic protease Plasmepsin V (PMV) which exposes a mature N-terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing by PMV, reinforcing that features of the mature N-terminus, and not PEXEL cleavage, identify cargo for export cargo. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

The PTEX Pore Component EXP2 Is Important for Intrahepatic Development during the Plasmodium Liver Stage.

During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3' untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface. IMPORTANCE After the mosquito bite that initiates a Plasmodium infection, parasites first travel to the liver and develop in hepatocytes. This liver stage is asymptomatic but necessary for the parasite to transition to the merozoite form, which infects red blood cells and causes malaria. To take over their host cells, avoid immune defenses, and fuel their growth, these obligately intracellular parasites must import nutrients and export effector proteins across a vacuole membrane in which they reside. In the blood stage, these processes depend on a translocon called PTEX, but it is unclear if PTEX also functions during the liver stage. Here, we adapted the glmS ribozyme to control expression of EXP2, the membrane pore component of PTEX, during the liver stage of the rodent malaria parasite Plasmodium berghei. Our results show that EXP2 is important for intracellular development in the hepatocyte, revealing that PTEX components are also functionally important during liver-stage infection.

Promising antimalarials targeting apicoplast DNA polymerase from Plasmodium falciparum.

Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it replicates and repairs the genome. apPOL has no direct orthologs in mammals and is considered a promising drug target for the treatment and/or prevention of malaria. We previously reported screening the Malaria Box to identify MMV666123 as an inhibitor of apPOL. Herein we extend our studies and report structure-activity relationships for MMV666123 and identify key structural motifs necessary for inhibition. Although attempts to crystallize apPOL with the inhibitor were not fruitful, kinetic analysis and crystal structure determinations of WT and mutant apo-enzymes, facilitated model building and provided insights into the putative inhibitor binding site. Our results validate apPOL as an antimalarial target and provide an avenue for the design of high potency, specific inhibitors of apPOL and other A-family DNA polymerases.

TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei.

Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups - Nup176, 269, 335, 390, and 434 - are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position. IMPORTANCE The nuclear pore complex (NPC) is a platform for constant evolution and has been used to study the evolutionary patterns of early-branching eukaryotes. The Plasmodium NPC is poorly defined due to its evolutionary divergent nature making it impossible to characterize it via homology searches. Although 2 decades have passed since the publication of the Plasmodium genome, 30% of the genes still lack functional annotation. Our study demonstrates the ability of proximity labeling using TurboID to assign function to orphan proteins in the malaria parasite. We have identified a total of 10 Nups that will allow further study of NPC dynamics, structural elements, involvement in nucleocytoplasmic transport, and unique non-transport functions of nucleoporins that provide adaptability to this malaria parasite.

Discovery of small molecule inhibitors of Plasmodium falciparum apicoplast DNA polymerase.

Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs.

Dramatic Consequences of Reducing Erythrocyte Membrane Cholesterol on Plasmodium falciparum.

Cholesterol is the most abundant lipid in the erythrocyte. During its blood-stage development, the malaria parasite establishes an active cholesterol gradient across the various membrane systems within the infected erythrocyte. Interestingly, some antimalarial compounds have recently been shown to disrupt cholesterol homeostasis in the intraerythrocytic stages of Plasmodium falciparum. These studies point to the importance of cholesterol for parasite growth. Previously, reduction of cholesterol from the erythrocyte membrane by treatment with methyl-ß-cyclodextrin (MßCD) was shown to inhibit parasite invasion and growth. In addition, MßCD treatment of trophozoite-stage P. falciparum was shown to result in parasite expulsion from the host cell. We have revisited these phenomena by using live video microscopy, ultrastructural analysis, and response to antimalarial compounds. By using time-lapse video microscopy of fluorescently tagged parasites, we show that MßCD treatment for just 30 min causes dramatic expulsion of the trophozoite-stage parasites. This forceful expulsion occurs within 10 s. Remarkably, the plasma membrane of the host cell from which the parasite has been expelled does not appear to be compromised. The parasitophorous vacuolar membrane (PVM) continued to surround the extruded parasite, but the PVM appeared damaged. Treatment with antimalarial compounds targeting PfATP4 or PfNCR1 prevented MßCD-mediated extrusion of the parasites, pointing to a potential role of cholesterol dynamics underlying the expulsion phenomena. We also confirmed the essential role of erythrocyte plasma membrane cholesterol for invasion and growth of P. falciparum. This defect can be partially complemented by cholesterol and desmosterol but not with epicholesterol, revealing stereospecificity underlying cholesterol function. Overall, our studies advance previous observations and reveal unusual cell biological features underlying cholesterol depletion of the infected erythrocyte plasma membrane. IMPORTANCE Malaria remains a major challenge in much of the world. Symptoms of malaria are caused by the growth of parasites belonging to Plasmodium spp. inside the red blood cells (RBCs), leading to their destruction. The parasite depends upon its host for much of its nutritional needs. Cholesterol is a major lipid in the RBC plasma membrane, which is the only source of this lipid for malaria parasites. We have previously shown that certain new antimalarial compounds disrupt cholesterol homeostasis in P. falciparum. Here, we use live time-lapse video microscopy to show dramatic expulsion of the parasite from the host RBC when the cholesterol content of the RBC is reduced. Remarkably, this expulsion is inhibited by the antimalarials that disrupt lipid homeostasis. We also show stereospecificity of cholesterol in supporting parasite growth inside RBC. Overall, these results point to a critical role of cholesterol in the physiology of malaria parasites.

Structured to conquer: transport across the Plasmodium parasitophorous vacuole.

During the vertebrate stage of the Plasmodium life cycle, obligate intracellular malaria parasites establish a vacuolar niche for replication, first within host hepatocytes at the pre-patent liver-stage and subsequently in erythrocytes during the pathogenic blood-stage. Survival in this protective microenvironment requires diverse transport mechanisms that enable the parasite to transcend the vacuolar barrier. Effector proteins exported out of the vacuole modify the erythrocyte membrane, increasing access to serum nutrients which then cross the vacuole membrane through a nutrient-permeable channel, supporting rapid parasite growth. This review highlights the most recent insights into the organization of the parasite vacuole to facilitate the solute, lipid and effector protein trafficking that establishes a nutrition pipeline in the terminally differentiated, organelle-free red blood cell.

Native structure of the RhopH complex, a key determinant of malaria parasite nutrient acquisition.

The RhopH complex is implicated in malaria parasites' ability to invade and create new permeability pathways in host erythrocytes, but its mechanisms remain poorly understood. Here, we enrich the endogenous RhopH complex in a native soluble form, comprising RhopH2, CLAG3.1, and RhopH3, directly from parasite cell lysates and determine its atomic structure using cryo-electron microscopy (cryo-EM), mass spectrometry, and the cryoID program. CLAG3.1 is positioned between RhopH2 and RhopH3, which both share substantial binding interfaces with CLAG3.1 but make minimal contacts with each other. The forces stabilizing individual subunits include 13 intramolecular disulfide bonds. Notably, CLAG3.1 residues 1210 to 1223, previously predicted to constitute a transmembrane helix, are embedded within a helical bundle formed by residues 979 to 1289 near the C terminus of CLAG3.1. Buried in the core of the RhopH complex and largely shielded from solvent, insertion of this putative transmembrane helix into the erythrocyte membrane would likely require a large conformational rearrangement. Given the unusually high disulfide content of the complex, it is possible that such a rearrangement could be initiated by the breakage of allosteric disulfide bonds, potentially triggered by interactions at the erythrocyte membrane. This first direct observation of an exported Plasmodium falciparum transmembrane protein-in a soluble, trafficking state and with atomic details of buried putative membrane-insertion helices-offers insights into the assembly and trafficking of RhopH and other parasite-derived complexes to the erythrocyte membrane. Our study demonstrates the potential the endogenous structural proteomics approach holds for elucidating the molecular mechanisms of hard-to-isolate complexes in their native, functional forms.

Transport mechanisms at the malaria parasite-host cell interface.

Obligate intracellular malaria parasites reside within a vacuolar compartment generated during invasion which is the principal interface between pathogen and host. To subvert their host cell and support their metabolism, these parasites coordinate a range of transport activities at this membrane interface that are critically important to parasite survival and virulence, including nutrient import, waste efflux, effector protein export, and uptake of host cell cytosol. Here, we review our current understanding of the transport mechanisms acting at the malaria parasite vacuole during the blood and liver-stages of development with a particular focus on recent advances in our understanding of effector protein translocation into the host cell by the Plasmodium Translocon of EXported proteins (PTEX) and small molecule transport by the PTEX membrane-spanning pore EXP2. Comparison to Toxoplasma gondii and other related apicomplexans is provided to highlight how similar and divergent mechanisms are employed to fulfill analogous transport activities.

Contacting domains segregate a lipid transporter from a solute transporter in the malarial host-parasite interface.

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.

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

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane. Although the PVM is the site of several transport activities essential to parasite survival, the basis for organisation of this membrane system is unknown. Here, we performed proximity labeling at the PVM with BioID2, which highlighted a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. Importantly, although EXP1 was required for intraerythrocytic development, a previously reported in vitro glutathione S-transferase activity could not account for this essential EXP1 function in vivo. EXP1 knockdown was accompanied by profound changes in vacuole ultrastructure, including apparent increased separation of the PVM from the parasite plasma membrane and formation of abnormal membrane structures. Furthermore, although activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 but not the HSP101 unfoldase was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper organisation of EXP2 within the PVM.

The parasitophorous vacuole of the blood-stage malaria parasite.

The pathology of malaria is caused by infection of red blood cells with unicellular Plasmodium parasites. During blood-stage development, the parasite replicates within a membrane-bound parasitophorous vacuole. A central nexus for host-parasite interactions, this unique parasite shelter functions in nutrient acquisition, subcompartmentalization and the export of virulence factors, making its functional molecules attractive targets for the development of novel intervention strategies to combat the devastating impact of malaria. In this Review, we explore the origin, development, molecular composition and functions of the parasitophorous vacuole of Plasmodium blood stages. We also discuss the relevance of the malaria parasite's intravacuolar lifestyle for successful erythrocyte infection and provide perspectives for future research directions in parasitophorous vacuole biology.

Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu.

X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis.

Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX.

Intraerythrocytic malaria parasites reside within a parasitophorous vacuolar membrane (PVM) generated during host cell invasion1. Erythrocyte remodelling and parasite metabolism require the export of effector proteins and transport of small molecules across this barrier between the parasite surface and host cell cytosol2,3. Protein export across the PVM is accomplished by the Plasmodium translocon of exported proteins (PTEX) consisting of three core proteins, the AAA+ ATPase HSP101 and two additional proteins known as PTEX150 and EXP24. Inactivation of HSP101 and PTEX150 arrests protein export across the PVM5,6, but the contribution of EXP2 to parasite biology is not well understood7. A nutrient permeable channel in the PVM has also been characterized electrophysiologically, but its molecular identity is unknown8,9. Here, using regulated gene expression, mutagenesis and cell-attached patch-clamp measurements, we show that EXP2, the putative membrane-spanning channel of PTEX4,10-14, serves dual roles as a protein-conducting channel in the context of PTEX and as a channel able to facilitate nutrient passage across the PVM independent of HSP101. Our data suggest a dual functionality for a channel operating in its endogenous context.

Malaria parasite translocon structure and mechanism of effector export.

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.

Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes.

Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting membranes. Parasite Ca2+ , kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. Here, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live-cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite [Ca2+ ]i , the PV began rounding. Then after ~1.5 min, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next ~6 min, there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress programme when newly formed parasites mobilised and erythrocyte membranes permeabilised and then ruptured-a dramatic finale to the parasite cycle of replication.