{"doi":"10.31274/td-20240329-397","title":"Identification of novel factors important for the organization and function of the Plasmodium falciparum parasitophorous vacuole","abstract":"Malaria is a global disease that impact hundreds of millions of people a year with deadly consequences. There is hope for global initiatives to combat and eradicate this disease, but a deeper understanding of the causative parasite, Plasmodium falciparum, is required in order to develop the needed therapeutics. This apicomplexan parasite develops in a complex, dual-host lifecycle involving transition between humans and an invertebrate mosquito vector. Human pathological symptoms arise exclusively from the asexual blood stage in which the parasite invades and grows within host red blood cells, a process that requires substantial host cell remodeling. During intraerythrocytic development, the parasite is enclosed by a parasitophorous vacuole membrane generated during invasion. Survival within this vacuolar barrier necessitates several transport and trafficking processes, including nutrient uptake, waste efflux, effector protein translocation, and endocytosis of host cytosol. Effector protein export out of the vacuole is mediated by the Plasmodium Translocon of EXported Proteins (PTEX) which consists of the AAA+ chaperone HSP101, the adaptor protein PTEX150, and a membrane pore formed by EXP2. The EXP2 pore also serves a second role as a vacuolar nutrient channel, but little is known about this unique dual functionality is controlled. Here, we performed proximity labeling using multiple PTEX components as bait to identify candidate protein co-factors for the EXP2 nutrient channel. Top hits enriched by EXP2 proximity labeling included the vacuole membrane protein EXP1. We characterize EXP1 with a TetR-DOZI-aptamers knockdown system, revealing it performs an essential role in intraerythrocytic development and that a previously hypothesized glutathione S-transferase activity cannot account for this essential function. Notably, depletion in EXP1 caused major changes to vacuole ultrastructure and the distribution of EXP2 but does not impact protein export, consistent with a specific role in supporting the nutrient channel. To further characterize EXP2 nutrient channel function, we attempted to develop a nutrient import sensor based on the promiscuous biotin ligase TurboID targeted to the vacuole lumen. We quantified biotinylation as a metric for biotin import through the nutrient channel following dimerizable Cre recombinase (diCre)-mediated conditional knockout of EXP2, HSP101, or PTEX150. Finally, we adapted a split-TurboID system to detect protein compartmentalization and topology in the complex membrane systems of the parasite-infected erythrocyte. We demonstrate the functionality of this system by generating an N_TurboID fragment fusion to the endogenous C-terminus of either EXP1 or EXP2 in parasites expressing the C_TurboID fragment trafficked to multiple parasite compartments. Notably, expression of an exported C_TurboID fragment in EXP1-N_TurboID parasites enables proximity labeling experiments at the EXP1 C-terminus exposed on the vacuolar surface for the first time. The establishment of these tools provides novel strategies for proximity labeling of parasite compartments and new approaches for studying the trafficking and topology of parasite proteins. Taken together, our work provides new insights into the biology of the parasite vacuole and the interactions of the EXP2 nutrient channel which may help in the fight against this deadly parasite.","journal":null,"year":2022,"id":314512,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9532,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":344708,"name":"Timothy Nessel","orcid":"0000-0002-8827-3905","position":0,"is_corresponding":true}],"reference_count":177,"raw_metadata":null,"created_at":"2026-07-19T00:33:52.047924Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}