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The difficulty of faithfully recapitulating malarial protein complexes in heterologous expression systems has long impeded structural study for much of the Plasmodium falciparum proteome. However, recent advances in single-particle cryo electron microscopy (cryoEM) now enable structure determination at atomic resolution with significantly reduced requirements for both sample quantity and purity. Combined with recent developments in gene editing, these advances open the door to structure determination and structural proteomics of macromolecular complexes enriched directly from P. falciparum parasites. Furthermore, the combination of cryoEM with the rapidly emerging use of in situ cryo electron tomography (cryoET) to directly visualize ultrastructures and protein complexes in the native cellular context will yield exciting new insights into the molecular machinery underpinning malaria parasite biology and pathogenesis. With half the world's population currently at risk, malaria remains a significant global health burden. The difficulty of expressing many malarial protein complexes in heterologous systems has precluded structural and biochemical studies, impeding efforts to elucidate the functions and molecular mechanisms of many important but poorly understood biological pathways, including potential therapeutic targets. Recent and ongoing advances in structure determination of macromolecular complexes using cryo electron microscopy (cryoEM) provide new avenues for structural study of the P. falciparum proteome, much of which previously resisted structure determination. CryoEM of endogenously derived macromolecular complexes, enabled by the significantly reduced sample requirements of cryoEM studies, will lead to the discovery of so far unknown native substrates, binding partners, and modifications. [ABSTRACT FROM AUTHOR]

The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that theymight play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC. [ABSTRACT FROM AUTHOR]