Identification of proteins in proximity to VAP and VPS13L1 in P. falciparum blood stages

ABSTRACT: P. falciparum blood stage parasites maintain an intricate system of membranes, requiring precise mechanisms of lipid synthesis and trafficking. Lipid transfer proteins (LTPs) at interorganelle membrane contact sites (MCSs) have emerged as key for ensuring proper lipid distribution in cells but so far remain largely unexplored in protozoans. In many cases this involves the ER which is a central hub for lipid synthesis and contacts all other organelles. In this project, we used the ER adaptor VAP, which recruits LTPs in other organisms, to identify essential mechanisms of lipid transfer at ER-MCSs in P. falciparum blood stages. Using proximity biotinylation techniques, combined with LC-MS/MS, proteins in proximity to P. falciparum VAP (PfVAP) were identified. The raw data for these experiments is here deposited. Using microscopy and cell biology techniques the proteins identified as hits through this experiment were filtered to uncover direct interacting partners of PfVAP, of which one was a bridge-like LTP homologous to opisthokont VPS13, hence renamed PfVPS13L1. This protein folds as a ~22nm rod with an internal hydrophobic groove that allows bulk flow of lipids between two adjacent membranes and localizes to hotspots within the inner membrane complex (IMC), a de novo generated organelle required for proper schizogony. While the ER-resident PfVAP binds the N-terminal end of the PfVPS13L1 bridge, we hypothesized its C-terminal end binds early IMC membranes. To confirm this, we performed another proximity biotinylation experiment to label proteins in proximity to the C-terminal end of PfVPS13L1, and identified several known IMC proteins as hits. The raw data for this experiment is deposited here as well. Further studies of parasites with PfVPS13L1 loss of function revealed a defect in IMC membrane extension and parasite segmentation, which in turn led to the complete inhibition of parasite propagation in RBCs. Our data supports a model in which PfVPS13L1 is critical for the formation of invasive stages by mediating bulk flow of lipids from the ER to the growing IMC. This function is homologous to what was previously reported for other VPS13 or VPS13-like opisthokont proteins, where their functions include the expansion of the isolation membrane during autophagosome formation or the expansion of the sporulation membrane during yeast meiosis. Our study thus reveals that bulk transfer of lipids via bridge-like LTPs is a conserved mechanism for membrane expansion throughout eukaryotes, including protozoa.In the files uploaded, the code P3190 corresponds to the DiQ-BioIDof PfVAP (Fig. 2b), and P3872 and P3754 to DiQ-BioID of C-terminal PfVPS13L1 (Fig. 4g and Extended Data Fig. 7c, respectively).

INSTRUMENT(S):

ORGANISM(S): Plasmodium Falciparum (isolate 3d7)

DISEASE(S): Malaria

SUBMITTER: Frank Stein  

LAB HEAD: Tobias Spielmann

PROVIDER: PXD066159 | Pride | 2025-07-28

REPOSITORIES: Pride