BPS2026 – Helix-loop-helix domain facilitates protein targeting to membrane contact sites at the malaria parasite–host red blood cell interface

The parasitophorous vacuole (PV) separates the malaria parasite from its host-red blood cell cytoplasm, forming a protein and nutrient trafficking hub at the host-parasite interface and providing a controlled microenvironment for Plasmodium to thrive. The two membranes defining the PV, the Parasitophorous vacuole membrane and parasite plasma membrane, form μm-sized membrane contact site-regions, leaving only ∼3-4 nm aqueous space in between the membranes. The Plasmodium falciparum Nieman-Pick C1 related protein (NCR1) localizes to those contact site-regions to facilitate cholesterol homeostasis of the parasite yet other possibly essential functions of the regions are unknown. To gain insight into the contact site-regions we aimed at understanding possible mechanisms of protein targeting to the regions. Noticing an (amphipathic) helix- loop- (amphipathic) helix (HLH) domain NCR1 unique to malaria parasites, we investigated its importance for NCR1’s malaria parasite specific localization. Colocalizing the endogenous protein with that of a modified second copy, we demonstrate the whole HLH domain is necessary for efficient NCR1 targeting and loss of targeting correlates with loss of function. We then go on to demonstrate that the HLH domain itself is sufficient for contact site targeting when anchored to the parasite plasma membrane, mimicking the topology of NCR1. Further, swapping the amphipathic helices for unrelated motifs with matching physical properties but different amino acid sequence indicates that NCR1 contact site targeting exploits direct membrane binding via the amphipathic helices rather than a specific protein-protein interaction. With a contact site targeting mechanism unlocked, we hope to uncover the proteome of the contact site-regions, characterize the set of functions requiring close membrane contact for malaria parasite proliferation, and devise anti-malarial strategies disrupting membrane contact site functions.