Plasmodium falciparum Niemann-Pick Type C1-Related protein relies on its physicochemical properties for membrane contact site localization required for cholesterol homeostasis.

The Niemann-Pick Type C1-Related protein of the malaria parasite Plasmodium falciparum, PfNCR1, is a promising anti-malarial drug target facilitating cholesterol homeostasis at the interface of the malaria parasite with its host-red blood cell. PfNCR1 is localized to otherwise functionally uncharacterized regions covering ~half of the host-parasite interface. These regions are defined by exceptionally narrow membrane contact sites, leaving only ~3-4 nm vertical aqueous space in between the membranes. Determining the origin and functional consequence of localization to the closely apposed membrane is central for our understanding of PfNCR1 as drug target but also offers a window into the mechanism of the group of homologous proteins, associated with congenital conditions and cancer. Here we define the mechanism of PfNCR1's membrane contact site localization and its implication for cholesterol transport. We identified a 141 amino acid long (amphipathic) helix - linker - (amphipathic) helix domain ("HLH domain") unique to Plasmodium spp., that is necessary for efficient localization of PfNCR1 to the narrow membrane contact sites. Mechanistically, we show that this localization relies on the HLH domain's physicochemical properties. GPI-anchoring the isolated HLH domain or a version of the HLH domain in which the helices are replaced by the amphipathic helix of human ATG3 are sufficient to target a fluorescent protein to regions of endogenous PfNCR1. Functionally, we demonstrate that the degree of PfNCR1 localization to narrow contact sites qualitatively correlates with its ability to maintain cholesterol homeostasis, linking PfNCR1's membrane contact to its recognized transport function. Collectively, the results establish the HLH domain as key element for PfNCR1's localization and effectiveness in cholesterol transport while also opening avenues to probe the narrow membrane contact site regions with engineered proteins.