Amphipathic helices repurpose a Patched/NPC1-like cholesterol transporter to function at Plasmodium –erythrocyte membrane contact sites

The interface between the malaria parasite Plasmodium spp. and its host erythrocyte critically mediates nutrient uptake, lipid exchange, and protein export required for parasite replication and thus pathogenesis. Approximately half of this interface consists of narrow membrane contact sites, a region defined by only 3 to 4 nm of aqueous space between the parasite plasma membrane and the parasitophorous vacuole membrane. Despite its extent, the region remains largely uncharacterized and inaccessible, not least due to its narrowness. The sole identified resident is the Plasmodium falciparum Niemann-Pick Type C1-Related protein, PfNCR1, an essential Patched/NPC1-like cholesterol transporter, suggesting this conserved transporter family has evolved an unconventional adaptation for localizing to membrane contact sites that can reveal a mechanism to access the region. Here, using live-cell microscopy and assessment of cholesterol homeostasis, we identify a helix–linker–helix (HLH) domain as the necessary modification for efficient PfNCR1 localization. We find that correct localization is required for the parasite to maintain low cholesterol levels, connecting targeting to function. Using a glycosylphosphatidylinositol-anchored fluorescent reporter as a minimal membrane-associated protein, we show that the HLH domain is sufficient to drive localization to the narrow membrane contact site region. Mechanistically, we demonstrate that targeting depends on the physicochemical character of the targeting domain rather than a specific primary sequence, identifying amphipathicity as a determining property of the HLH domain. Taken together, we propose physicochemical partitioning as a fundamental principle for protein targeting at the Plasmodium –erythrocyte interface, and for driving direct functional interactions of a Patched/NPC1-like protein with a second membrane.