Trafficking of the Plasmodium falciparum RhopH3 rhoptry protein through the Maurer’s clefts in Pf130 associated compartments in infected erythrocytes

Rhoptry proteins of the RhopH complex (RhopH1/Clag, RhopH2 and RhopH3) participate in the formation of new permeability pathways for nutrient uptake in Plasmodium falciparum infected erythrocytes. RhopH proteins associate with the Maurer’s clefts (MC) during transport to the host cell membrane and interact with the cytoskeleton. However, the role of specific MC proteins facilitating rhoptry protein interaction and trafficking is unknown. The major interaction with MCs occurs with RhopH3. Dual roles ascribed to RhopH3 include functions in host cell invasion and formation of the plasmodial surface anion channel (PSAC). The topological orientation and organization of the RhopH3-MC membrane interaction in the host cell cytoplasm has not been described. In this study, we performed biochemical analysis to investigate the topology and trafficking of RhopH3 and Pf130 from the parasite to the erythrocyte cytoplasm using streptolysin O (SLO) permeabilization and protease treatment of infected erythrocytes. We investigated Pf130, a soluble protein previously identified by monoclonal antibodies and shown by immunoelectron microscopy (IEM) to be distributed underneath knobs and associated with the MC in infected erythrocytes. Pf130 is expressed early in the erythrocytic cycle and persists to the schizont stage. Immunofluorescence and confocal microscopy show that Pf130 is a resident MC protein due to its colocalization with the MC protein REX-1. Immunofluorescence assay (IFA) showed colocalization of Pf130 with RhopH3 in the cytoplasm of SLO permeabilized and trypsin treated infected erythrocytes. These novel findings show that both proteins were protected from protease digestion demonstrating that conformational changes are required to transport the RhopH proteins through association with the MC as integral membrane proteins.