In 2017, the Apicomplexan Molecular Physiology Section examined structure and function of a conserved high-molecular weight protein complex, termed the RhopH complex, in malaria parasites. We previously reported that one protein member of this complex, CLAG3, contributes to an essential nutrient uptake channel (plasmodial surface anion channel, PSAC), the target of an active drug discovery and development program. Here, we found that the other two members of this complex, RhopH2 and RhopH3 are essential as gene knockouts are not viable in the human P. falciparum pathogen. We therefore used two types of conditional knockdowns for both members along with stage-specific biochemical studies to examine their functions. This work determined that the three members of the RhopH complex assemble co-translationally before trafficking initially to rhoptries, specialized organelles within invasive merozoites. These organelles are secreted onto new host erythrocytes at the time of invasion. We therefore examined and excluded long-presumed roles for RhopH2 and CLAG3 in invasion, but implicated a RhopH3 contribution either through ligand-receptor interactions or signaling to mediate parasite internalization by erythrocytes. The three-protein complex also does not serve roles in formation or function of the parasitophorous vacuolar membrane, but transits through an export translocon on this vacuole to reach the host membrane. All three proteins are integral to the host membrane, but only CLAG3 is exposed at the host cell surface based on protease susceptibility studies. Knockdown of either RhopH2 or RhopH3 disrupts the entire complex and PSAC activity, accounting for the essentiality of their genes in all examined malaria parasite species. This dual-function protein complex appears to be an attractive target for development of new antimalarial therapies. eLife 6:e23485 (2017).
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