Mutational analysis reveals how the essential interactions of a ClpP proteolytic system regulate the apicoplast proteome in malaria parasites

Abstract The human malaria parasite, Plasmodium falciparum, contains a plastid called the apicoplast that functions to produce essential metabolites. Most of apicoplast proteins are encoded by the nuclear genome and it is unclear how the apicoplast controls its own proteome. Here, we investigated the apicoplast caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Our data show that interfering with expression, activity or interactions of the Clp protease (PfClpP), chaperone (PfClpC), non-catalytic subunit (PfClpR), or potential substrate adaptor molecule (PfClpS) results in parasite death because of apicoplast loss. Using conditional null-mutants, we demonstrated that PfClpP is essential due to its role in apicoplast biogenesis. Conditional knockdown mutants of PfClpP revealed the robustness of its proteolytic activity and its interaction with the chaperone PfClpC. A CRISPR/Cas9 based system was developed and used for the expression of a PfClpC variant with a mutation in the putative protease binding site, which led to the discovery of its essential interaction with PfClpP. A combination of several tagged Clp proteins was used for affinity purification and determined complex composition as well as identify putative substrates. Expression of a catalytically-dead variant of PfClpP in the background of PfClpP conditional mutants showed that PfClpP oligomerizes as a zymogen and that the pro-domain is removed via trans-autocatalysis. Conditional knockdown mutants of PfClpS demonstrated its essential function in plastid biogenesis and its interaction with PfClpR as well as putative substrates. This comprehensive study reveals the molecular interactions driving the function of the apicoplast PfClpP/R/C/S system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.