The Clp proteolytic system acts as an essential nexus for Plasmodium plastid biogenesis

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 prokaryotic 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 substrate adaptor molecule (PfClpS) results in apicoplast loss. Using null-mutants, we demonstrated that PfClpP is essential due to its role in apicoplast biogenesis. Conditional mutants of PfClpP revealed the robustness of its proteolytic activity and its interaction with the chaperone PfClpC. Expression of a PfClpC variant with a mutation in the protease binding site demonstrated its role as an essential regulator of complex activity. A combination of several tagged Clp proteins was used for affinity purification and enabled us to determine complex composition. A CRISPR/Cas9 based system was developed and used for the expression of a catalytically-dead form of PfClpP in the background of PfClpP conditional mutants. These double-mutant parasites were used to determine the mechanisms of PfClpP oligomerization and maturation, and revealed its trans-autocatalytic properties. Further, we investigated the functions of the substrate adaptor molecule, PfClpS, and showed that it is essential for plastid biogenesis. This comprehensive study reveals the molecular mechanisms 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.