Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.

Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction, and ubiquitination assay confirmed the presence of a functional Skp1-Cullin-1-Fbox (PfSCF) complex, comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1, and calcyclin binding protein. Immunoprecipitation, sequence analysis, and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specificity factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. The protein levels of several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were significantly altered upon PfCullin-2 depletion, which likely reflects association of PfCRL4 with multiple pathways. PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had a significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. We demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity. Author summary: Plasmodium falciparum is the major contributor to global burden of malaria. The parasite produces and disposes a number of its cellular contents during its development, which would require degradation machineries. Ubiquitin proteasome system (UPS) is a major degradation machinery in eukaryotes, which tags proteins by ubiquitin and degrades the tagged-proteins. Ubiquitin E3 ligases recognize the proteins to be tagged with ubiquitin, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group of these enzymes. CRLs have crucial roles in a variety of processes and are also targets of ongoing drug discovery projects. Hence, we investigated CRLs of P. falciparum, and show that it has two functional CRLs: PfSCF and PfCRL4. Both these enzymes are present during parasite's development in erythrocytes, the malaria-causing phase of the parasite. Both the enzymes are present in cytoplasmic and nuclear compartments, suggesting roles in these two compartments. Depletion of PfCullin-2, a constituent of PfCRL4, at protein level decreased parasite growth during disease-causing and transmission phases. The depletion also caused damage to parasite membranes and blocked nuclear division, indicating a crucial role of PfCRL4. Overall, we have identified two major enzymes of the parasite UPS, which could be used as drug targets. [ABSTRACT FROM AUTHOR]