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

Abstract

AbstractProtein 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 ubiquitin E3 Ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated inPlasmodiumand related parasites. To investigate the CRLs of human malaria parasitePlasmodium falciparum, we generated parasites expressing taggedP. falciparumcullin-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 vitroprotein-protein interaction and ubiquitination assay confirmed the presence of a functional SCF (PfSCF), 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 specific 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. Several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were dysregulated upon PfCullin-2-depletion, which likely reflects association of PfCRL4 with multiple pathways. Consistent with dysregulation of 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 significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. Taken together, we for the first time demonstrate the presence of functional CRLs inP. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity. This study will benefit investigation of similar ligases in related parasites.