Gorde, S. M. | bioRxiv Science

bioRxiv2024

The apicomplexan DedA superfamily protein VMP1 is crucial for the biogenesis and function of secretory organelles

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, contain specialized secretory organelles such as micronemes, rhoptries, and dense granules, which are essential for parasite motility, host cell invasion, development, and egress. DedA superfamily proteins are implicated in lipid mobilization, which is a key requirement for organelle biogenesis. Herein, we identified and investigated the vacuole membrane protein 1 (VMP1), a DedA superfamily member, of P. falciparum (PfVMP1) and T. gondii (TgVMP1). PfVMP1 and TgVMP1 are ER-localized lipid scramblases. TgVMP1 depletion adversely affected parasite development, motility, host cell invasion, and egress. These phenotypes were consistent with impaired rhoptry and dense granule biogenesis, and decreased secretion of micronemes, rhoptries, and dense granules in TgVMP1-depleted parasites, indicating a crucial role for TgVMP1 in the biogenesis and function of these organelles. TgVMP1 depletion impaired lipid droplet homeostasis and ER organization. Restoration of the ER-localized lipid scramblase by complementing TgVMP1-depleted parasites with PfVMP1 or a homolog as distant as human VMP1 rescued the depleted parasites, indicating their functional conservation and a crucial role for ER-resident lipid scramblase activity in the biogenesis and function of secretory organelles. The essentiality of TgVMP1 for parasite development and likely functional conservation of apicomplexan VMP1 proteins highlight their drug-target potential.

cell biology↗

bioRxiv2023

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 ubiquitin E3 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 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 in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity. This study will benefit investigation of similar ligases in related parasites.

microbiology↗