Biology subjects

Dewangan, P. S.

Publications and source records attributed to Dewangan, P. S..

5 recordsLinked to original sources

bioRxiv2026

Atypical RanGAP drives nucleocytoplasmic transport in a parasitic Alveolate

Transport of macromolecules between the nucleus and cytoplasm requires a gradient of the small GTPase, Ran. Ran:GTP marks the nucleus because Ran activity requires a cytoplasmic GTPase activating protein (RanGAP) for GTP hydrolysis. As expected for such central and essential cellular machinery, both Ran and RanGAP are conserved across the vast majority of eukaryotes. Many alveolates, including apicomplexan parasites, however, lack a canonical RanGAP. Here, we biochemically purify RanGAP activity from the model alveolate Toxoplasma gondii. We demonstrate that this activity is provided by a neofunctionalized RabGAP-fold protein, called TBC9. We find that TBC9 is sufficient to provide RanGAP activity in yeast, and is absolutely required to maintain active nucleocytoplasmic transport in Toxoplasma. By purifying Toxoplasma Ran and TBC9, we demonstrate that TBC9 is a robust and specific GAP for Ran. Finally, we delineate an essential, conserved low complexity motif in the C-terminus of TBC9 that drives interaction with Ran and is required for its full RanGAP activity. We use this C-terminal motif to identify TBC9 orthologs in all non-ciliate clades of Alveolata, which suggests that the canonical RanGAP has been replaced by the neofunctionalized TBC9 RanGAP in these lineages.

cell biology↗

bioRxiv2025

Identification of divergent Toxoplasma Nuclear Pore Complex components highlights speciation of mRNA export machinery

BackgroundA hallmark of the eukaryotic cell is the regulated transport between the nucleus and cytoplasm, which is mediated by a multi-subunit protein assembly called the nuclear pore complex (NPC). While its overall architecture has been preserved across eukaryotes, the NPC structure varies in different organisms, which appears to have tuned its function. Outside of a handful of model systems, the NPC has not been comprehensively studied. This is particularly true of species that are not closely related to well-studied models, such as apicomplexan parasites. Indeed, the evolutionary divergence of Apicomplexa has complicated facile prediction of NPC proteins in these organisms. Because of this, the NPC components remain largely unidentified, and therefore NPC cellular function in Apicomplexa is poorly understood. Principal FindingsHere we identified, experimentally validated, and functionally characterized protein components of the NPC in the apicomplexan parasite Toxoplasma gondii. By combining proximity biotinylation with careful bioinformatic analysis, we identified 16 previously uncharacterized proteins that localize to the Toxoplasma NPC. We demonstrated 8 of these proteins are essential to parasite replication. Importantly, we defined components of the mRNA export machinery, as well as Nups required for the stability and/or assembly of specific NPC subcomplexes. Consistent with the evolutionary distance between Toxoplasma and well-studied models, the majority of our newly validated NPC components show no clear homology to NPC proteins in yeast, animals, or plants. Moreover, we demonstrated that the Toxoplasma mRNA export machinery has a distinct composition from other well-established systems. Intriguingly, Sus1, a well-defined protein of the TREX-2 and SAGA complexes, is missing from the Toxoplasma genome. In contrast, others, such as Centrin-3, have been conserved in Toxoplasma, but are not required for mRNA export in the parasite. ConclusionOur work highlights the distinct composition of multiple subcomplexes of the Toxoplasma NPC and paves the way for future studies to provide high-resolution structural information on the parasites unusual NPC architecture.

cell biology↗

bioRxiv2022

Divergent kinase WNG1 is regulated by phosphorylation of an atypical activation sub-domain

Apicomplexan parasites like Toxoplasma gondii grow and replicate within a specialized organelle called the parasitophorous vacuole. The vacuole is decorated with parasite proteins that integrate into the membrane after trafficking through the parasite secretory system as soluble, chaperoned complexes. A regulator of this process is an atypical protein kinase called WNG1. Phosphorylation by WNG1 appears to serve as a switch for membrane integration. However, like its substrates, WNG1 is secreted from the parasite dense granules, and its activity must therefore be tightly regulated until the correct membrane is encountered. Here we demonstrate that, while another member of the WNG family can adopt multiple multimeric states, WNG1 is monomeric and therefore not regulated by multimerization. Instead, we identify two phosphosites on WNG1 that are required for its kinase activity. Using a combination of in vitro biochemistry and structural modeling, we identify basic residues that are also required for WNG1 activity and therefore appear to recognize the activating phosphosites. Among these coordinating residues are the "HRD" Arg, which recognizes activation loop phosphorylation in canonical kinases. WNG1, however, is not phosphorylated on its activation loop, and its activating phosphosites instead appear to lock the kinase C-lobe into an activated conformation. These data suggest a simple model for WNG1 activation by increasing ATP concentration above a critical threshold once WNG1 traffics to the parasitophorous vacuole.

biochemistry↗

bioRxiv2022

Multivalent interactions drive the Toxoplasma AC9:AC10:ERK7 complex to concentrate ERK7 in the apical cap

The Toxoplasma inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the MAP kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast-2-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC10:AC9:ERK7 complex and demonstrate that the interactions among them are critical to maintain the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap. IMPORTANCEThe phylum Apicomplexa consists of obligate, intracellular parasites including the causative agents of toxoplasmosis, malaria, and cryptosporidiosis. Hallmarks of these parasites are the IMC and the apical complex, both of which are unique structures that are conserved throughout the phylum and required for parasite survival. The apical cap portion of the IMC has previously been shown to stabilize the apical complex. Here, we expand on those studies to determine the precise protein-protein interactions of the apical cap complex that confer this essential function. We describe the multivalent nature of these interactions and show that the resulting protein oligomers likely tether ERK7 in the apical cap. This study represents the first description of the architecture of the apical cap at a molecular level, expanding our understanding of the unique cell biology that drives Toxoplasma infections.

microbiology↗

bioRxiv2020

Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis

Apicomplexan parasites use a specialized cilium structure called the apical complex to organize their secretory organelles and invasion machinery. The apical complex is integrally associated with both the parasite plasma membrane and an intermediate filament cytoskeleton called the inner membrane complex (IMC). While the apical complex is essential to the parasitic lifestyle, little is known about the regulation of apical complex biogenesis. Here, we identify AC9 (apical cap protein 9), a largely intrinsically disordered component of the Toxoplasma gondii IMC, as essential for apical complex development, and therefore for host cell invasion and egress. Parasites lacking AC9 fail to successfully assemble the tubulin-rich core of their apical complex, called the conoid. We use proximity biotinylation to identify the AC9 interaction network, which includes the kinase ERK7. Like AC9, ERK7 is required for apical complex biogenesis. We demonstrate that AC9 directly binds ERK7 through a conserved C-terminal motif and that this interaction is essential for ERK7 localization and function at the apical cap. The crystal structure of the ERK7:AC9 complex reveals that AC9 is not only a scaffold, but also inhibits ERK7 through an unusual set of contacts that displaces nucleotide from the kinase active site. ERK7 is an ancient and auto-activating member of the mitogen-activated kinase family and we have identified its first regulator in any organism. We propose that AC9 dually regulates ERK7 by scaffolding and concentrating it at its site of action while maintaining it in an "off" state until the specific binding of a true substrate. Significance StatementApicomplexan parasites include the organisms that cause widespread and devastating human diseases such as malaria, cryptosporidiosis, and toxoplasmosis. These parasites are named for a structure, called the "apical complex," that organizes their invasion and secretory machinery. We found that two proteins, apical cap protein 9 (AC9) and an enzyme called ERK7 work together to facilitate apical complex assembly. Intriguingly, ERK7 is an ancient molecule that is found throughout Eukaryota, though its regulation and function are poorly understood. AC9 is a scaffold that concentrates ERK7 at the base of the developing apical complex. In addition, AC9 binding likely confers substrate selectivity upon ERK7. This simple competitive regulatory model may be a powerful but largely overlooked mechanism throughout biology.

microbiology↗