Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in<i>Toxoplasma gondii</i>

O’Shaughnessy, William; Hu, Xiaoyu; Beraki, Tsebaot; McDougal, Matthew B.; Reese, Michael L.

doi:10.1101/2020.02.02.931022

“…Usually, genetic ablation of an inhibitor would be expected to present the opposite phenotype from its target. Because ERK7 kinase activity is required for conoid formation (17), our data suggest that AC9 inhibition of the kinase is not permanent. Instead, we propose that AC9 binding to ERK7 represents an unusual mechanism of ensuring kinase specificity ( Figure 6D).…”

Section: Discussionmentioning

confidence: 78%

“…We recently demonstrated that ERK7 localizes to the apical cap, and that its loss-of-function phenotype is essentially identical to what we have observed for AC9 (17). We therefore prioritized investigating the function of this interaction.…”

Section: Proximity Biotinylation Reveals Ac9 Interacts With the Map Kmentioning

confidence: 74%

“…These data provide new insight into the functions of the IMC apical cap in regulating parasite development. Using proximity biotinylation, we defined the AC9 interaction network, which includes ERK7, a conserved MAPK that regulates ciliogenesis in metazoa (15,16), and which we have recently shown is required for conoid formation (17). We demonstrate that AC9 is required for the correct localization of ERK7 at the apical cap, and that this scaffolding interaction is essential for apical complex maturation.…”

Section: Introductionmentioning

confidence: 89%

“…Apicomplexa (17), though little is known about its signaling cascade and regulatory interactions in any organism. AC9 is conserved among coccidian parasites, though the overall protein sequences are highly divergent (21-55% identity).…”

Section: Proximity Biotinylation Reveals Ac9 Interacts With the Map Kmentioning

confidence: 99%

“…Notably, ERK7 family members are unusual in the MAPK family as they are able to autophosphorylate their activation loops (18), and thus bypass the need for a MAPK kinase for their activation. We have previously found that ERK7 kinase activity is required for conoid formation (17).…”

Section: Ac9 Binds Erk7 In An Unusual Inhibitory Conformationmentioning

confidence: 99%

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

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Moon

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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 2 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.

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Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required forToxoplasmaapical complex biogenesis

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¹

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O’Shaughnessy

²

,

Moon

³

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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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 theToxoplasma gondiiIMC, 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 extracellular signal-regulated kinase 7 (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 autoactivating member of the mitogen-activated kinase (MAPK) family and its regulation is poorly understood in all organisms. 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.

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Two alveolin network proteins are essential for the subpellicular microtubules assembly and conoid anchoring to the apical pole of matureToxoplasma gondii

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Pacheco

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Bertiaux

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Toxoplasma gondii belongs to the coccidian sub-group of Apicomplexa that possess an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic and dynamic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR), a microtubule-organizing center for the 22 subpellicular microtubules (SPMTs). The SPMTs are linked to the Inner Membrane Complex (IMC), a patchwork of flattened vesicles, via an intricate network of small filaments composed of alveolins proteins. Here, we capitalize on super-resolution techniques including stimulated emission depletion (STED) microscopy and ultrastructure expansion microscopy (U-ExM) to localize the Apical Cap protein 9 (AC9) and its close partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Conditional depletion of AC9 or AC10 using the Auxin-induced Degron (AiD) system uncovered a severe loss of fitness. Parasites lacking AC9 or AC10 replicate normally but are defective in microneme secretion and hence fail to invade and egress from infected cells. Remarkably, a series of crucial apical complex proteins (MyoH, AKMT, FRM1, CPH1, ICMAP1 and RNG2) are lost in the mature parasites although they are still present in the forming daughter cells. Electron microscopy on intracellular or deoxycholate-extracted parasites revealed that the mature parasite mutants are conoidless. Closer examination of the SPMTs by U-ExM highlighted the disassembly of the SPMTs in the apical cap region that is presumably at the origin of the catastrophic loss of APR and conoid. AC9 and AC10 are two critical components of the alveolin network that ensure the integrity of the whole apical complex in T. gondii and likely other coccidians.

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Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure inToxoplasma gondii

Cited by 14 publications

References 36 publications

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

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

Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required forToxoplasmaapical complex biogenesis

Two alveolin network proteins are essential for the subpellicular microtubules assembly and conoid anchoring to the apical pole of matureToxoplasma gondii

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