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

10.1101/2020.02.02.931089 ◽

2020 ◽

Cited By ~ 1

Author(s):

Peter S. Back ◽

William J. O’Shaughnessy ◽

Andy S. Moon ◽

Pravin S. Dewangan ◽

Xiaoyu Hu ◽

...

Keyword(s):

Specific Binding ◽

Interaction Network ◽

Apicomplexan Parasites ◽

Intrinsically Disordered ◽

Membrane Complex ◽

Complex Development ◽

Parasitic Lifestyle ◽

Apical Complex ◽

Inner Membrane Complex ◽

And Function

AbstractApicomplexan 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.

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

10.1101/2022.01.04.474968 ◽

2022 ◽

Author(s):

Peter S Back ◽

William J O'Shaughnessy ◽

Andy S Moon ◽

Pravin S Dewangan ◽

Michael L Reese ◽

...

Keyword(s):

Map Kinase ◽

Kinase Activity ◽

Pairwise Interaction ◽

Inner Membrane ◽

Membrane Complex ◽

Apical Complex ◽

Multivalent Interactions ◽

Inner Membrane Complex ◽

Parasite Motility ◽

The Stability

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.

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

10.1101/2020.02.25.962589 ◽

2020 ◽

Author(s):

Nicolò Tosetti ◽

Nicolas Dos Santos Pacheco ◽

Eloïse Bertiaux ◽

Bohumil Maco ◽

Lorène Bournonville ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Super Resolution ◽

Stimulated Emission ◽

Microtubule Organizing Center ◽

Membrane Complex ◽

Catastrophic Loss ◽

Organizing Center ◽

Apical Complex ◽

Critical Components ◽

Inner Membrane Complex

AbstractToxoplasma 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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Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes

Journal of Cell Science ◽

10.1242/jcs.099002 ◽

2012 ◽

Vol 125 (8) ◽

pp. 2053-2063 ◽

Cited By ~ 65

Author(s):

M. K. Dearnley ◽

J. A. Yeoman ◽

E. Hanssen ◽

S. Kenny ◽

L. Turnbull ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Inner Membrane ◽

Membrane Complex ◽

Inner Membrane Complex ◽

And Function

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A novel Toxoplasma IMC sutures-associated protein regulates suture protein targeting and colocalizes with membrane trafficking machinery

10.1101/2021.06.22.449166 ◽

2021 ◽

Author(s):

Jessica H Chern ◽

Andy S Moon ◽

Allan L Chen ◽

Jihui Sha ◽

James A Wohlschlegel ◽

...

Keyword(s):

Host Cell ◽

Cell Invasion ◽

Membrane Trafficking ◽

Protein Targeting ◽

Gene Knockout ◽

Host Cell Invasion ◽

Membrane Complex ◽

Inner Membrane Complex ◽

And Function

The cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identify here a novel protein that localizes to discrete punctae that are embedded in the parasite's cytoskeleton along the IMC sutures. Gene knockout analysis shows that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then use deletion analyses to identify a domain in the N-terminus of the protein that is critical for both localization and function. Finally, we use the protein as bait for in vivo biotinylation which identifies several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, this data reveals an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggests that the sutures punctae are likely a portal for trafficking cargo across the IMC.

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The Dynamic Roles of the Inner Membrane Complex in the Multiple Stages of the Malaria Parasite

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.611801 ◽

2021 ◽

Vol 10 ◽

Author(s):

Josie Liane Ferreira ◽

Dorothee Heincke ◽

Jan Stephan Wichers ◽

Benjamin Liffner ◽

Danny W. Wilson ◽

...

Keyword(s):

De Novo ◽

Morphological Changes ◽

Protein Composition ◽

Cell Types ◽

Structural Element ◽

Inner Membrane ◽

Cell Functions ◽

Membrane Complex ◽

Inner Membrane Complex ◽

And Function

Apicomplexan parasites, such as human malaria parasites, have complex lifecycles encompassing multiple and diverse environmental niches. Invading, replicating, and escaping from different cell types, along with exploiting each intracellular niche, necessitate large and dynamic changes in parasite morphology and cellular architecture. The inner membrane complex (IMC) is a unique structural element that is intricately involved with these distinct morphological changes. The IMC is a double membrane organelle that forms de novo and is located beneath the plasma membrane of these single-celled organisms. In Plasmodium spp. parasites it has three major purposes: it confers stability and shape to the cell, functions as an important scaffolding compartment during the formation of daughter cells, and plays a major role in motility and invasion. Recent years have revealed greater insights into the architecture, protein composition and function of the IMC. Here, we discuss the multiple roles of the IMC in each parasite lifecycle stage as well as insights into its sub-compartmentalization, biogenesis, disassembly and regulation during stage conversion of P. falciparum.

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