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

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921245117 ◽

2020 ◽

Vol 117 (22) ◽

pp. 12164-12173 ◽

Cited By ~ 3

Author(s):

Peter S. Back ◽

William J. O’Shaughnessy ◽

Andy S. Moon ◽

Pravin S. Dewangan ◽

Xiaoyu Hu ◽

...

Keyword(s):

Specific Binding ◽

Interaction Network ◽

Intrinsically Disordered ◽

Membrane Complex ◽

Extracellular Signal ◽

Complex Development ◽

Parasitic Lifestyle ◽

Apical Complex ◽

Inner Membrane Complex ◽

And Function

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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Toxoplasma gondii myosins B/C

The Journal of Cell Biology ◽

10.1083/jcb.200012116 ◽

2001 ◽

Vol 155 (4) ◽

pp. 613-624 ◽

Cited By ~ 66

Author(s):

Frédéric Delbac ◽

Astrid Sänger ◽

Eva M. Neuhaus ◽

Rolf Stratmann ◽

James W. Ajioka ◽

...

Keyword(s):

Cell Division ◽

Toxoplasma Gondii ◽

Daughter Cell ◽

Gliding Motility ◽

Apicomplexan Parasites ◽

Daughter Cells ◽

Membrane Complex ◽

Alternatively Spliced ◽

Butanedione Monoxime ◽

Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

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Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii

The Journal of Cell Biology ◽

10.1083/jcb.200311137 ◽

2004 ◽

Vol 165 (3) ◽

pp. 383-393 ◽

Cited By ~ 185

Author(s):

Elizabeth Gaskins ◽

Stacey Gilk ◽

Nicolette DeVore ◽

Tara Mann ◽

Gary Ward ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Complex ◽

Integral Membrane Protein ◽

Gliding Motility ◽

Host Cells ◽

Inner Membrane ◽

Apicomplexan Parasites ◽

Membrane Complex ◽

Inner Membrane Complex ◽

Two Stages

Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

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A Novel Actin-Related Protein Is Associated with Daughter Cell Formation in Toxoplasma gondii

Eukaryotic Cell ◽

10.1128/ec.00064-08 ◽

2008 ◽

Vol 7 (9) ◽

pp. 1500-1512 ◽

Cited By ~ 20

Author(s):

Jennifer L. Gordon ◽

Wandy L. Beatty ◽

L. David Sibley

Keyword(s):

Cell Division ◽

Toxoplasma Gondii ◽

Daughter Cell ◽

Cell Formation ◽

The Body ◽

Apicomplexan Parasites ◽

Daughter Cells ◽

Membrane Complex ◽

Transport Vesicles ◽

Inner Membrane Complex

ABSTRACT Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.

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Daughter Cell Assembly in the Protozoan ParasiteToxoplasma gondii

Molecular Biology of the Cell ◽

10.1091/mbc.01-06-0309 ◽

2002 ◽

Vol 13 (2) ◽

pp. 593-606 ◽

Cited By ~ 120

Author(s):

Ke Hu ◽

Tara Mann ◽

Boris Striepen ◽

Con J. M. Beckers ◽

David S. Roos ◽

...

Keyword(s):

Cell Division ◽

Fluorescent Protein ◽

Molecular Genetic ◽

Inner Membrane ◽

Apicomplexan Parasites ◽

Membrane Complex ◽

Ideal System ◽

Animal Pathogens ◽

Inner Membrane Complex

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii.Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites.

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Structural role of essential light chains in the apicomplexan glideosome

10.1101/867499 ◽

2019 ◽

Cited By ~ 2

Author(s):

Samuel Pazicky ◽

Karthikeyan Dhamotharan ◽

Karol Kaszuba ◽

Haydyn Mertens ◽

Tim Gilberger ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Light Chain ◽

Structural Information ◽

Host Cells ◽

Light Chains ◽

Apicomplexan Parasites ◽

Membrane Complex ◽

C Terminus ◽

Inner Membrane Complex

AbstractApicomplexan parasites, such as Plasmodium falciparum and Toxoplasma gondii, traverse the host tissues and invade the host cells exhibiting a specific type of motility called gliding. The molecular mechanism of gliding lies in the actin-myosin motor localized to the intermembrane space between the plasma membrane and inner membrane complex (IMC) of the parasites. Myosin A (MyoA) is a part of the glideosome, a large multi-protein complex, which is anchored in the outer membrane of the IMC. MyoA is bound to the proximal essential light chain (ELC) and distal myosin light chain (MLC1), which further interact with the glideosome associated proteins GAP40, GAP45 and GAP50. Whereas structures of several individual glideosome components and small dimeric complexes have been solved, structural information concerning the interaction of larger glideosome subunits and their role in glideosome function still remains to be elucidated. Here, we present structures of a T. gondii trimeric glideosome sub complex composed of a myosin A light chain domain with bound MLC1 and TgELC1 or TgELC2. Regardless of the differences between the secondary structure content observed for free P. falciparum PfELC and T. gondii TgELC1 or TgELC2, the proteins interact with a conserved region of TgMyoA to form structurally conserved complexes. Upon interaction, the essential light chains undergo contraction and induce α-helical structure in the myosin A C-terminus, stiffening the myosin lever arm. The complex formation is further stabilized through binding of a single calcium ion to T. gondii ELCs. Our work provides an important step towards the structural understanding of the entire glideosome and uncovering the role of its members in parasite motility and invasion.Author summaryApicomplexans, such as Toxoplasma gondii or the malaria agent Plasmodium falciparum, are small unicellular parasites that cause serious diseases in humans and other animals. These parasites move and infect the host cells by a unique type of motility called gliding. Gliding is empowered by an actin-myosin molecular motor located at the periphery of the parasites. Myosin interacts with additional proteins such as essential light chains to form the glideosome, a large protein assembly that anchors myosin in the inner membrane complex. Unfortunately, our understanding of the glideosome is insufficient because we lack the necessary structural information. Here we describe the first structures of trimeric glideosome sub complexes of T. gondii myosin A bound to two different light chain combinations, which show that T. gondii and P. falciparum form structurally conserved complexes. With an additional calcium-free complex structure, we demonstrate that calcium binding does not change the formation of the complexes, although it provides them with substantial stability. With additional data, we propose that the role of the essential light chains is to enhance myosin performance by inducing secondary structure in the C-terminus of myosin A. Our work represents an important step in unveiling the gliding mechanism of apicomplexan parasites.

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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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Identification of PhIL1, a Novel Cytoskeletal Protein of the Toxoplasma gondii Pellicle, through Photosensitized Labeling with 5-[125I]Iodonaphthalene-1-Azide

Eukaryotic Cell ◽

10.1128/ec.00114-06 ◽

2006 ◽

Vol 5 (10) ◽

pp. 1622-1634 ◽

Cited By ~ 31

Author(s):

Stacey D. Gilk ◽

Yossef Raviv ◽

Ke Hu ◽

John M. Murray ◽

Con J. M. Beckers ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Membrane Proteins ◽

Transmembrane Domain ◽

Protozoan Parasite ◽

Integral Membrane Protein ◽

Apicomplexan Parasites ◽

Membrane Complex ◽

Inner Membrane Complex ◽

Complex Integral ◽

Host Cell Recognition

ABSTRACT The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[125I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [125I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

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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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