AC9AID tagging constructs were generated using a PCR-amplified homology-directed repair template (P1-2) and a CRISPR-Cas9 pU6-Universal plasmid with a protospacer against the 3 untranslated region of the gene of the interest (P3-4)

AC9AID tagging constructs were generated using a PCR-amplified homology-directed repair template (P1-2) and a CRISPR-Cas9 pU6-Universal plasmid with a protospacer against the 3 untranslated region of the gene of the interest (P3-4). 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. 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 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. 8-Hydroxyguanosine The crystal structure of the ERK7CAC9 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. Cilia are ancient eukaryotic organelles that organize signal-transduction cascades and mediate cell motility. These functions are driven by the cooperation of cytoskeleton and membrane structures and require specialized signaling and trafficking machinery for their biogenesis and maintenance (1C3). In apicomplexan parasites, the cilium is thought to have evolved to form the apical complex (4C7), which organizes the parasites 8-Hydroxyguanosine invasion machinery and for which the phylum is named. Apicomplexa include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis, which all invade mammalian cells to cause disease. Like more typical eukaryotic cilia, the apical complex is composed of specialized microtubule structures inserted into the plasma membrane (8). In addition, the apical complex is the site of secretion of specialized organelles called micronemes and rhoptries that mediate the parasites attachment to and invasion of host cells. In the asexual stage of most apicomplexans, secretion is thought to occur through a tubulin-rich structure in the apical complex called the conoid (8, 9). The apical complex is also intimately associated with an intermediate filament cytoskeleton called the inner-membrane complex (IMC) that scaffolds the apicomplexan cell, ensuring its correct morphology. The IMC anchors the parasite actin-based motility machinery (10), powering the parasites motility as it glides across and invades host cells. While the IMC extends the length of the parasite, it has clearly segregated apical, medial, and basal subdomains that are defined by specific protein S1PR4 localization (11, 12). In apical IMC, apical cap protein 9 (AC9), as essential to the parasite lytic cycle. We found that loss of AC9 results in parasites that are entirely 8-Hydroxyguanosine unable to egress from their host cells or invade new cells. These deficiencies are attributable to the failure of the parasites to form a functional apical complex, as 8-Hydroxyguanosine the conoids are entirely missing in mature parasites and regulated secretion is disrupted. These data provide insight into the functions of the IMC apical cap in regulating 8-Hydroxyguanosine parasite development. Using proximity biotinylation, we defined the AC9 interaction network, which includes extracellular signal-regulated kinase 7 (ERK7), a conserved mitogen-activated protein kinase (MAPK) that regulates ciliogenesis in Metazoa (15, 16), and which we have recently shown is required for conoid formation (17). We demonstrated 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. Finally, we solved the crystal structure of the ERK7CAC9 complex, which revealed that the.