AC9

AC9 is an alveolin-network component of the inner membrane complex (IMC) that functions as an essential regulatory scaffold for apical complex assembly in Toxoplasma gondii, localizing to the apical cap intercalated between the subpellicular microtubules (SPMTs) (PMID:32379047). Its central mechanistic role is to control the MAP kinase ERK7: AC9 directly binds ERK7 through a conserved C-terminal motif, and the crystal structure of the ERK7–AC9 complex shows AC9 acting as an inhibitory scaffold that displaces nucleotide from the kinase active site while concentrating ERK7 at the apical cap (PMID:32409604). AC9 operates within an AC9:AC10:ERK7 ternary complex assembled through multivalent pairwise interactions that oligomerize and tether ERK7 kinase activity to the apical cap cytoskeleton (PMID:35130732). Conditional depletion of AC9 dismantles this system, causing loss of the conoid and apical polar ring, SPMT disorganization, defective microneme secretion, and failure of invasion and egress; depletion of ERK7 phenocopies these defects, placing AC9 and ERK7 in a single pathway for apical complex integrity (PMID:32379047, PMID:32409604, PMID:34607461).

1. 2020 High

   Establishing where AC9 resides addressed whether it is a bona fide apical cytoskeletal protein and pinpointed it to the alveolin network between SPMTs, the structural context needed to interpret its function.

   Evidence Ultrastructure expansion microscopy combined with BioID proximity biotinylation in T. gondii

   PMID:32379047

   Open questions at the time

   • Localization alone does not reveal molecular activity
   • Does not identify binding partners at the apical cap
2. 2020 High

   Conditional depletion tested whether AC9 is functionally required, showing it is essential for conoid, APR, and SPMT integrity and for the parasite lytic cycle.

   Evidence Conditional knockdown with EM, U-ExM, and invasion/egress/microneme secretion assays in T. gondii

   PMID:32379047 PMID:32409604

   Open questions at the time

   • Phenotype does not by itself define the molecular mechanism linking AC9 to apical assembly
3. 2020 High

   Identifying ERK7 as a direct AC9 binding partner and solving the complex structure explained how AC9 works mechanistically — as an inhibitory scaffold that both localizes and switches off the kinase by displacing nucleotide from its active site.

   Evidence BioID, direct binding assays, X-ray crystallography of the ERK7–AC9 complex, and kinase activity assays with interaction-motif mutagenesis

   PMID:32409604

   Open questions at the time

   • Does not resolve how the AC9-imposed 'off' state is relieved or regulated in vivo
   • In vitro nucleotide displacement not directly tracked in the living parasite
4. 2021 High

   Testing ERK7 depletion asked whether AC9 and ERK7 act in the same pathway, and the phenocopy established genetic epistasis for apical complex maintenance.

   Evidence Conditional depletion with U-ExM/NHS-ester staining, EM, comparative proteomics, and microneme secretion assays in T. gondii

   PMID:34607461

   Open questions at the time

   • Does not define the ERK7 substrates whose phosphorylation drives apical assembly
5. 2022 High

   Interaction mapping addressed how the scaffold is organized, showing AC9, AC10, and ERK7 form a multivalent ternary complex that oligomerizes and tethers ERK7 to the apical cap cytoskeleton.

   Evidence Deletion analysis, yeast two-hybrid, and co-immunoprecipitation in T. gondii

   PMID:35130732

   Open questions at the time

   • Stoichiometry and higher-order architecture of the assembled complex not resolved
   • Does not establish how the complex couples ERK7 regulation to conoid/APR biogenesis

• The physiological ERK7 substrates and the trigger that relieves AC9-mediated kinase inhibition during apical complex assembly remain unidentified.
• No ERK7 substrate at the apical cap identified in the corpus
• Mechanism regulating the AC9-imposed inhibitory state in vivo unknown