PROJECT SUMMARY Toxoplasma gondii and related apicomplexan parasites contain a specialized organelle called the inner membrane complex (IMC) that plays essential roles in host cell invasion and daughter cell formation. The IMC consists of flattened membrane vesicles and a supporting cytoskeletal meshwork that is flanked by the plasma membrane and subpellicular microtubules at the periphery of the parasite. Recent in vivo biotinylation (BioID) studies have revealed a surprising level of compartmentalization within the IMC organelle, with distinct groups of proteins segregating to the cone-shaped apical cap, the body, or to the basal complex of both the cytoskeletal and membrane subcompartments. Analyses from our group and others have exposed new essential functions of the IMC including the role of the apical cap complex AC9/AC10/ERK7 in mounting the conoid which is essential for organelle secretion and invasion as well as the conserved early daughter bud protein IMC32 that is essential for replication. In this renewal, we will expand on these studies to determine precisely how these and other critical IMC components are able to carry out their functions. First, we will determine the role of novel apical cap proteins that are putative interactors of the AC9/AC10/ERK7 complex and assess their role in apical cap function and conoid assembly. We will then explore how IMC32 collaborates with the newly discovered partner IMC48 to function at the earliest stages of parasite division and use these proteins to further explore the early daughter bud proteome. Finally, we will exploit our recently developed photoreactive unnatural amino acid system as well as cryo-electron tomography to determine how critical alveolins of the parasite cytoskeleton are organized to serve as scaffolds for the organelle. Together, this project will promote a much deeper understanding of the architecture and function of the Toxoplasma IMC. As this organelle is parasite-specific and not present in its human host, determining precisely how these essential IMC components function promises to enable the design of novel therapies against T. gondii and other apicomplexan parasites.