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. Determining the precise functions of the IMC has been hampered by a limited understanding of its protein constituents, which have predominantly focused on the motor components of the glideosome that powers parasite motility and the family of alveolins that are embedded within the IMC cytoskeletal network underlying the flattened membrane vesicles of the organelle. We have recently overcome this gap in our knowledge using in vivo biotinylation (BioID) to identify over forty new IMC components, more than doubling the known IMC proteome. These new proteins have revealed a surprising level of compartmentalization within the organelle, with distinct components segregating to the cytoskeletal or membrane subregions of the apical cap, IMC body, or the recently discovered IMC sutures, which organize the IMC membranes into their characteristic rectangular plates. Analyses of a few of these proteins have revealed new important roles in controlling parasite shape, cellular division, and invasion. In this proposal, we will leverage our new cohort of IMC proteins to first provide a temporal and spatial understanding of how these components participate in the dynamic process of internal budding known as endodyogeny. This will reveal groups of proteins that function to assemble the IMC at specific steps of endodyogeny and also provide insight into the dynamic process of distinguishing maternal and daughter IMCs during division. We will then explore how the key IMC suture proteins ISC3 and ISC6 regulate proper IMC membrane biogenesis, parasite morphology and replication. Finally, we exploit the recent genome-wide CRISPR screen in Toxoplasma to focus on a subset of our new IMC proteins that are likely to play crucial roles within distinct suborganellar compartments of the organelle. Together, this project will fundamentally transform our understanding of the organization and function of the Toxoplasma IMC. As this organelle is parasite-specific and is not present in its human host, elucidating critical IMC components and their respective functions promises to reveal ideal targets for the design of novel therapies against T. gondii and other apicomplexan parasites.