Project Summary / Abstract Endosomal function depends on constant membrane remodeling, stemming from a need for organization and maintenance of specialized subdomains and for the sorting and trafficking required for the protein and lipid cargoes that pass through, or are delivered to, the endosomal network. Membrane remodeling is achieved by the concerted actions of several families of dedicated remodeling proteins, including the SNX-BARs. SNX-BARs are endosome-centered proteins with pivotal roles in several trafficking pathways. The defining feature of all SNX-BARs is their PX-BAR module, a tight association of a typically lipid-binding PX domain and a BAR domain, which, when homo- or hetero-dimerized, is a membrane curvature-sensing or remodeling module. Prevailing models of SNX-BAR function suggest that recruitment to target membranes can generate and/or stabilize tubular transport carriers, which, functionally, are coupled to cargo selection and sorting. Specific SNX-BARs have been shown to be required for endocytosis, retrograde trafficking, autophagy and other processes. Consequently, defects in SNX-BAR-dependent pathways are associated with a range of diseases, particularly neurodegenerative. Given the central importance of SNX-BARs in endosomal trafficking, several pressing questions about SNX-BAR biology must be addressed. How is SNX-BAR membrane remodeling activity regulated? SNX-BARs all have extensive membrane-engaging interfaces and it is unknown how membrane attachment and detachment are controlled in the cell. Using cryo-electron microscopy, we recently demonstrated a novel, autoinhibited tetrameric configuration of the SNX-BAR Mvp1, where its lipid binding surfaces are sequestered into the interior of the tetramer. Mvp1 has an N-terminal Intrinsically Disordered Region (IDR) which is essential for tetramerization and its presence is required for Mvp1 trafficking functions in vivo. How do SNX- BARs select pathway-specific cargoes and how is cargo binding coordinated with SNX-BAR remodeling activity? Many cargoes are known to interact with specific SNX-BARs but how they do so is not understood. In many cases, SNX-BARs are known to functionally cooperate with dynamin superfamily proteins (DSPs) within the same remodeling pathway, but their respective contributions to remodeling are unclear. We will address these questions using a comprehensive and multi-disciplinary approach, targeting selected SNX-BARs involved in several pathways. We will explore the functions of their uncharacterized IDRs using structural, in vitro and in vivo approaches, using both yeast and mammalian model systems. Our key, driving hypothesis is that, as all known SNX-BAR homo- and heterodimers have at least one IDR, the IDR is an essential regulatory module that controls membrane remodeling, membrane access and couples SNX-BAR function with cargo recruitment and the activities of other cellular machines. These concepts are novel and innovative and will contribu...