The Endosomal Sorting Complex Required for Transport (ESCRT) machinery is a set of interacting protein complexes responsible for cargo selection and biogenesis of intralumenal vesicles inside endosomal multivesicular bodies, also known as late endosomes. Evolutionary conservation and the discovery that ESCRTs are required for topologically equivalent processes including viral budding, cytokinetic abscission, and nuclear envelope closure led to the now widely accepted concept that ESCRTs are uniquely involved in membrane fission for reactions that share this topology. ESCRT-III proteins act by changing conformation and polymerizing into membrane-remodeling filaments that spiral on the inside – negatively curved – surface of membrane tubules, ultimately pulling the tubules closed to drive membrane fission and release. Intriguingly, there are twelve ESCRT-III proteins in humans that are not functionally interchangeable. Beyond identification of different molecular binding partners, there has been little structural distinction among these proteins to explain their unique physiological importance. We recently made the surprising discovery that two human ESCRT-III proteins – CHMP1B and IST1 – assemble into filaments that spiral around the outside – positively curved – surface of membrane tubules, forming external coats in vitro and in vivo. This unexpected preference for positively curved membrane tubules challenges the dogma that the membrane deforming and fission activity associated with ESCRT-III filaments is limited to a single topology and prompts us to reconsider established paradigms for ESCRT-III function. This project will (1) define the topology preference of particular ESCRT-III homo- and heteropolymers polymers and the corresponding distribution of endogenous ESCRT-III proteins across the endosomal system, (2) establish the role(s) of representative ESCRT-III proteins in distinct endosomal cargo trafficking pathways, and (3) compare the effects of depleting different ESCRT-III proteins on endosomal and lysosomal morphology. This work will expand our understanding of the ESCRT-III membrane remodeling system with significant implications for future studies of trafficking and organization within the endolysosomal system.