Project Summary The endosomal system is composed of a continuum of organelles that are derived by vesicle-mediated trafficking from the plasma membrane and Golgi apparatus which mature until fusing with the lysosome. Molecular sorting reactions that take place during endosome maturation direct molecules into the lysosomal degradation pathway, or into pathways that export the molecules from the endosome for subsequent trafficking to another organelle for re-use. The latter ‘endosomal recycling’ and ‘retrograde’ trafficking pathways provide a key means for regulating the composition of the PM, and hence cellular identity, and of internal organelles in response to metabolic and environmental cues. Importantly, genetic perturbations to these pathways result in lysosomal storage diseases, Parkinson’s disease, Alzheimer’s disease, cancer, and other diseases, indicating that proper sorting at the endosome is essential for protection from disease. The broad goal of this research project is to elucidate the mechanisms by which proteins and lipids are sorted and trafficked from the endosome. In previous funding cycles we discovered that Golgi-directed retrograde pathways are used to modulate the composition of the plasma membrane in response to environmental and nutritional cues, and we established roles for a soluble protein sorting complex called ‘retromer’ and effectors of phosphatidylinositol 3- kinase, called sorting nexin proteins, in this process. These components function together to capture and package integral membrane protein and lipid cargo into transport carriers that bud and fission from the endosome. The proposed research will elucidate the mechanistic principles and events that underlie sorting of integral membrane proteins and lipids within the endo-lysosomal system. Diverse experimental approaches will be applied to identify and characterize regulatory mechanisms that control retromer-mediated sorting in cells, and these will be complemented with biochemical reconstitution studies that will elucidate and order the molecular functions of the components of retromer and associated proteins. Additional studies will elucidate the functions and mechanistic principles of retromer-independent recycling pathways by sorting nexins.