PROJECT SUMMARY The lysosome is an important organelle responsible for digesting and recycling materials, nutrient sensing, and controlling cell proliferation. It is also linked to neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's diseases. Despite its importance, the regulation of lysosomal membrane protein composition and the function of its transporters and ion channels are not well understood. My lab has made significant progress in studying the turnover mechanisms of lysosomal membrane proteins by identifying a conserved ubiquitin- and ESCRT-dependent pathway for protein degradation. However, several key questions remain, including the identification of substrates of lysosomal E3 ligases in humans (future direction 1), the assembly of the ESCRT machinery to the lysosome membrane for internalizing ubiquitinated cargoes (future direction 2), and identifying other proteins involved in the quality control process. Additionally, there is a lack of understanding of the substrates, direction of transport, and energy dependence of many lysosomal transporters and ion channels (future direction 3). My lab has developed a reconstitution platform to study lysosomal membrane transport proteins, which will be combined with conventional methods such as metabolomics of isolated lysosomes to better understand these processes.