Project Summary The lysosome is an essential organelle responsible for the digestion and recycling of materials delivered by endocytosis and autophagy. It also plays important role in nutrient sensing and control of cell growth by regulating the localization and activity of mTORC1 signaling complex. Because of its importance, lysosome dysfunction leads to ~ 50 types of lysosomal storage diseases (LSDs) and contributes to many aging-related neurodegenerative diseases such as Alzheimer’s, Huntington’s, and Parkinson’s diseases. Despite exhaustive research on how proteins are delivered to lysosomes, how lysosomes regulate their own membrane proteins remains poorly understood. However, studying this question will reveal how cells maintain a healthy lysosome during stresses and aging. Our long-term goal is to understand these fundamental questions using both yeast and mammalian cells as model systems. Recently, we discovered a ubiquitin- and ESCRTdependent down-regulation pathway for lysosome (vacuole) membrane proteins in yeast. Follow-up investigations in our laboratory led us to hypothesize that the ubiquitin- and ESCRTdependent degradation pathway is a general conserved mechanism to regulate the lysosome membrane composition from yeast to human. Consistently, recent proteomic studies identified multiple E3 ubiquitin ligases on the human lysosome membrane. Furthermore, the ESCRT machinery was shown to be recruited to the human lysosome membrane. In this proposed research, we plan to expand our initial findings by pursuing three specific aims. Our Aim 1 will investigate how TORC1 regulates the vacuole membrane proteome via the ubiquitin- and ESCRT-dependent pathway in yeast. Our Aim 2 will study how yeast vacuole membrane E3 ligases recognize their membrane substrates at both structure and function levels. Our Aim 3 will study how human lysosomes turnover their membrane proteins. Our research will shed light on the development of new treatment strategies for LSDs and lysosome- related neurodegenerative diseases.