Mechanisms of Lysosome Biogenesis and Regulation

Abstract

Lysosomes are the central catabolic organelles that contain acidic hydrolases to break down macromolecules. On the membrane surface, lysosomal transporters and channels shuttle nutrients and metabolites across the membrane to maintain cellular homeostasis. Pathogenic mutations of these lysosomal proteins lead to inherited disorders called lysosomal storage diseases. In addition, lysosomes serve as signaling hubs that sense nutrient availability and environmental cues to control cellular activities, including protein synthesis, cell growth & differentiation, and autophagy. Thus, dysregulation of lysosomal function is frequently associated with neurodegeneration diseases and cancers. Despite the significance of lysosomal proteins in maintaining lysosome integrity and cellular hemostasis, the mechanisms of their regulation and turnover are largely unknown. Previously, my research mentor Dr. Ming Li showed that transporters on the yeast vacuole (functionally equivalent to human lysosome) were selectively downregulated in response to a specific substrate level. These pioneering discoveries laid the foundation to understand how cells regulate their lysosomal membrane proteins (LMPs) in response to environmental cues. For this dissertation, we sought to explore the mechanisms of lysosome regulation in both yeast and human cells. In Chapter 2, we asked how cells regulate lysosome proteome in response to environmental stresses, such as starvation or TORC1 inactivation. In yeast, we showed that TORC1 inactivation leads to upregulation of vacuole biogenesis and autophagy pathways; however, it also triggers the downregulation of many vacuolar membrane proteins to support vacuole remodeling. We further demonstrated that the degradation of these vacuolar membrane proteins is mediated by the ubiquitin- and ESCRT-dependent microautophagy process. As our yeast studies consistently demonstrated the importance of the ubiquitination process and ESCRT machinery in regulating LMPs, we wondered if such mechanisms are evolutionarily conserved in human cells. In Chapter 3, we performed a cycloheximide chase screen and identified two short-living lysosomal membrane proteins, RNF152 and LAPTM4A. We showed that the degradation of RNF152 is triggered by its autoubiquitination and the LAPTM4A is ubiquitinated by NEDD4. Furthermore, the ESCRT-machinery is required to internalize the ubiquitinated RNF152 and LAPTM4A into the lysosome lumen. In Chapter 4, we conducted a CRISPR-Cas9 screen to identify uncharacterized genes involved in the degradation of LMPs and lysosome function. Our screen revealed a novel factor, TMEM251, required for lysosome function. Ablation of TMEM251 leads to defective mannose-6-phosphate (M6P) formation of lysosomal enzymes, a critical process for targeting acidic hydrolases to lysosomes. Our study further indicated that TMEM251 is necessary for the processing and activation of the GlcNAc-1-phosphotransferase, which catalyzes the first-step reaction of M6P formation. In this dissertation, we present novel mechanisms of lysosome biogenesis and regulation. We discovered a conserved ubiquitin- and ESCRT-dependent pathway to turnover LMPs in yeast and human cells. Our studies on LMP regulation provide a better understanding of lysosomal protein quality control and how lysosomes adapt to environmental changes. In addition, our discovery of TMEM251 sheds light on understanding the mechanisms of lysosome biogenesis.