Lysosomes are essential for degrading macromolecules, removing faulty organelles, and regulating cellular homeostasis, with dysfunction linked to severe diseases. This study uses advanced subcellular proteomics, organelle purification techniques, and structural biology to elucidate complex protein interactions within and around lysosomes. We will isolate intact lysosomes using lysosomal immunopurification (LysoIP) and apply membrane-penetrating and impermeable cross-linkers to stabilize protein interactions. This allows us to capture a detailed snapshot of these interactions as they occur naturally within the cell. The cross-linked proteins will be analyzed using high-resolution mass spectrometry to map interactions in the lysosomal lumen and on its membrane. Identified interactions will be verified and functionally investigated under different nutrient conditions to understand their regulatory mechanisms. Bioinformatics tools will be employed for data analysis and structural biology, providing a comprehensive view of lysosomal protein dynamics.

Our research will focus on both the internal and external protein interactions of lysosomes, which are crucial for their role in maintaining cellular health. By integrating bioinformatics for structural biology, we will gain detailed insights into the 3D arrangements and interactions of lysosomal proteins. To verify the identified protein interactions and investigate their specific functions, we will use spatial proteomics, particularly fluorescence microscopy, to better understand their regulatory mechanisms. By exploring how these protein interactions change under varying nutrient levels, we aim to gain insights into how lysosomes help regulate the cell"s response to its environment. This interdisciplinary research will significantly enhance our understanding of lysosome functions, potentially leading to new treatments for diseases related to lysosomal malfunction and innovative strategies to maintain cellular health. Through this study, we aim to contribute foundational knowledge that will advance the field of cellular homeostasis and disease treatment.