Visualization of lysosomal membrane proteins by cryo-electron tomography

Dysfunction in lysosomal membrane proteins and their associated complexes impairs the lysosome's essential role as a key signaling hub within the cell. This disruption underlies severe neurological disorders, such as lysosomal storage diseases and cancer. While direct visualization of the proteomic membrane environment of isolated lysosomes is crucial for advancing our understanding of its functions, it remains a significant challenge. To overcome this, we developed a method to enrich intact lysosomes using the essential lysosomal ion channel, transient receptor potential mucolipin 1. This enabled us to employ cryo electron tomography to reveal the heterogeneous molecular landscape of lysosomes. Notably, in concordance with quantitative mass spectrometry we identified protein densities on lysosomal membranes consistent with V-ATPase, Flotillin, clathrin-coated vesicles, mTORC1, HOPS, VPS13C, and dynein-dynactin. These findings demonstrate that our method offers a robust platform for advancing the structural and functional understanding of individual lysosomes, facilitating the visualization and resolution of endogenous protein complexes.