Lysosomes are essential degradative and metabolic organelles that maintain cellular homeostasis through macromolecular turnover, nutrient sensing, and signaling. Lysosomal dysfunction underlies numerous human diseases, including lysosomal storage disorders, neurodegenerative diseases, and cancers. However, how lipid metabolism regulates lysosomal homeostasis remains poorly understood.

Here, we performed a genome-wide CRISPR screen in Mucolipidosis V (LYSET-deficient) cells and identified ether lipid synthesis as a key regulator of lysosomal homeostasis. Genetic or pharmacological inhibition of ether lipid synthesis reduced lysosome accumulation and partially restored lysosomal degradative capacity. Mechanistically, ether lipid reduction partially restored the maturation of a subset of lysosomal hydrolases and enhanced lysosomal exocytosis, thereby promoting lysosomal substrate clearance. These findings uncover a previously unrecognized mechanism by which lipid metabolism regulates lysosomal homeostasis and suggest new therapeutic opportunities for lysosomal storage disorders.