Lysosomes, central hydrolytic organelles, are regulated by ion flow, including calcium and protons, via transporters and channels to maintain an acidified lumen for hydrolytic activity. TRPML1, a lysosomal ion channel, effluxes cations upon activation, promoting rapid conjugation of ATG8 proteins to the lysosomal membrane in a process known as conjugation of ATG8 to single membranes (CASM). However, our understanding of how TRPML1 activation reorganizes the lysosomal proteome is poorly understood. Here, we identify DMXL1 as a key regulator of lysosomal homeostasis through quantitative proteomics of lysosomes during TRPML1 activation by the agonist MLSA5. DMXL1 is recruited to lysosomes and Salmonella-containing vacuoles, both in a CASM-dependent manner. As the mammalian ortholog of yeast Rav1, DMXL1 assembles with Rav2 ortholog ROGDI and WDR7, and associates with V0 and V1 subunits of the lysosomal V-ATPase. TRPML1 activation drives V1 subunit recruitment to lysosomes in a DMXL1- and DMXL2-dependent manner. DMXL1- and DMXL2-deficient cells display reduced V1-ATPase recruitment, increased lysosomal pH and diminished hydrolytic capacity. Using AlphaFold modeling supported by cross-linking proteomics, we identify interaction interfaces within the DMXL1-ROGDI-WDR7 complex, as well as an ATP6V1A binding interface in DMXL1, whose mutation affects interaction and function. Our findings suggest CASM-dependent DMXL1 recruitment, coupled with V-ATPase assembly, is critical for maintaining lumenal pH and lysosomal function in response to TRPML1 activation.
© 2025. The Author(s).