biostudies-literatureLee CNINDS NIH HHSNIGMS NIH HHS2060-2075https://www.ebi.ac.uk/biostudies/studies/S-EPMC12246789biostudies-literatureUnknown32(10)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 RNature structural & molecular biologyDMXL1 promotes recruitment of V1-ATPase to lysosomes upon TRPML1 activation.PMC12246789R01 GM132129R01 NS083524R01 NS110395Harper JWLee CFu TMoran MMBresnahan TPaulo JAHelaine SGonzalez-Lozano MANiday ZEldridge MJGDel Camino DfalseDMXL1 promotes recruitment of V1-ATPase to lysosomes upon TRPML1 activation.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 R2025-01-01T00:00:00Z2025 Oct2026-06-11T05:06:01.119Z2026-06-11T03:08:24.35ZS-EPMC122467894052798810.1038/s41594-025-01581-x