Abstract The proper function of lysosomes depends on their ability to store and release calcium. While several lysosomal calcium release channels have been described, how mammalian lysosomes replenish their calcium stores has not been determined. Using genetic depletion and overexpression techniques combined with electrophysiology and visualization of subcellular ion concentrations and their fluxes across the lysosomal membrane, we show here that TMEM165 imports calcium to the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. Accordingly, TMEM165 accelerates the recovery of cells from cytosolic calcium overload thereby enhancing cell survival while causing a significant acidification of the perilysosomal area and the entire cytosol. These data indicate that in addition to its essential role in the glycosylation in the Golgi, a small but significant fraction of TMEM165 localizes on the lysosomal limiting membrane, where it protects cells against cytosolic calcium overload, preserves lysosomal function by refilling lysosomal calcium stores and regulates perilysosomal and lysosomal pH.

TMEM165 replenishes lysosomal calcium stores, protects cells against calcium overload, and mediates calcium-induced lysosomal H+ leakage