Lysosomes are acidic intracellular vesicles containing hydrolases that degrade intracellular and extracellular debris delivered through endocytic trafficking and autophagy.Lysosome function requires the establishment of luminal ionic homeostasis for ions including H+ and Ca2+, which are 1,000-5,000 times more concentrated in the lysosome lumen than in the cytosol.Lysosomal H+ homeostasis is required to activate hydrolases and Ca2+ efflux through lysosomal ion channels serves as signals required for precise delivery of hydrolases and cargo and the timely removal of catabolites. Impaired lysosomal Ca2+ homeostasis results in lysosomal dysfunction, lysosomal storage diseases (LSDs), and has been implicated more broadly in neurodegenerative phenotypes.The molecular mechanisms by which lysosomes acquire and refill Ca2+ are unknown.We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channel TRPML1 to repeatedly induce lysosomal Ca2+ release.In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling.Instead, pharmacological and genetic depletion or chelation of endoplasmic reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling.More specifically, antagonists of ER IP3 receptors rapidly and completely blocked Ca2+ refilling to lysosomes.Reducing ER Ca2+ or blocking IP3 receptors resulted in a dramatic lysosome storage phenotype.By closely apposing each other, the ER may serve as a direct and primary source of Ca2+ to the lysosome.These findings may clarify seemingly overlapping ER and lysosome Ca2+ stores in some studies and shed light on why ER Ca2+ homeostasis is often involved in LSDs and neurodegenerative diseases.
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