PIKFYVE inhibition induces endosome- and lysosome-derived vacuole enlargement via ammonium accumulation

Abstract FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), that is essential for PtdIns(3,5)P 2 production, is an important regulator of lysosomal homeostasis. PIKFYVE dysfunction leads to cytoplasmic vacuolization; however, the underlying mechanism remains unknown. In this study, we explored the cause of vacuole enlargement upon PIKFYVE inhibition in DU145 prostate cancer cells. Enlargement of vacuoles by PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Addition of ammonia, a metabolite of glutamine, was sufficient to enlarge vacuoles via PIKFYVE inhibition. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome–lysosome permeabilization resulted in ammonium leakage from the cells, indicating ammonium accumulation in the endosomes and lysosomes. Ammonium accumulation and vacuole expansion were suppressed by the lysosomal lumen neutralization. It is therefore assumed that PIKFYVE inhibition interferes with the efflux of NH 4 + , which is protonated NH 3 in the lysosomal lumen, leading to osmotic swelling of vacuoles. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagic flux. In conclusion, this study showed that PIKfyve inhibition disrupts lysosomal homeostasis via ammonium accumulation. Summary statement Inhibition of the phosphoinositide kinase PIKFYVE results in endosome/lysosome enlargement and impaired lysosomal function. This study showed that the accumulation of glutamine-derived ammonium is the cause of these events.