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

FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), that is essential for PtdIns(3,5)P2 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 cell lines. The observed vacuolization was consistent with the localization of endosome and lysosome markers, indicating that the vacuoles were enlarged endosomes and lysosomes. Enlargement of vacuoles by PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Here, addition of ammonia resulted in vacuole enlargement even in the absence of glutamine, suggesting that ammonia generated by glutamine metabolism promotes vacuole enlargement. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome–lysosome permeabilization resulted in ammonia leakage from the cells, indicating ammonia 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 NH4+, which is protonated NH3 formed after entering into the lumen, leading to vacuole enlargement due to osmotic swelling. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagy flux. Over all, this study showed that vacuole enlargement caused by PIKFYVE inhibition was due to the ammonium accumulation in endosomes and lysosomes. Our findings indicate the involvement of PIKFYVE in glutamine–ammonia metabolism and the possibility of pharmacological intervention in glutamine–ammonia metabolism by sequestering ammonia in endosomes and lysosomes. ### Competing Interest Statement The authors have declared no competing interest.