ABSTRACT
FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), which is essential for phosphatidylinositol 3,5-bisphosphate [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 cells. Enlargement of vacuoles upon 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 NH4+, which formed through protonation of NH3 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 shows that PIKFYVE inhibition disrupts lysosomal homeostasis via ammonium accumulation.
Footnotes
Author contributions
Conceptualization: J.U.; Methodology: J.U., T.K.; Formal analysis: J.U., H.N.; Investigation: J.U.; Resources: T.Y.; Writing - original draft: J.U.; Writing - review & editing: H.N., T.Y., I.M., T.M.; Project administration: J.U.; Funding acquisition: J.U., T.M.
Funding
This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number 22K06637) and granted by Akiyama Life Science Foundation and The Hokkoku Cancer Foundation.
Data availability
All relevant data can be found within the article and its supplementary information.
