A selective PIKfyve inhibitor blocks PtdIns(3,5)P 2 production and disrupts endomembrane transport and retroviral budding

Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small‐molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5‐bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug‐resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5‐bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux.