A novel imaging method revealed phosphatidylinositol 3,5-bisphosphate-rich domains in the endosome/lysosome membrane

We developed a new method to observe distribution of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P 2 ] using electron microscopy. In freeze-fracture replicas of quick-frozen samples, PtdIns(3,5)P 2 was labeled specifically using recombinant ATG18 tagged with glutathione S-transferase and 4×FLAG, which was mixed with an excess of recombinant PX domain to suppress binding of ATG18 to phosphatidylinositol 3-phosphate. Using this method, PtdIns(3,5)P 2 was found to be enriched in limited domains in the yeast vacuole and mammalian endosomes. In the yeast vacuole exposed to hyperosmolar stress, PtdIns(3,5)P 2 was distributed at a significantly higher density in the intramembrane particle (IMP)-deficient liquid-ordered domains than in the surrounding IMP-rich domains. In mammalian cells, PtdIns(3,5)P 2 was observed in endosomes of tubulo-vesicular morphology labeled for RAB5 or RAB7. Notably, distribution density of PtdIns(3,5)P 2 in the endosome was significantly higher in the vesicular portion than in the tubular portion. The nano-scale distribution of PtdIns(3,5)P 2 revealed in the present study is important to understand its functional roles in the vacuole and endosomes.