title: Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.
abstract: Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality.
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.|Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, 113-8654, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Takatori
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Tsuyako
authors.initials: T
authors.lastname: Tatematsu
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Jinglei
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Matsumoto
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Akano
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Toyoshi
authors.initials: T
authors.lastname: Fujimoto
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014617:Vacuoles / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism
chemical_list: D018129:Phosphatidylinositol Phosphates; C106336:phosphatidylinositol 3,5-diphosphate; C055525:phosphatidylinositol 3-phosphate
keywords: electron microscopy; endosome; freeze-fracture; hyperosmotic stress; lysosome; membrane domain; phosphatidylinositol 3,5-bisphosphate; vacuole