title: Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique.
abstract: Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.
authors.affiliation: Department of Anatomy and Molecular Cell Biology; Nagoya University Graduate School of Medicine; Nagoya, Japan.
authors.forename: Toyoshi
authors.initials: T
authors.lastname: Fujimoto
authors.affiliation: Frontier Research Center; Tokyo Institute of Technology; Yokohama, Japan.
authors.forename: Hayashi
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: Frontier Research Center; Tokyo Institute of Technology; Yokohama, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohsumi
mesh_terms: D000818:Animals; D001343:Autophagy; D005614:Freeze Fracturing; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D008322:Mammals; D008854:Microscopy, Electron / Q000379:methods*; D010588:Phagosomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013194:Staining and Labeling / Q000379:methods; D013237:Stereoisomerism
chemical_list: D018129:Phosphatidylinositol Phosphates; C055525:phosphatidylinositol 3-phosphate
keywords: autophagic body; autophagosome; electron microscopy; freeze-fracture; phosphatidylinositol 3-kinase; phosphatidylinositol 3-phosphate; phosphatidylinositol 3-phosphate phosphatase; quick-freezing