title: 3-methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions.
abstract: Tobacco (Nicotiana tabacum) culture cells perform autophagy and degrade cellular proteins in response to sucrose starvation. When protein degradation is blocked by the cysteine protease inhibitor E-64c, lysosomes containing particles of cytoplasm (autolysosomes) accumulate in the cells. Therefore, using light microscopy, we can determine whether cells have performed autophagy. In this study, we investigated whether or not 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, blocks autophagy in tobacco culture cells. The accumulation of autolysosomes was blocked by the addition to the culture media of 5 mM 3-MA together with E-64c. We did not detect autolysosomes or structures thought to be involved with autophagy, such as autophagosomes, accumulating in these cells, as observed by electron microscopy. 3-MA blocked cellular protein degradation without any effect on cellular protease activity. In mammalian cells, phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a putative target of 3-MA. The PtdIns 3-kinase inhibitors wortmannin and LY294002 also inhibited the accumulation of autolysosomes in tobacco culture cells. These results suggest that (1) 3-MA inhibits autophagy by blocking the formation of autophagosomes in tobacco culture cells, and (2) PtdIns 3-kinase is essential for autophagy in tobacco cells.
authors.affiliation: School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526 Japan RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Yokohama, 230-0045 Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Takatsuka
authors.forename: Yuko
authors.initials: Y
authors.lastname: Inoue
authors.forename: Ken
authors.initials: K
authors.lastname: Matsuoka
authors.forename: Yuji
authors.initials: Y
authors.lastname: Moriyasu
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D008854:Microscopy, Electron; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010940:Plant Proteins / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology*; D014026:Nicotiana / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D014617:Vacuoles / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010940:Plant Proteins; C025946:3-methyladenine; D013395:Sucrose; D010450:Endopeptidases; D000225:Adenine; D000077191:Wortmannin