title: Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.
abstract: Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.
authors.affiliation: a Graduate School of Food and Nutritional Sciences; University of Shizuoka ; Shizuoka , Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Yano
authors.affiliation: b Department of Regulatory Biology ; Graduate School of Science and Engineering; Saitama University ; Saitama , Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yanagisawa
authors.affiliation: b Department of Regulatory Biology ; Graduate School of Science and Engineering; Saitama University ; Saitama , Japan.
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Mukae
authors.affiliation: a Graduate School of Food and Nutritional Sciences; University of Shizuoka ; Shizuoka , Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Niwa
authors.affiliation: b Department of Regulatory Biology ; Graduate School of Science and Engineering; Saitama University ; Saitama , Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Inoue
authors.affiliation: b Department of Regulatory Biology ; Graduate School of Science and Engineering; Saitama University ; Saitama , Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Moriyasu
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000648:ultrastructure; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology*; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology*; D014026:Nicotiana / Q000166:cytology*; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D018942:Macrolides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C025946:3-methyladenine; D013395:Sucrose; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; C036978:concanamycin A; D025262:Vacuolar Proton-Translocating ATPases; D007930:Leucine; D000225:Adenine
keywords: E-64c; autolysosome/autophagosome; autophagy; concanamycin; tobacco BY-2; vacuole