title: Isolation of Autolysosomes from Tobacco BY-2 Cells.
abstract: Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H+-ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.
authors.affiliation: Tokai University Junior College, Shizuoka, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Takatsuka
authors.affiliation: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama-shi, Saitama, 338-8570, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Inoue-Aono
authors.affiliation: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama-shi, Saitama, 338-8570, Japan. moriyasu@mail.saitama-u.ac.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Moriyasu
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification; D001343:Autophagy; D015415:Biomarkers / Q000737:chemistry; D018929:Cell Culture Techniques; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008247:Lysosomes / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification; D020675:Peroxisomes / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D059828:Plant Cells / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D059748:Proteolysis; D013395:Sucrose / Q000737:chemistry; D014026:Nicotiana / Q000737:chemistry / Q000166:cytology; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification; D014617:Vacuoles / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure
chemical_list: D015415:Biomarkers; D003470:Culture Media; D015853:Cysteine Proteinase Inhibitors; D010940:Plant Proteins; D013395:Sucrose; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; D000135:Acid Phosphatase; D010447:Peptide Hydrolases; D025262:Vacuolar Proton-Translocating ATPases; D007930:Leucine
keywords: Acid phosphatase; Autolysosome; Autophagy; Cysteine protease inhibitor; H+-ATPase; Protease; Protein degradation; Sucrose starvation; Vacuole