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.
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References
Seglen PO, Bohley P (1992) Autophagy and other vacuolar protein degradation mechanisms. Experientia 48:158–172
Klionsky DJ, Cuervo AM, Seglen PO (2007) Methods for monitoring autophagy from yeast to human. Autophagy 3:181–206
Mizushima N (2007) Autophagy: process and function. Genes Dev 21:2861–2873
Suzuki K, Ohsumi Y (2007) Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae. FEBS Lett 581:2156–2161
Bassham DC, Laporte M, Marty F et al (2006) Autophagy in development and stress responses of plants. Autophagy 2:2–11
Marty F (1997) The biogenesis of vacuoles: insights from microscopy. Adv Bot Res 25:1–42
Rose TL, Bonneau L, Der C et al (2006) Starvation-induced expression of autophagy-related genes in Arabidopsis. Biol Cell 98:53–67
Takatsuka C, Inoue Y, Higuchi T et al (2011) Autophagy in tobacco BY-2 cells cultured under sucrose starvation conditions: isolation of the autolysosome and its characterization. Plant Cell Physiol 52:2074–2087
Yano K, Yanagisawa T, Mukae K et al (2015) 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. Plant Signal Behav 10 (11): e1086859
Moriyasu Y, Ohsumi Y (1996) Autophagy in tobacco suspension-cultured cells in response to sucrose starvation. Plant Physiol 111:1233–1241
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Moriyasu Y, Inoue Y (2008) Use of protease inhibitors for detecting autophagy in plants. Method Enzymol 451:557–580
Acknowledgements
Research reported in this publication was supported by the Institute for Space and Aeronautical Sciences [“Grant for Basic Biology Study oriented to utilization of Space station” to Y.M.]; Japan Space Forum [“Ground-based Research Announcement for Space Utilization” to Y.M.]; KAKENHI [grant number 23120504, 23570222, and 25120704 to Y.M.].
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Takatsuka, C., Inoue-Aono, Y., Moriyasu, Y. (2017). Isolation of Autolysosomes from Tobacco BY-2 Cells. In: Taylor, N., Millar, A. (eds) Isolation of Plant Organelles and Structures. Methods in Molecular Biology, vol 1511. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6533-5_12
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DOI: https://doi.org/10.1007/978-1-4939-6533-5_12
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6533-5
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