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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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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