A 60 kDa Plasma Membrane Protein Changes its Localization to Autophagosome and Autolysosome Membranes during Induction of Autophagy in Rat Hepatoma Cell Line, H-4-II-E Cells.

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  • Tagawa Yoshihiro
    Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
  • Yamamoto Akitsugu
    Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan Liver Research Center (Cell Biology), Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
  • Yoshimori Tamotsu
    Department of Cell Biology, National Institute for Basic Biology, Okawaki 444-8585, Japan
  • Masaki Ryuichi
    Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan Liver Research Center (Cell Biology), Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
  • Omori Koichiro
    Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan Liver Research Center (Cell Biology), Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
  • Himeno Masaru
    Division of Physiological Chemistry, Faculty of Pharmaceutical Science, Kyushu University, Fukuoka 812-8582, Japan
  • Inoue Kyoichi
    Internal Medicine, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
  • Tashiro Yutaka
    Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan

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  • 60 kDa Plasma Membrane Protein Changes its Localization to Autophagosome and Autolysosome Membranes during Induction of Autophagy in Rat Hepatoma Cell Line H-4-II-E Cells

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We previously reported the preparation and characterization of an antibody against membrane fraction of autolysosomes from rat liver (J. Histochem. Cytochem. 38, 1571-1581, 1990). Immunoblot analyses of total membrane fraction of a rat hepatoma cell line, H-4-II-E cells by this antibody suggested that H-4-II-E cells expressed several autolysosomal proteins, including a protein with apparent molecular weight of 60 kDa. It was suggested that this 60 kDa protein was a peripheral membrane protein, because it was eluted from the membrane by sodium carbonate treatment. We prepared an antibody against this 60 kDa protein by affinity purification method, and examined its behavior during induction of autophagy. Autophagy was induced by transferring the cells from Dulbecco's modified Eagle medium(DMEM) containing 12% fetal calf serum into Hanks' balance salt solution. In DMEM, the 60 kDa protein showed diffused immunofluorescence pattern, and immunoelectron microscopy suggested that this protein was located on the extracellular side of the plasma membrane. After inducing autophagy, the immunofluorescence configuration of the 60 kDa protein changed from the diffused pattern to a granulous one. Immunoelectron microscopy showed that the 60 kDa protein was localized on the luminal side of the limiting membrane of autolysosomes and endosomes. in the presence of bafilomycin A1 which prevents fusion between autophagosomes and lysosomes, the 60 kDa protein was localized on the limiting membrane of the autophagosomes and endosomes. These results suggest that the 60 kDa protein is transported from the plasma membrane to the autophagosome membrane through the endosomes.

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