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

Cell Struct Funct. 1999 Apr;24(2):59-70. doi: 10.1247/csf.24.59.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Bacterial Agents / pharmacology
  • Antibodies
  • Autophagy / drug effects
  • Autophagy / physiology*
  • Biomarkers / analysis
  • Blotting, Western
  • Carbonates / pharmacology
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cell Membrane / ultrastructure
  • Culture Media
  • Endosomes / drug effects
  • Endosomes / metabolism
  • Endosomes / ultrastructure
  • Fluorescent Antibody Technique
  • Intracellular Membranes / drug effects
  • Intracellular Membranes / metabolism
  • Intracellular Membranes / ultrastructure
  • Isoelectric Point
  • Liver / cytology
  • Liver / metabolism
  • Liver Neoplasms, Experimental
  • Lysosomes / drug effects
  • Lysosomes / metabolism*
  • Lysosomes / ultrastructure
  • Macrolides*
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism*
  • Microscopy, Immunoelectron
  • Molecular Weight
  • Phagosomes / drug effects
  • Phagosomes / metabolism*
  • Phagosomes / ultrastructure
  • Rats
  • Solubility / drug effects
  • Tumor Cells, Cultured

Substances

  • Anti-Bacterial Agents
  • Antibodies
  • Biomarkers
  • Carbonates
  • Culture Media
  • Macrolides
  • Membrane Proteins
  • sodium carbonate
  • bafilomycin A1