As a first step towards isolation of autophagic sequestering membranes (phagophores), we have purified autophagosomes from rat hepatocytes. Lysosomes were selectively destroyed by osmotic rupture, achieved by incubation of hepatocyte homogenates with the cathepsin C substrate glycyl-phenylalanyl-naphthylamide (GPN). Mitochondria and peroxisomes were removed by Nycodenz gradient centrifugation, and cytosol, microsomes and other organelles by rate sedimentation through metrizamide cushions. The purified autophagosomes were bordered by dual or multiple concentric membranes, suggesting that autophagic sequestration might be performed either by single autophagic cisternae or by cisternal stacks.
