PURIFICATION AND CHARACTERIZATION OF AUTOPHAGOSOMES FROM RAT HEPATOCYTES

To investigate the properties and intracellular origin of autophagosom es, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubate d with vinblastine to induce autophagosome accumulation; the cells wer e then homogenized and treated with the cathepsin C substrate glycyl-L -phenylalanine 2-naphthylamide to cause osmotic disruption of the lyso somes. Nuclei were removed by differential centrifugation, and the pos tnuclear supernatant was fractionated on a discontinuous Nycodenz dens ity gradient. The autophagosomes, recognized by their content of autop hagocytosed lactate dehydrogenase (LDH), could be recovered in an inte rmediate-density fraction, free from cytosol and mitochondria. Finally , the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal de nsity gradient, followed by flotation in iodixanol to remove the Perco ll particles. The final autophagosome preparation represented a 24-fol d purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cyto plasm with a normal ultrastructure, including mitochondria, peroxisome s and endoplasmic reticulum in the same proportions as in intact cells . However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein mar kers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal gl ycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1 , cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protei n of 78 kDa, protein disulphide isomerase), suggesting that the seques tering membranes are not derived directly from any of these organelles , but rather represent unique organelles (phagophores).