Absence of cytochrome P-450 and presence of autolysosomal membrane antigens on the isolation membranes and autophagosomal membranes in rat hepatocytes

We wished to determine if phenobarbital (PB)-inducible cytochrome P-450 P-450(PB)] and autolysosomal membrane antigens could be localized immunocytochemically on the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by the post-embedding protein A-gold method. P-450(PB) was maximally induced by PB treatment; then formation of autophagosomes and accumulation of autolysosomes were induced by cessation of PB treatment and by injection of leupeptin, respectively. P-450(PB) was detected neither on the isolation membranes nor on the limiting membranes of autophagosomes and autolysosomes. Autolysosomal membrane antigens, which were localized by the immunogold technique exclusively in post-Golgi compartments such as lysosomes, endosomes, and plasma membrane but were not found in pre-Golgi compartments such as endoplasmic reticulum (ER) and nuclear envelope, were detected in large amounts on the isolation membranes. These results suggest that the isolation membranes originate not from ER membranes but from post-Golgi membranes. We also present direct immunoelectron microscopic evidence that P-450(PB) is indeed degraded in the autolysosomes: when rats were treated with leupeptin, P-450(PB) was detected not only within the autophagosomes but also within the autolysosomes, whereas without leupeptin treatment, P-450(PB) was detectable only within the autophagosomes.