Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)-phthalate treatment. III. Fusion of early autophagosomes with lysosomal compartments.

Fusion of early autophagosomes containing peroxisomes with endosomal and lysosomal structures in rat liver cells was investigated. Male Wistar rats were administered di-(2-ethylhexyl)phthalate (DEHP) for 14 days to induce proliferation of peroxisomes, and then the animals were injected intravenously with horseradish peroxidase (HRP)-conjugated asialofetuin to label the lysosomal compartment. Either 30 min or 60 min after the injection, the animals were treated with leupeptin for 20, 40 and 60 min, respectively. Most of autophagic vacuoles containing peroxisomes were stained with diaminobenzidine (DAB) endocytosed HRP-asialofetuin 60 min after leupeptin injection, whereas many of them were negative for DAB reaction 20 min after leupeptin injection. Between 20 to 40 min after leupeptin treatment many autophagic vacuoles fused with DAB-positive lysosomal compartments, including late endosomes. Percoll gradient centrifugation showed that particles containing HRP activity migrated from a density of 1.09 g/ml to that of 1.14 g/ml as the time after leupeptin injection passed. Acid phosphatase activity migrated in the same manner. These results clearly show that early autophagosomes obtain the lysosomal proteinases by fusion with lysosomal compartments, including the late endosomes and that peroxisomes trapped in autophagosomes are degraded by these proteinases.