Mechanism and regulation of lysosomal sequestration and proteolysis.

Abstract

Lysosomes and autolysosomes were isolated from the livers of dextran-treated and leupeptin-injected rats, respectively. The contents of sequestered cytoplasmic proteins were much higher in autolysosomes, and more than 50% of them were found in the sediment. Isolated dextran-lysosomes showed high proteolytic activity toward sequestered cytoplasmic enzymes following incubation at pH 5. E-64 caused a complete inhibition of their degradation. Leupeptin treatment caused enrichment of ubiquitin protein conjugates of high molecular mass in the sediment of autolysosomes, which is comparable to enrichment of carbamyl-phosphate synthetase as a marker of bulk cytoplasmic sequestration in autolysosomes. The results suggest that protein ubiquitination may not be a signal for sequestration of proteins into the autophagic pathway.