Uptake and degradation of proteins by isolated rat liver lysosomes. Suggestion of a microautophagic pathway of proteolysis.
The aim of this work was to study possible uptake of electron-dense marker particles and uptake and degradation of proteins in vitro by two types of lysosomes, namely, secondary lysosomes and iron-laden "residual" bodies. The sedimentability and latency of lysosomal enzymes were measured to evaluate whether or not the different lysosomes were suitable for in vitro incubation experiments. Residual bodies displayed leakage and enzyme inactivation with increasing incubation times, whereas secondary lysosomes were stable. The two types of lysosomes were incubated in vitro with isotopically labeled methemoglobin or insulin. Secondary lysosomes, but not iron-laden residual bodies, were active in the degradation of the exogenously added proteins. Control experiments were performed to evaluate possible extra-lysosomal proteolysis of the substrates due to leakage of lysosomal enzymes during incubation. The enzymes released during incubation could only account for a minor portion of the degradation of the added labeled proteins. Secondary lysosomes were preincubated with radiolabeled protein, followed by washing and reincubation. The radioactivity in the lysosomal pellet decreased during reincubation concomitant with an increase in trichloroacetic acid-soluble label. Chloroquine decreased the release of degradation products from the lysosomes. Further support for the interpretation that lysosomes take up proteins was gained from ultrastructural observations. Secondary lysosomes were incubated with electron-dense Percoll particles in vitro. Morphologic analysis revealed Percoll particles inside intralysosomal vesicles which appeared to be formed during incubation. The results indicate that lysosomes are able to internalize their membranes during in vitro incubation followed by degradation of the micropinocytosed proteins.
D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D001343:Autophagy; D002402:Cathepsin D; D002403:Cathepsins / Q000032:analysis; D002458:Cell Fractionation; D002738:Chloroquine / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008705:Methemalbumin / Q000494:pharmacology; D010587:Phagocytosis; D010873:Pinocytosis; D011506:Proteins / Q000378:metabolism*; D051381:Rats
D007328:Insulin; D008705:Methemalbumin; D011506:Proteins; D002738:Chloroquine; D000135:Acid Phosphatase; D002403:Cathepsins; D002402:Cathepsin D