Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts.
We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.
D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008845:Microinjections; D010446:Peptide Fragments / Q000378:metabolism*; D011887:Raffinose / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*
D010446:Peptide Fragments; C023560:ribonuclease S-peptide; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D011887:Raffinose