Covalent linkage of ribonuclease S-peptide to microinjected proteins causes their intracellular degradation to be enhanced during serum withdrawal.
The amino-terminal 20 amino acids are required for microinjected ribonuclease A (RNase A) to be taken up by lysosomes and degraded at an enhanced rate during serum withdrawal. We used water-soluble carbodiimides to covalently attach the RNase S-peptide (residues 1-20) to [3H]RNase S-protein (residues 21-124) at unspecified locations. We then measured catabolism of the [3H]S-protein-S-peptide conjugate after its microinjection into human diploid fibroblasts. The attached S-peptide caused the degradation of S-protein to be enhanced 2-fold in the absence of serum. Control experiments showed that degradation of [3H]RNase S-protein remained unresponsive to serum after conjugation with the inactive fragment, RNase S-peptide (residues 1-10). Covalent attachment of RNase S-peptide had a similar effect on the catabolism of two other proteins. Degradation rates of microinjected 125I-labeled lysozyme and 125I-labeled insulin A chain are normally unresponsive to serum withdrawal. However, breakdown rates of microinjected 125I-labeled lysozyme-S-peptide and 125I-labeled insulin A chain-S-peptide conjugates were increased 2-fold during serum deprivation. We suggest that RNase S-peptide acts as a "single sequence" that directs cytosolic proteins to lysosomes through a pathway that is activated by deprivation conditions.
D002460:Cell Line; D003470:Culture Media; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008845:Microinjections; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000008:administration & dosage / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*
D003470:Culture Media; D007328:Insulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011506:Proteins; C008147:insulin, iodo-; C023560:ribonuclease S-peptide; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic