Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0° by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0°, although they are totally digested at 37° or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0°. A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of ∼39 amino acid residues, as estimated by chromatography on Sephadex G-50.
CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS : I. Location of the Polypeptides within Ribosomes
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G. Blobel, D. D. Sabatini; CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS : I. Location of the Polypeptides within Ribosomes . J Cell Biol 1 April 1970; 45 (1): 130–145. doi: https://doi.org/10.1083/jcb.45.1.130
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