ARTICLE

CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS : II. Location of the Polypeptides in Rough Microsomes

D. D. Sabatini ¹ and G. Blobel ¹

¹ From The Rockefeller University, New York 10021

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

• Hebert, D. N., Molinari, M. (2007). In and Out of the ER: Protein Folding, Quality Control, Degradation, and Related Human Diseases. Physiol. Rev. 87: 1377-1408 [Abstract] [Full Text]  
• Shim, J.-H., Xiao, C., Hayden, M. S., Lee, K.-Y., Trombetta, E. S., Pypaert, M., Nara, A., Yoshimori, T., Wilm, B., Erdjument-Bromage, H., Tempst, P., Hogan, B. L.M., Mellman, I., Ghosh, S. (2006). CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis.. JCB 172: 1045-1056 [Abstract] [Full Text]  
• Wickner, W., Schekman, R. (2005). Protein Translocation Across Biological Membranes. Science 310: 1452-1456 [Abstract] [Full Text]  
• Wickner, W., Lodish, H. (1985). Multiple mechanisms of protein insertion into and across membranes. Science 230: 400-407 [Abstract]  
• Wickner, W (1980). Assembly of proteins into membranes. Science 210: 861-868 [Abstract]  
• Rothman, J., Lenard, J (1977). Membrane asymmetry. Science 195: 743-753 [Abstract]  
• Palade, G (1975). Intracellular aspects of the process of protein synthesis. Science 189: 347-358  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents