Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma

Fractionation of MOPC 41 DL-I tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a beterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same tool wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section. THE JOURNAL OF CELL BmLOCV 9 VOLUME 67, 1975 . pages 835-851 835 Biological membranes present a diffusion barrier for macromolecules such as proteins, but transfer of a large number of specific proteins across membranes is an important physiological activity of virtually all cells. Segregation by a membrane is required not only for secretory proteins but also for lysosomal and peroxysomal proteins and for certain mitochondrial or chloroplast proteins synthesized in the cytoplasm. Transfer of proteins across membranes may even be required for some intramembrane proteins, e.g., if the site of insertion into the membrane were separated from the site of synthesis by the lipid bilayer. The discovery of an abundance of ribosome membrane junctions, particularly in secretory cells in the mid 50's (24, 25) and the demonstration in the mid 60's (1, 26, 27, 29) that nascent chains synthesized on membrane-bound ribosomes are vectorially discharged across the membrane, suggested that the ribosome membrane junction may function in the transfer of proteins across the membrane: by topologically linking the site of synthesis with the site of transfer, the protein would transverse the membrane only in status nascendi in an extended form before assuming its native structure, thus maintaining the membrane 's role as a diffusion barrier