Previous reports have shown that rough microsomes treated with high salt (Warren and Dobberstein, 1978, Nature, 273:569-571) or proteases (Walter et al ., 1979, Proc. Nati. Acad. Sci. U. S. A., 76:1,795) are unable to vectorially translocate nascent proteins. Readdition of the high salt or protease extracts restored activity to such inactive rough microsomes. A detailed study was carried out to determine how this factor interacts with the rough microsomal membrane. Proteolytic cleavage was found to be necessary but not sufficient to remove this factor from the membrane. A subsequent treatment with high salt had to be carried out . Endogenous (pancreatic) protease could effect the required cleavage, but low levels of trypsin, clostripain, or elastase were far more efficient. Several proteases were not effective. The minimum level of salt (after proteolysis) required to solubilize the active factor was -200 mM KCI. Salt extracts prepared by treatment with one of the effective proteases were capable of restoring activity to inactive microsomes produced by treatment with one of the others .
When rough microsomes are subjected to limited proteolysis and high salt, a soluble fraction can be separated from the membrane . Neither fraction alone is capable of vectorially translocating nascent peptides . When the soluble extract is recombined with the residual membrane fraction, translocating activity is restored . Standard biochemical techniques were used to identify and characterize the active component derived by treating rough microsomes with elastase and high salt. The active factor is a peptide fragment with an apparent molecular weight of 60,000. It represents the cytoplasmic domain of a larger membrane protein. The fragment is basic and has at least one accessible sulfhydryl group. These characteristics facilitated its purification and identification as a membrane component required for translocation of nascent peptides across microsomal membranes.