Interaction of secreted nascent chains with surrounding membrane in Bacillus subtilis

To determine the length of secreted nascent polypeptide chain that is surrounded by membrane, we digested labeled nascent chains protruding from protoplasts of Bacillus subtilis with Pronase and isolated the residual ribosome-attached chains from the membrane-polysome fraction. Gel chromatography revealed a sharp major peak that had been protected by membrane plus bound ribosomes. The ribosomes themselves protected half as great a length. Because no free chain between the ribosome and the membrane was detected by Pronase treatment, the difference between the two protected lengths should measure the length protected by the membrane. More accurate measurements of these lengths, obtained by dansylation of the exposed NH2 terminus of the isolated fragments, yielded a difference of 21 amino acids. This value corresponds to an extended chain of 75 A, which is approximately the thickness of the bacterial cell membrane. We earlier presented evidence that bacterial ribosomes are attached to membrane solely by their secreted chain. The present results further show that after loss of the extracellular segment of the chain its attachment persists, at 37 degrees as well as 0 degrees C. These findings suggest that the chain does not slip through a passive membrane but is actively held within a channel.