In 1975, Günter Blobel and Bernhard Dobberstein combined cell-free translation with dog-pancreas microsomes. Density-gradient centrifugation isolated the membranes; removal of resident ribosomes allowed reconstitution with rabbit-reticulocyte ribosome subunits. Added messenger RNA encoded an immunoglobulin light chain; globin provided a comparison. Products made with or without membranes were challenged with proteases and analyzed by SDS-PAGE and autoradiography. Membranes present during synthesis yielded processed, protected light chain; globin remained susceptible. Adding membranes after precursor synthesis failed to reproduce import. These comparisons support selective passage during synthesis in this preparation.[ref]
The companion paper specifies radioactive amino acid incorporation, ice-cold trypsin plus chymotrypsin treatment, a slab-gel electrophoresis apparatus and dried-gel exposure to X-ray film. Product-size analysis distinguishes a preserved full-size product from residual labeled fragments.[ref]
Transfer of Proteins across Membranes I by Günter Blobel and Bernhard Dobberstein. Methods pp. 837–838: proteolysis, SDS-PAGE and autoradiography; Discussion p. 847
Protection was incomplete. The authors considered leaky vesicles, and warned that heat treatment could damage processing as well as ribosome attachment. Their experiments did not identify SRP or the Sec61 complex. Nor does failure of late membrane addition establish that all organelle protein import must occur during synthesis.[ref]
Transfer of Proteins across Membranes II by Günter Blobel and Bernhard Dobberstein. pp. 858–859: incomplete protection; p. 861: heat-inactivation caveat; p. 862: alternatives for failed processing
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