= Microsomal protein import experiment
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.<Transfer of Proteins across Membranes II by Günter Blobel and Bernhard Dobberstein>[Transfer of proteins across membranes. II, Methods p. 853; Figure 6 and text pp. 856–859; Figure 7 pp. 860–861; Figure 8 and text pp. 861–862]{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.<Transfer of Proteins across Membranes I by Günter Blobel and Bernhard Dobberstein>[Methods pp. 837–838: proteolysis, SDS-PAGE and autoradiography; Discussion p. 847]{ref}
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.<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]{ref}
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