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Cell biologist studying protein targeting across membranes. With Bernhard Dobberstein, Blobel reconstructed aspects of cotranslational protein translocation, connecting information in a growing polypeptide with delivery to the secretory pathway.^[ref]

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]

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]

Protein cell biology studies how proteins function within biological cells: where they are delivered, which partners they encounter, and how these interactions affect cellular processes. A protein's protein primary structure and abundance do not by themselves specify its location or available partners. RNA-binding proteins, G proteins and protein targeting illustrate different aspects of that problem.

The microsomal protein import experiment makes location experimentally testable. In 1975, Günter Blobel and Bernhard Dobberstein combined cell-free translation components with dog-pancreas microsomes. An immunoglobulin light chain product made while suitable membranes were present became processed and resistant to external proteolysis, whereas globin did not enter the same protected compartment. Adding membranes only after synthesis did not reproduce the effect in this preparation.^[ref]

The important observation is controlled accessibility, not just recovery near a membrane. A protein can bind a vesicle's outside without crossing it; enclosure can protect it from an external protease. Protease protection assays therefore complement measurements of total product and membrane topology.

For an original two-compartment example, suppose a fixed total of100 competent cargo molecules is divided between20 enclosed and80 exposed molecules. Redistributing the same total to70 enclosed and30 exposed changes accessibility even though total abundance stays100. If every enclosed molecule is protected and every exposed molecule is digested in the stipulated model, the protected fraction changes from0.20 to0.70. Real measurements also need checks for membrane integrity, protease activity and product recovery; the fractions alone do not identify every transport mechanism. This is why cellular organization belongs alongside the isolated molecule's chemistry.

A signal peptide is a short targeting segment of a newly made polypeptide chain. A familiar ER signal sequence lies near the N-terminus and is removed during entry; Günter Blobel and Bernhard Dobberstein's 1975 experiments linked a larger immunoglobulin light chain precursor to a processed product.^[ref]

Cleavage is a separate question from recognition. Not every targeting signal is a cleaved signal peptide: an internal signal anchor can remain part of a transmembrane protein. Conversely, loss of a segment does not by itself prove membrane crossing. Compare the independent size and accessibility readouts in the microsomal protein import experiment.

By Günter Blobel, Bernhard Dobberstein. Journal of Cell Biology 67(3), 835–851 (1975). The University of California, Berkeley hosts this journal scan.

Inspected text: mcb.berkeley.edu/labs/krantz/pdf/blobel%26dobberstein-transfer_of_proteins_across_membranes_I-JCB-1975.pdf

DOI: doi.org/10.1083/jcb.67.3.835

Cited passages: Introduction p. 836; Methods pp. 837–838: cell-free translation, proteolysis, slab SDS-PAGE and dried-gel autoradiography; Figures 5,7,14; Discussion p. 847: size-resolved protection versus sedimentation.

By Günter Blobel, Bernhard Dobberstein. Journal of Cell Biology 67(3), 852–862 (1975). SciSpace hosts this journal scan.

Inspected text: scispace.com/pdf/transfer-of-proteins-across-membranes-ii-reconstitution-of-5pau33unje.pdf

DOI: doi.org/10.1083/jcb.67.3.852

Cited passages: Methods p. 853: dog-pancreas microsomes, centrifugation, stripped preparations; Figure 6 and text pp. 856–859: light chain, globin, plus/minus protease; Figure 7 and text pp. 860–861: reconstitution and heat-inactivation caveat; Figure 8 and text pp. 861–862: membranes after synthesis, failed detergent-solubilized processing.