Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.
Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global analysis of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the large majority of secretory proteins to be cotranslationally translocated, including substrates capable of posttranslational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately after or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol after translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient cotranslational recruitment.
These authors contributed equally to this work.
Calvin H
CH
Jan
These authors contributed equally to this work.
Christopher C
CC
Williams
Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, California Institute for Quantitative Biosciences, Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. These authors contributed equally to this work. jonathan.weissman@ucsf.edu.
Jonathan S
JS
Weissman
D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism; D002477:Cells / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C084842:SEC66 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D010749:Phosphoprotein Phosphatases; C484329:SSH1 protein, human; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli