Article

The structure of ribosome-channel complexes engaged in protein translocation.

Department of Physiology and Biophysics Boston University School of Medicine, Boston, MA 02118, USA.
Molecular Cell (Impact Factor: 14.46). 12/2000; 6(5):1219-32. DOI: 10.1016/S1097-2765(00)00118-0
Source: PubMed

ABSTRACT Cotranslational translocation of proteins requires ribosome binding to the Sec61p channel at the endoplasmic reticulum (ER) membrane. We have used electron cryomicroscopy to determine the structures of ribosome-channel complexes in the absence or presence of translocating polypeptide chains. Surprisingly, the structures are similar and contain 3-4 connections between the ribosome and channel that leave a lateral opening into the cytosol. Therefore, the ribosome-channel junction may allow the direct transfer of polypeptides into the channel and provide a path for the egress of some nascent chains into the cytosol. Moreover, complexes solubilized from mammalian ER membranes contain an additional membrane protein that has a large, lumenal protrusion and is intercalated into the wall of the Sec61p channel. Thus, the native channel contains a component that is not essential for translocation.

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    ABSTRACT: mRNAs encoding cytosolic and signal sequence-bearing proteins are translated by free and endoplasmic reticulum (ER)-bound ribosomes, respectively. Recent ribosome footprinting studies have also demonstrated translation of cytosolic protein-encoding mRNAs on ER-bound ribosomes, findings that raise important questions regarding the mechanism of ribosome and mRNA localization and association with the ER. Using a semi- intact HeLa cell model, we performed a polysome solubilization screen and identified conditions that biochemically distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. RNA-protein UV photocrosslinking studies revealed numerous ER integral membrane proteins with RNA binding activity, consistent with direct RNA anchoring functions. Quantitative proteomic analyses of HeLa cytosolic and two classes of ER-bound polysomes identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins whereas whereas translocon accessory proteins such as ribophorin I were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocrosslinked protein-RNA adducts identified Sec61α , β and ribophorin I, suggesting roles for the protein translocation and modification machinery in mRNA anchoring to the ER. We propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable a transcriptome-wide function for the ER in cellular protein synthesis.
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    ABSTRACT: Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies.

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May 19, 2014