The ER-associated ribosome caught in the act
In eukaryotic cells, co-translational protein translocation across the endoplasmic reticulum (ER) membrane requires an elaborate macromolecular machinery comprising the 80S ribosome and the translocon complex. Here, we used cryoelectron tomography and subtomogram averaging to provide a three-dimensional reconstruction of ER membrane associated 80S ribosomes and a description of their spatial organization in situ.
HFSP Career Development Award holder Friedrich Förster and colleaguesauthored on Thu, 08 November 2012
The 80S ribosome, a 3.6 MDa riboprotein-complex in mammals, translates messenger RNA (mRNA) to polypeptides. In mammalian cells, ribosomes that synthesize proteins of the secretory pathway are typically associated with the rough endoplasmic reticulum (ER). Most nascent chains are co-translationally translocated across the ER membrane or are integrated into it through the hetero-trimeric Sec61-translocation channel which forms the translocon complex together with more than 20 additional membrane proteins. Due to its complexity and the involvement of membrane proteins the structure of the ribosome-translocon complex remained elusive.
Figure: Structure and 3D arrangement of ER membrane associated ribosomes. (A) The subtomogram-average of the ER membrane associated ribosome includes the 60S (blue) and the 40S (yellow) ribosomal subunit, the ER membrane (gray), and ER-lumenal electron densities (red). While one lumenal density was identified as the TRAP complex, the second lumenal density awaits unambiguous identification. The ER membrane was cut for better visibility of the ER-lumenal densities. (B) Preferred 3D arrangement of membrane-bound ribosomes visualized by replicates of the tomographic map. A putative mRNA molecule was fitted to mRNA entry and exit site and the codon recognition site of adjacent ribosomes. Small ribosomal subunits are depicted transparently for visualization of the mRNA pathway.
We have used cryoelectron tomography (CET) to resolve the endogenous ER membrane associated 80S ribosome and the protein translocation machinery of dog pancreatic ER-derived microsomes in situ. In contrast to other structural biology techniques, such as X-ray crystallography or single particle cryo-EM analysis, CET does not require solubilization and purification of complexes. Averaging of approximately 1,000 subtomograms that were extracted in silico from the tomograms yielded a 31 Å resolution reconstruction of the ribosome-translocon complex in the native ER membrane. In the tomographic map we observe unexpected ribosome-membrane interaction sites, most strikingly a direct contact of ribosomal expansion segment ES27L (an insertion of ribosomal RNA in eukaryotic ribosomes compared to their prokaryotic counterparts) with the ER membrane. We speculate that this interaction is responsible for eukaryotic ribosomes remaining associated to the membrane after translation termination.
Furthermore, we were able to resolve membrane-embedded and lumenal complexes of the protein translocation machinery, most prominently a complex protruding 9 nm into the microsomal lumen. Analysis of the three-dimensional distribution of ribosomes on the ER membrane reveals that membrane-associated ribosomes adopt a preferred arrangement that is likely specific for ER-associated polyribosomes. Non-translating ribosomes would retain this near-range order due to their low mobility on the crowded ER membrane, which may explain the high translation efficiency of ER membrane associated ribosomes compared to their cytosolic counterparts.
Structure and 3D Arrangement of Endoplasmic Reticulum Membrane-Associated Ribosomes. Stefan Pfeffer, Florian Brandt, Thomas Hrabe, Sven Lang, Matthias Eibauer, Richard Zimmermann and Friedrich Förster. Structure 20, 1508-18 (2012). DOI: 10.1016/j.str.2012.06.010