Birgitta Beatrix

Cryo-EM structure of the ribosome-SecYE complex in the membrane environment.

The ubiquitous SecY–Sec61 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor–gated PCC in the membrane.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

Structures of the Sec61 Complex Engaged in Nascent Peptide Translocation or Membrane Insertion.

The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for co-translational protein transport. The Sec complex consists of three subunits (Sec61α, β and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains. A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides. Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes. However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel.

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.

Coordination of a histidine residue of the protein-component S to the cobalt atom in coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

Electron paramagnetic resonance (EPR) spectroscopy of glutamate mutase from Clostridium cochlearium was performed in order to test the idea, that a histidine residue of component S replaces the dimethylbenzimidazole ligand of the Co‐atom during binding of coenzyme B12 to the enzyme. The shapes and the superhyperfine splitting of the g z‐lines of the Co(II) EPR spectra were used as indicators of the interaction of the axial base nitrogen with the Co‐atom. A mixture of completely 15N‐labelled component S, unlabelled component E, coenzyme B12 and glutamate gave slightly sharper g z‐lines than that with unlabelled component S. A more dramatic change was observed in the Co(II) spectrum of the inactivated enzyme containing tightly bound cob(II)alamin, in which unlabelled component S caused a threefold superhyperfine‐splitting of the g z‐line, whereas the 15N‐labelled protein only caused a twofold splitting, as expected for a direct interaction of a nitrogen of the enzyme with the Co‐atom. By using a sample of 15N‐labelled component S, in which only the histidines were 14N‐labelled, the EPR spectra showed no difference to those with unlabelled component S. The experiments indeed demonstrate a replacement of the dimethylbenzimidazole ligand in coenzyme B12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skeleton rearranging mutases and methionine synthase.

Analysis of RovA, a Transcriptional Regulator of Yersinia pseudotuberculosis Virulence That Acts through Antirepression and Direct Transcriptional Activation

The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.

αNAC depletion as an initiator of ER stress-induced apoptosis in hypoxia

Accumulation of unfolded proteins triggers endoplasmic reticulum (ER) stress and is considered a part of the cellular responses to hypoxia. The nascent polypeptide-associated complex (NAC) participates in the proper maturation of newly synthesized proteins. However, thus far, there have been no comprehensive studies on NAC involvement in hypoxic stress. Here, we show that hypoxia activates glycogen synthase kinase-3β (GSK-3β) and that the activated GSK-3β destabilizes αNAC with the subsequent apoptosis of the cell. Hypoxia of various cell types and the mouse ischemic brain was associated with rapid downregulation of αNAC and ER stress responses involving PERK, ATF4, γ-taxilin, elF2α, Bip, and CHOP. Depletion of αNAC by RNA interference specifically activated ER stress responses and caused mitochondrial dysfunction, which resulted in apoptosis through caspase activation. Interestingly, we found that the hypoxic conditions activated GSK-3β, and that GSK-3β inhibition prevented αNAC protein downregulation in hypoxic cells and rescued the cells from apoptosis. In addition, αNAC overexpression increased the viability of hypoxic cells. Taken together, these results suggest that αNAC degradation triggers ER stress responses and initiates apoptotic processes in hypoxic cells, and that GSK-3β may participate upstream in this mechanism.

Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretory polypeptides to the Sec61 complex

Nascent polypeptide associated complex (NAC) interacts with nascent polypeptides emerging from ribosomes. Both signal recognition particle (SRP) and NAC work together to ensure specificity in co‐translational targeting by competing for binding to the ribosomal membrane attachment site. While SRP selects signal‐containing ribosomes for targeting, NAC prevents targeting of signal peptide‐less nascent chains to the endoplasmic reticulum membrane. Here we show that the ribosome binding that occurs in NACs absence delivers signal‐less nascent chains to the Sec61 complex, underscoring the danger of unregulated exposure of the ribosomal M‐site. Recently, the idea that NAC prevents ribosome binding has been challenged. By carefully examining the physiologic NAC concentration in a variety of tissues from different species we here demonstrate that the discrepancy resulted from subphysiologic NAC concentrations.

Dual binding mode of the nascent polypeptide-associated complex reveals a novel universal adapter site on the ribosome.

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.