The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1
The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.
The atomic coordinates for the Rea1 AAA+ ring and the Rea1 linker in the ADP state have been deposited with PDB IDs 6HYP and 6HYD, respectively. The accession codes for the Rea1 and Rea1_ΔAAA2L-H2α models in the AMPPNP state are 6I26 and 6I27, respectively. The accession codes for the cryoEM maps of the Rea1 AAA+ ring and the Rea1 linker in the ADP state are EMD-0309 and EMD-0308, respectively. The accession code for the unsharpened cryoEM map of the Rea1 AAA+ ring in the ADP state is EMD-0330. The cryoEM maps of the Rea1 and Rea1_ΔAAA2L-H2α AMPPNP states have the EMD accession codes EMD-0328 and EMD-0329, respectively.
Rea1 AAA+ ring in the ADP stateProtein Data Bank, 6HYP.
Rea1 linker in the ADP stateProtein Data Bank, 6HYD.
Rea1 AMPPNP structureProtein Data Bank, 6I26.
Rea1_ΔAAA2L-H2α AMPPNP structureProtein Data Bank, 6I27.
Sharpened map of the Rea1 AAA+ ring ADP stateElectron Microscopy Data Bank, EMD-0309.
Unsharpened map of the Rea1 linker ADP stateElectron Microscopy Data Bank, EMD-0308.
Unsharpened map of the Rea1 AAA+ ring ADP stateElectron Microscopy Data Bank, EMD-0330.
Sharpened map of the Rea1 AMPPNP stateElectron Microscopy Data Bank, EMD-0328.
Sharpened map of the Rea1_ΔAAA2L-H2α AMPPNP stateElectron Microscopy Data Bank, EMD-0329.
Article and author information
HS-ATIP-avenir grant (CDP 0B1INSB-HS-9ADO1051)
- Helgo Schmidt
LabEX startup grant (GIE I-LABEX-HS)
- Helgo Schmidt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Andres E Leschziner, University of California, San Diego, United States
- Received: June 12, 2018
- Accepted: November 8, 2018
- Accepted Manuscript published: November 21, 2018 (version 1)
- Accepted Manuscript updated: November 26, 2018 (version 2)
- Version of Record published: December 7, 2018 (version 3)
© 2018, Sosnowski et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
- Page views
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
- Structural Biology and Molecular Biophysics
P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1–4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.
- Structural Biology and Molecular Biophysics
Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein’s RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1’s RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles.