Crystal structures of Rea1-MIDAS bound to its ribosome assembly factor ligands resembling integrin-ligand-type complexes

Abstract

The Rea1 AAA⁺-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin α-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell-cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional β-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes.

Fig. 1

The crystal structure of the extended CtRea1-MIDAS reveals novel Rea1-specific elements. a Domain architecture of Rea1. The domain boundaries are indicated by the residue numbers below for S. cerevisiae (black) and C. thermophilum (red). b Crystal structure of the Rea1-MIDAS domain from C. thermophilum in two orientations. The close-up view highlights the DxSxS motif required for coordination of the divalent metal ion (left-hand structures). A ConSurf analysis of the CtRea1-MIDAS domain is shown in the two right-hand structures. Conserved amino acids, maroon; variable amino acids, turquoise. c Structure comparison of the CtRea1-MIDAS domain and the integrin A-domain (PDB ID: 1IDO) in two orientations. The integrin A-domain, green; the classical MIDAS fold of Rea1, blue; Rea1-specific elements I–III, yellow, purple, and red, respectively

Fig. 2

The crystal structure of the CtRea1-MIDAS ∆loop with its ligands CtRsa4-UBL and CtYtm1-UBL. a, b Yeast two-hybrid analysis of the interactions between the indicated Rea1-MIDAS constructs and full-length Rsa4 from C. thermophilum (a) and S. cerevisiae (b). The Rea1-MIDAS constructs were fused to an N-terminal GAL4-BD (binding domain) and the Rsa4 constructs were fused to an N-terminal GAL4-AD (activation domain). Plasmids were co-transformed into the PJ69-4A yeast two-hybrid strain and representative transformants were spotted in tenfold serial dilutions on SDC (SDC-Leu-Trp) and SDC-His (SDC-Leu-Trp-His) plates. Cell growth was monitored after incubation for 3 days at 30 °C. Co-transformation of p53 (residues 72–390) fused to the GAL4-BD and SV40 (residues 84–708) fused to the GAL4-AD served as a positive control. c, d Crystal structures of the Rea1-MIDAS domain lacking the protruding element II loop in complex with the UBL domains of Ytm1 (c) and Rsa4 (d) from C. thermophilum. The domain organization of Ytm1 and Rsa4 are shown above the structures. The residue numbers indicate the domain boundaries for S. cerevisiae (black) and C. thermophilum (red). Zoomed-in views of the Mg²⁺-coordinating residues are shown left of the X-ray structures. The Mg²⁺ ion is shown in green, the amino acids of the MIDAS consensus motif in red and the glutamate within Ytm1 and Rsa4, which is essential for binding to the MIDAS, in orange (c) and purple (d), respectively. e Comparison of the Rea1-MIDAS structure and the Rea1-MIDAS ∆loop structure in complex with the Rsa4-UBL. The Rea1-specific element III is shown in red, and the rearrangement from its disordered state in the MIDAS apo structure to a β-hairpin in the complex with the Rsa4-UBL is shown

Fig. 3

The Rea1-MIDAS-specific element III forms a novel interaction site with the UBL domain. a Crystal structure of the CtRea1-MIDAS ∆loop CtRsa4-UBL complex (left panel) and close-up of the interaction between the MIDAS element III and the Rsa4-UBL domain (right panels). The residues selected for mutational analysis are indicated. b Multiple sequence alignment of Rea1 showing the Rea1-MIDAS-specific element III. Sequences of Chaetomium thermophilum (Ct), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Kluyveromyces lactis (Kl), Arabidopsis thaliana (At), Dictyostelium discoideum (Dd), Mus musculus (Mm), and Homo sapiens (Hs) were aligned with Clustal Omega and visualized with Jalview. The element III with its two β-sheets are indicated in red, and residues mutated in this study are shown above the alignment (red: C. thermophilum, black: S. cerevisiae). c, d Yeast two-hybrid analysis between the indicated Rea1-MIDAS constructs and full-length Rsa4 from C. thermophilum (c) and S. cerevisiae (d). The MIDAS constructs were fused to an N-terminal GAL4-BD and the Rsa4 constructs to an N-terminal GAL4-AD. The constructs were co-transformed into yeast (PJ69-4A) and cells were spotted on SDC (SDC-Leu-Trp) and SDC-His (SDC-Leu-Trp-His) plates. Cell growth was inspected after 3 days at 30 °C. e The indicated Rea1 constructs were transformed in a rea1∆ shuffle strain. After plasmid shuffling on SDC plates containing 5-FOA, cells were spotted on YPD plates and growth was monitored at the indicated temperatures and times. f The rea1∆ rsa4∆ and rea1∆ ytm1∆ double shuffle strains were transformed with the indicated wild-type and mutant constructs. Transformants were spotted on SDC (SDC-Leu-Trp) and SDC + FOA plates and cell growth at 30 °C was monitored after 3 and 5 days, respectively. g Electron density map of the Rix1–Rea1 particle (EMD-3199) and the models of Rsa4 and Rea1 (PDB ID: 5JCS) in purple and orange, respectively. A density not occupied by the model is highlighted with a red circle (upper close-up). The lower close-up views show the rigid body fit of the Rea1-MIDAS ∆loop–Rsa4-UBL complex from C. thermophilum within the electron density map of the Rix1–Rea1 particle (EMD-3199). The β-hairpin formed by the Rea1-specific element III is highlighted with a red circle

Fig. 4

The conserved Rea1-MIDAS element II loop is required for Rea1’s essential function. a The indicated constructs were transformed in a rea1∆ shuffle strain and the viability of the respective mutants assessed on 5-FOA-containing plates. Cells were grown at 30 °C for 3 (SDC-Leu) and 5 days (SDC + FOA). b The indicated constructs under control of the GAL1-10 promoter were transformed in a wild-type strain (expressing endogenous REA1) and cell growth was monitored after incubation at 30 °C on plates containing glucose (SDC-Leu, left) or galactose (SGC-Leu, right) for 2 and 3 days, respectively. c Affinity purification of the indicated Rea1 constructs fused to an N-terminal TAP-Flag tag and under control of the endogenous REA1 promoter. The constructs were transformed into an AID-HA-REA1 degron strain. The endogenous Rea1 was depleted by the addition of auxin (final concentration 500 µM) to the cultures 2 h before cells were harvested. Final eluates were analyzed by SDS-PAGE and Coomassie staining or by western blot analysis against pre-60S assembly factors and ribosomal protein L3, using the indicated antibodies. d The subcellular localization of the indicated full-length Rea1 constructs N-terminally fused to GFP was monitored by fluorescence microscopy. Scale bar: 5 µm. Source data are provided as a Source Data file

Fig. 5

The Rea1 MIDAS element II loop harbors a conserved PY-NLS that interacts with Kap104. a Multiple sequence alignment of Rea1 showing the MIDAS loop region (purple bar). The PY-NLS is highlighted and the mutated residues are marked with an asterisk. The PY-NLS consensus sequence is shown below the alignment. The sequences of Chaetomium thermophilum (Ct), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Kluyveromyces lactis (Kl), Arabidopsis thaliana (At), Dictyostelium discoideum (Dd), Mus musculus (Mm), and Homo sapiens (Hs) were aligned with Clustal Omega and visualized with Jalview. b The ScMIDAS loop (residues 4655–4701) or the CtMIDAS loop (residues 4732–4778) were fused to a 3×GFP reporter and the subcellular localization of fusion proteins was monitored by fluorescence microscopy. Nomarski (DIC), GFP and merged pictures are shown. Scale bar: 5 µm. c, d Binding assay between GST-CtKap104 and CtRea1-MIDAS, CtMIDAS ∆loop, the indicated CtMIDAS PY-NLS point mutants, and CtSyo1. After incubation with GSH-agarose, GST-CtKap104 and bound proteins were eluted with GSH and analyzed by SDS-PAGE and Coomassie staining. e The subcellular localization of the CtMIDAS loop (left), the ScMIDAS loop (right), or indicated PY-NLS point mutants fused to 3×GFP was monitored by fluorescence microscopy. Nomarski (DIC), GFP and merged pictures are shown. Scale bars: 5 µm. Source data are provided as a Source Data file

Fig. 6

Nuclear import of Rea1 can be restored by a heterologous PY-NLS but still lacks Rea1-MIDAS loop-specific functions. a The PY-NLS containing Rea1-MIDAS element II loop (residues 4657–4696) was exchanged with PY-NLS-containing regions derived from either L4, Syo1, or Hrp1, whose domain organizations are shown. The different PY-NLS-containing fragments used to insert into the Rea1-MIDAS Δloop construct are highlighted in red and their amino acid sequences are shown below. Additional linker sequences are labeled and marked in gray. b Yeast two-hybrid analysis of the interactions between Rsa4 and the indicated Rea1-MIDAS constructs from S. cerevisiae. c Subcellular localization of the indicated N-terminally GFP-tagged Rea1 constructs under control of the endogenous REA1 promotor was monitored by fluorescence microscopy. Nomarski (DIC), GFP and merged images are shown. Scale bar: 5 µm. d Affinity purification of the Rea1-MIDAS Δloop constructs containing foreign PY-NLSs from L4, Syo1, or Hrp1. The indicated Rea1 constructs were fused to an N-terminal TAP-Flag tag under the endogenous REA1 promotor, which were transformed into an AID-HA-REA1 degron strain. Rea1 depletion was induced by addition of auxin (500 µM final concentration) for 2 h before harvesting and subsequent affinity purification. Final eluates were analyzed by SDS-PAGE following Coomassie staining or by western blot analysis using the indicated antibodies. e The indicated Rea1 constructs were transformed in a rea1∆ shuffle strain and the viability of the respective mutants assessed on 5-FOA-containing plates. f The indicated Rea1 constructs under control of the GAL1-10 promoter or an empty vector control were transformed in a wild-type strain (expressing endogenous REA1) and cell growth was monitored on plates containing glucose (SDC-Leu, left) or galactose (SGC-Leu, right). g Rix1-TAP pre-60S particles isolated after overexpression of the indicated Rea1 constructs under control of the GAL1-10 promotor (lanes 3–8) were incubated with 2.5 mM ATP (lanes 2, 4, 6, 8) or left untreated (lanes 1, 3, 5, 7) before re-isolation via L3-Flag. Final Flag-eluates were analyzed by Coomassie staining or by western blotting using the indicated antibodies. Source data are provided as a Source Data file

Fig. 7

The MIDAS element II loop is required for interaction of the Rea1-MIDAS domain with the Rea1-AAA⁺ ring. a GST-tagged CtRsa4-UBL was incubated with CtRea1-MIDAS or MIDAS∆loop and the CtRea1-NAAA⁺ domain. Indicated nucleotides were added to a final concentration of 2 mM. The resulting complexes were isolated by incubation with GSH-agarose. GST-CtRsa4 and bound material was eluted with GSH and analyzed with SDS-PAGE and Coomassie staining. Protein inputs are shown in lanes 1–4, final eluates in lanes 5–13. b Left: cryo-EM density and model of the AAA⁺ ATPase ring of SpRea1 (Mdn1) in presence of ATP and Rbin-1 (PDB ID: 6EES, EMD-9036). Right: close-up views highlighting the rigid-body fits of the CtMIDAS apo (turquoise) and the CtMIDAS∆loop (blue) structures into the SpRea1 density. The model of the SpRea1-MIDAS was omitted for clarity. No density was observed for the disordered loop of the Rea1-specific element III within the MIDAS apo structure. Instead, the formed β-hairpin within the MIDAS ∆loop structure fits into a density underneath the MIDAS domain. c Model of SpRea1 (PDB ID: 6EES), lacking the MIDAS domain, including the cryo-EM density (EMD-9036) of the MIDAS and contacting unaccounted-for density within the AAA⁺ pore in two orientations and as cutaway view (left three panels). The right view shows the rigid body fit of the CtMIDAS apo structure within the density. The unassigned density is directly connected to the densities of α1 and β1 of the MIDAS domain, strongly suggesting the loop (purple) of the Rea1-specific element II binds in the pore of the AAA⁺ ring. d Model of Rea1 import and assembly into pre-60S particles. In the cytoplasm, the PY-NLS within the loop of the MIDAS domain attached to the unstructured D/E-rich domain is recognized by the importin Kap104, which delivers Rea1 into the nucleus. Upon Kap104 dissociation, the accessible MIDAS loop tethers the MIDAS onto the AAA⁺ ring protruding into the ring pore, which together with formation of the β-hairpin-anchor primes the molecule for Rsa4-UBL interaction and release. At this stage, Rea1 is assembled to the nucleoplasmic pre-60S particle. Source data are provided as a Source Data file