Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes1,2,3,4,5,6. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails)7,8,9, whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails10,11,12. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis13. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.
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The cryo-EM density maps of the pre-state with the absence and presence of Arb1 and the post-state have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-4753, EMD-4751 and EMD-4752. The corresponding atomic models have been deposited in the Protein Data Bank under the accession codes 6R87, 6R84 and 6R86. For gel source images, see Supplementary Fig. 1.
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W.N. acknowledges funding from the Deutsche Forschungsgemeinschaft (NE 101/28-1) and from the Carl Friedrich von Siemens Foundation and thanks M. Kiebler for providing laboratory space and facilities. The authors thank S. Rieder and C. Ungewickel for technical assistance and M. Esaki for the Rip1 antibody. This research was supported by a grant from the Deutsche Forschungsgemeinschaft (GRK1721 to R.B.) and by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (grant numbers 26116003 to T. Inada and 19K16052 to T. Izawa). T.S. is supported by a DFG fellowship through the Graduate School of Quantitative Biosciences Munich (QBM).
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Sample preparation and cryo-EM analysis of Vms1–60S ribosomal subunit particles.
a, Vms1 and Vms1 mutants were immunoprecipitated from the lysates of vms1∆ cells expressing Vms1–3C–3×Myc or indicated Vms1 mutants from the GPD1 promoter. The precipitates were eluted by 3C cleavage and analysed by SDS–PAGE and Coomassie brilliant blue staining. b, Two-dimensional classification of Vms1(∆VIM/Q295L)–60S particles (pre-state). c, Two-dimensional classification of Vms1(∆VIM)–60S particles (post-state). d, Three-dimensional classification of Vms1(∆VIM/Q295L)–60S ribosome particles (pre-state). Reconstruction in blue and yellow mark final maps. e, Three-dimensional classification of Vms1(∆VIM)–60S particles (post-state). Reconstruction in green marks final map.
Extended Data Fig. 2 Resolution of Vms1 domains and their roles in ribosome binding, rescuing growth and preventing mitochondrial toxicity.
a, Final resolution of Vms1 pre-state and post-state 60S ribosome particles. b, Local resolution of Vms1 pre-state and post-state. Left, overviews; right, transverse views. The electron density for the pre-state is displayed at 1.68σ and for the post-state at 1.83σ. c, Tri-model position of Vms1 and its interaction with peptidyl-tRNA (pre-state, top; density of Vms1 LRS–VLRF1 and tRNA is displayed at 3.23σ, Vms1 AnkR–CC at 2.20σ, nascent chain at 2.10σ, and Vms1 ZnF and Tif6 at 1.29σ) and interaction with Tif6 (post-state, bottom; density of Vms1 LRS–ZnF–VLRF1 and Tif6 is displayed at 2.15σ, and Vms1 AnkR–CC at 0.75σ). Left, local resolution; right, density with docked models. d, Fit of models to maps. FSC curves calculated between the refined model and the final map (black), with the self- and cross-validated correlations in blue and orange, respectively. e, Comparison between pre- and post-state of VLRF1 in two views (left and middle). Atom-to-atom quantification of the VLRF1 state difference (right). f, Prospective transverse model for interaction of the Vms1 VIM domain and Cdc48 on the 60S ribosomal subunit. g, Vms1 and Vms1 mutants were immunoprecipitated from the lysates of vms1∆ cells expressing Vms1–3×Myc or indicated Vms1 mutants from the GPD1 promoter. The precipitates were analysed by SDS–PAGE and Coomassie brilliant blue staining. Asterisks indicate IgG heavy and light chains. h, Rescue of growth of vms1Δltn1Δ cells by expression of Vms1, Vms1(∆ZnF), Vms1(∆AnkR) or Vms1(∆ZnF/∆AnkR) from the VMS1 promoter. Cells were grown at 37 °C on YPD or YPG plates. i, Prevention of aggregation of the NS-mtGFP reporter in vms1∆ cells by overexpression of Vms1–3×Myc or indicated Vms1 mutants from the GPD1 promoter. The cell extracts were analysed by immunoblotting with the indicated antibodies. j, Analysis of the mitochondrial indicator protein Rip1 in vms1Δltn1Δ cells by expression of Vms1–3×Myc or the indicated Vms1 mutants from the VMS1 promoter. The cells were grown at 30 °C. Cell lysates were analysed by immunoblotting using Rip1 and Myc antibodies. k, Density and molecular models of Vms1, tRNAVms1, and uL16 loop in the pre-state. The density of uL16 is displayed at 4.06σ, tRNA at 3.76σ and Vms1 at 3.57σ. See Supplementary Fig. 1 for gel source images.
a, Final resolution of the Vms1(∆VIM/Q295L) pre-state in the presence of Arb1. b, Local resolution map of the pre-state in the presence of Arb1. Left, overview; right, transverse view. The electron density is displayed at 2.21σ. c, Local resolution map of Arb1 with peptidyl-tRNA. d, Density and docked models of Arb1 and tRNA in yellow and blue, respectively. The tRNA density in c, d is displayed at 3.59σ and the rest remains at 2.21σ. e, View of the Arb1 model surface in two orientations. The nucleotide binding domains (NBD1 and 2) and the LD are indicated. f, Comparison of Arb1 with ABCF-type ATPases in open and closed states. The models of the ADP-bound RLI from P. furiosus (PDB 1YQT), the AMP-PNP bound Rli1 from S. cerevisiae (PDB 5LL6) and the AMP-PNP bound MsrE from Pseudomonas aeruginosa (PDB 5ZLU) were compared to Arb1 by rigid-body fitting of NBD1 into the density of the Arb1 NBD1. g, Steric clash of Arb1–tRNAVms1 with Rqc2–P-tRNARqc2 on the 60S subunit. Arb1, tRNAVms1 and Vms1 LRS–VLRF1 domain are shown as models and Rqc2, A-tRNARqc2 and P-tRNARqc2 as densities. See Methods for the contour levels. h, Overexpression of Arb1–Myc does not lead to displacement of Rqc2 on 60S subunits. Lysates of cells expressing Rqc2–3HA from its endogenous promoter with or without overexpression of Arb1–1×Myc were analysed by sucrose-gradient centrifugation. Fractions were analysed by SDS–PAGE and immunoblotting using HA and Myc antibodies. 60S and 80S ribosomes were detected using anti-Rpl3 antibody. T, top and B, bottom of the gradient. i, Density and molecular model of Vms1, tRNAVms1, uL16 and Arb1 of the Vms1–60S pre-state in the presence of Arb1. The density of 25S A2971 is displayed at 5.07σ, Arb1 at 4.55σ, Vms1 at 4.34σ, tRNA at 3.87σ and uL16 at 3.76σ. j, Model illustrating the rotation of 25S A2971 (A2602 in E. coli) by 180° upon Arb1 binding. k, The vms1∆ arb1∆ shuffle strain was complemented either with an Arb1(WT) construct or an Arb1-sAid-HA degron plasmid (Arb1::degron). Growth on YPD and YPD + auxin (500 µM final concentration) plates was monitored after two days at 30 °C (left). Arb1 protein level of the vms1∆ arb1::degron strain before and after treatment with auxin (500 µM final concentration) for 60 min at 30 °C (right). Cell lysates were analysed by immunoblotting using the HA antibody. l, Yeast in vitro release reaction of arrested peptides translated from the NS-3×Flag RQC reporter mRNA in vms1∆ski2∆ lysate. Buffer (−), Arb1, Vms1 or Vms1 together with Arb1 were added to cycloheximide-stopped translation reactions. Incubation time: 5 min (lane 1 and 2) at 25 °C; 0, 2 or 5 min, respectively (lanes 3–12). Molar ratios of Vms1 and Arb1 are 1 to 25 (Vms1 + Arb1) and 1 to 50 (Vms1 1/5 + Arb1), respectively. Bottom, longer exposure of the relatively weak bands. CCA, CCA from tRNA 3′ end; pep: peptidyl or peptide; * and ** indicate peptidyl-tRNA and free peptide from the colliding ribosome, respectively. m, Overexpression of Arb1 suppresses aggregation of a NS-cGFP reporter construct. Cell extracts of ltn1∆ cells expressing NS-cGFP and overexpressing Myc-tagged Arb1 or indicated Arb1 mutants were analysed by immunoblotting using anti-GFP and anti-Myc antibodies. n, Cell lysates of vms1∆arb1∆ cells expressing Arb1–HA or indicated Arb1 mutants without or with expression of Vms1–3×Myc were analysed by immunoprecipitation using Myc antibody. The inputs and the immunoprecipitates were analysed by Coomassie brilliant blue staining and immunoblotting using HA antibody. Asterisks indicate IgG heavy and light chains. o, Densities and molecular models of Vms1, tRNAVms1 and uL16 loop in the two pre-states either without Arb1 (left) or with Arb1 (middle). Right, the superposition of the uL16 loop in the Arb1 containing pre-state and in the eIF5A-bound 60S ribosomal subunit (PDB 5GAK) in purple and pink, respectively. The density of uL16 left structure is displayed at 4.06σ; the rest remains the same as in i. p, Model of tRNA positioning and dislocation by Arb1. See Supplementary Fig. 1 for gel source images.
Extended Data Fig. 4 Schematic model of the structural roles of Vms1 and Arb1 in counteracting CAT-tailing of toxic faulty mitochondrial proteins by Rqc2 and promoting their release into the mitochondria.
Top two rows: in wild-type cells, 60S subunits accumulate in close proximity to the protein import complex (TOM) of the outer membrane because import can occur co-translationally, especially when translation is stalled. Vms1 and Arb1 interfere sterically with binding of Rqc2 on the 60S subunits and thereby with CAT-tailing, accelerating peptidyl-tRNA release. The released polypeptides are imported into the mitochondria and degraded by the intramitochondrial chaperone and protease system. Middle row: in the absence of Vms1 and Ltn1, CAT-tailed mitochondrial proteins are released into the mitochondria where they aggregate with pre-existing proteins, leading to breakdown of mitochondrial functions in oxidative phosphorylation and other essential processes such as mitochondrial protein synthesis. Bottom row: overexpression of Arb1 can partially compensate for deficiency of Vms1 and Ltn1 by impairing Rqc2 dependent CAT-tailing and promoting peptidyl-tRNA release.