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Dual interaction of the Hsp70 J-protein cochaperone Zuotin with the 40S and 60S ribosomal subunits

Abstract

Ribosome-associated J protein–Hsp70 chaperones promote nascent-polypeptide folding and normal translational fidelity. The J protein Zuo1 is known to span the ribosomal subunits, but understanding of its function is limited. Here we present new structural and cross-linking data allowing more precise positioning of Saccharomyces cerevisiae Zuo1 near the 60S polypeptide-exit site and suggesting interactions of Zuo1 with the ribosomal protein eL31 and 25S rRNA helix 24. The junction between the 60S-interacting and subunit-spanning helices is a hinge that positions Zuo1 on the 40S yet accommodates subunit rotation. Interaction between the Zuo1 C terminus and 40S occurs via 18S rRNA expansion segment 12 (ES12) of helix 44, which originates at the decoding site. Deletions in either ES12 or the Zuo1 C terminus alter readthrough of stop codons and –1 frameshifting. Our study offers insight into how this cotranslational chaperone system may monitor decoding-site activity and nascent-polypeptide transit, thereby coordinating protein translation and folding.

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Figure 1: Structure of the Zuotin homology domain (ZHD) of Zuo1.
Figure 2: Interaction of Zuo1 with the 60S subunit.
Figure 3: Interaction of the Zuo1 C terminus with the 40S ribosomal subunit and effect on translation fidelity.
Figure 4: Positioning of the Zuo1 C-terminal four-helix bundle on 40S.
Figure 5: Model of the network of interaction of Zuo1 with the ribosome.

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Acknowledgements

We thank A. Senes, J. Keck and S. Butcher for helpful discussions during the course of this work; D. Bedwell (University of Alabama), P. Farabaugh (University of Maryland, Baltimore County) and J. Dinman (University of Maryland) for dual-luciferase plasmids; K. Asano (Kansas State University) for the rDNA-deletion strain; and J. Warner (Albert Einstein College of Medicine) for anti-uL3. This work was supported by National Institutes of Health grants GM031107 and GM027870 (E.A.C.). C.A.B. was supported by NIH grants GM094584, GM094622 and GM098248. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006).

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Authors and Affiliations

Authors

Contributions

K.L. performed in vivo cross-linking experiments, analyses of ribosome association, generation of mutants and other molecular-biological experiments. R.S. performed in vivo misreading experiments, analyses of ribosome association, generation of mutants and other molecular-biological experiments. O.K.S. performed crystallographic analysis, analyses of ribosome association and generation of mutants. C.A.B. performed crystallographic analysis. E.A.C. oversaw all aspects of the experiments and manuscript preparation. All authors participated in interpreting the data and writing the paper.

Corresponding author

Correspondence to Elizabeth A Craig.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Structure and sequence analyses of the Zuotin homology domain (ZHD).

(a) Superimposition of the two molecules of Zuo1166-303 in the asymmetric unit. Chain A and B are colored blue and purple, respectively. No electron density was observed for residues 166-168 of chain A and 166-167 of chain B. The RMSD of alpha carbon positions is 2.8 Å. The major difference between chain A and B is the loop between helix II and helix III (residues 228-245). When these residues are excluded, the RMSD is 1.4 Å. Chain A and chain B have an average B-value of 35 and 40, respectively. Chain A was used throughout. Figure generated by PyMol. (b) Representative stereo image of the 2FO-FC electron density map of ZHD calculated after the final refinement contoured at 1.0 σ. Residues Tyr221, Asp283, Pro284 and Arg285 forming the hinge at the junction between ZHD and middle domain shown in stick representation. Figure generated by PyMol. (c) Model structures of the ZHD. S. cerevisiae and human Zuo1 and Jjj1 sequences were aligned by Clustal Omega. The resulting alignment was used for generating model structures by Modeller, employing the S. cerevisiae Zuo1 ZHD structure as a template. The ZHD of yeast Jjj1 contains an insertion between helix I and helix II that is not conserved in higher eukaryotes such as humans. This insertion and the lower conservation of helix I led to an initial definition of the ZHD (i.e. 205-285 of Zuo1) that lacked helix I. (d) Phylogenetic analysis of Zuo1 and Jjj1 and conservation of residues forming the hinge region in the two proteins. Amino acid sequences of Zuo1 and Jjj1 orthologs from the indicated organisms were aligned using ClustalW. The trees were constructed using the Maximum Likelihood method based on the JTT matrix-based model conducted using MEGA7. The three residues present at the hinge of Zuo1 and Jjj1 [Asp283(D), Pro284(P), Arg285(R) and Asp257(D), Lys258(K), Arg259(R), respectively] are shown indicating a high conservation of salt-bridge partners aspartic acid and arginine flanking a relatively variable residue.

Supplementary Figure 2 Interaction of Zuo1 ZHD with the 60S subunit.

(a) Bpa was incorporated into eL31a-HA and eL22a-HA at positions highlighted. Sites that cross-linked shown in ball and stick representation; those that did not by stick only. (b) Site-specific cross-linking between eL31a-HA and Zuo1. Cells expressing indicated variants were grown in the presence of Bpa and exposed to UV light (+), or as a control not exposed (-), before lysis. Samples, run in parallel, were separated by gel electrophoresis and analyzed using antibodies specific for Zuo1 or HA tag (eL31a). Migration of molecular weight standards (left, in kDa) and Zuo1 and HA reactive bands (right, arrows) are indicated. Asterisk indicates crosslink between Zuo1 and Ssz1 (see panel d for explanation). Crosslinking was carried out with two independent yeast transformants, with similar results. (c) Residues of Zuo1 ZHD tested by Bpa cross-linking are highlighted (top, cyan). Sites that crosslinked are shown in ball and stick representation; those that did not, stick representation only. Cross-linking to eL31a was analyzed as in b except Zuo1-Asp262Bpa samples were electrophoresed in gels having 15%, rather than 10%, acrylamide. Crosslinking was carried out with two independent yeast transformants, with similar results. (d) Control of site-specific Bpa cross-linking experiments for unidentified band indicated by asterisks in panel b above, and in Fig. 2b. Cells expressing HA-tagged eL31a wild type (WT) and Arg79Bpa (top) and ∆ssz1 cells carrying pRS315-Ssz1 or a pRS315-Ssz1 variant with stop codon altered to TGA from TAG (bottom) were grown in the presence of Bpa. Cross-linking was analyzed as in b except antibodies specific for Zuo1 or Ssz1 were used for immunoblot analysis. Band reacting with both Zuo1 and Ssz1 antibody indicated with asterisk (*). Zuo1 and eL31a cross-linked band indicated with arrow. Migration of molecular weight standards is indicated in kDa (left). (e) Validation of antibody directed against Zuo1 residues 166-284. Yeast cell lysates of wild type cells of DS10 (WT strain) or ∆zuo1 cells expressing Zuo1 (WT), no Zuo1 (---) or truncation Zuo1 variant lacking the N-terminal 91 residues (92-433) were subjected to electrophoresis and immunoblot analysis. Antibody raised to Zuo1 residues from 166 to 368 tagged with GST was used. (f) Lysates of cells expressing wild type (WT) or variant Zuo1 with alterations Asp262/Thr266/Val273Ala (DTV) were centrifuged through sucrose cushions. Supernatant (S) and pellet (P) fractions, as well as total lysates (T), were analyzed by immunoblotting with indicated antibodies after SDS-PAGE. One representative immunoblot (from three independent experiments, performed with different yeast cultures) is shown. (b-e) Uncropped gel images are shown in Supplementary Data Set 1. (g) Secondary structure of 25S rRNA. Close-up view of H24 and H59 shown, with red dashed lines indicating deletions. Adapted from http://apollo.chemistry.gatech.edu/RibosomeGallery.

Supplementary Figure 3 Docking of Zuo1 ZHD to the 60S subunit.

The atomic structure of the ribosome (PDB 3J78) was fit to the cryo-EM map (gray surface) of Zuo1-Ssz1 bound ribosome. Zuo1 ZHD 169-303 was then manually docked to eL31, positioning the cross-linked residues (Thr266 and Val273 of Zuo1 and Val7, Arg79 and Glu81 of eL31, shown as spheres) in close proximity. After manually pointing the Arg247/Arg251 (spheres) at the tip of helix III, either towards H24 (H24 model, ZHD purple) or H59 and eL22 (eL22/H59 model, ZHD red), Zuo1 ZHD was fit to the cryo-EM map by rigid-body docking. PTE: Polypeptide tunnel exit.

Supplementary Figure 4 Interaction of the C terminus of Zuo1 with the 40S subunit.

(a) Secondary structure and location of expansion segment 12 (ES12) of rRNA H44. Secondary structure of 18S rRNA was adapted from http://apollo.chemistry.gatech.edu/RibosomeGallery. Deleted base pairs of ES12 are indicated with red dashed lines in the close-up view. (b) Stability of interaction of Zuo1 and MD variant with ribosomes. ∆zuo1 cells expressing either wild type Zuo1 (WT) or variant Zuo1Lys341/342/344Ala (K341/342/344A3) were lysed and centrifuged through sucrose cushions. Equivalent amounts of total lysate (T), supernatant (S) and pellet (P) fractions were subjected to electrophoresis and immunoblot analysis. Antibodies specific for Zuo1, and for uL3 and Ssa, as controls for ribosomes and a soluble protein, respectively were used. One representative immunoblot from three independent experiments, performed with different yeast transformants, is shown. Uncropped gel images are shown in Supplementary Data Set 1. (c) Expression of Zuo1 from the MET3 promoter. ∆zuo1 cells harboring a plasmid with no insert (---), Zuo1 expressed from its endogenous promoter (PZUO1) or from the repressible MET3 promoter (PMET3) were grown in selective minimal glucose medium supplemented with 400 μg/ml methionine to 0.5 OD units. Equal number of cells were harvested and indicated amounts (1x, 2x, 10x or 20x) of whole cell lysates were subjected to electrophoresis and immunoblotting with antibody specific for Zuo1 or a cytoplasmic protein, Ssa, as a control. Zuo1 levels from PZUO1 and PMET3 were analyzed at least three times using different yeast transformants; one representative immunoblot is shown. Uncropped gel images are shown in Supplementary Data Set 1. (d) Growth analysis of strains with a deletion of ZUO1 (∆zuo1) or a deletion of all chromosomal rDNA genes, and expressing only one rDNA gene from a plasmid (rRNAsc). Ten-fold serial dilutions of yeast cells were spotted on selective medium plates and incubated at 30°C for 2 days or at 23°C for 3.5 days. Plates supplemented with 0.75 M NaCl (+cation) were incubated at 30°C for 3.5 days. ∆zuo1 cells contained plasmids encoding either wild-type Zuo1 (WT), no insert (---), Zuo1Lys348/352/353Ala (K3A3), residues 1-310 of Zuo1 (1-310) or reduced levels of Zuo1 expressed from the MET3 promoter (Low). In the case of rRNAsc, cells harboring either wild type (WT) rDNA or a copy of rDNA with a deletion of 10 base pairs from the stem of ES12 (ES12∆10) are shown. One representative experiment (from three independent experiments, performed with different yeast cultures) is shown. (e) in vivo analyses of readthrough and -1 frameshifting of wild type Zuo1 and Zuo1His128Gln, altering the conserved HPD motif of the J-domain. Relative levels in cells expressing wild type Zuo1 set at 1. Data shown are means and s.e.m. from values obtained for three independent yeast transformants each assayed in triplicate. (f) Effects of alteration of amino acids forming the ZHD-MD hinge on cell growth. ∆zuo1 cells that contained plasmids encoding either wild type Zuo1 (WT), no insert (---), or indicated variants were used. Cell growth was analyzed as in d.

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Supplementary Data Set 1

Uncropped western blots (PDF 804 kb)

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Lee, K., Sharma, R., Shrestha, O. et al. Dual interaction of the Hsp70 J-protein cochaperone Zuotin with the 40S and 60S ribosomal subunits. Nat Struct Mol Biol 23, 1003–1010 (2016). https://doi.org/10.1038/nsmb.3299

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