Global profiling of SRP interaction with nascent polypeptides (original) (raw)

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Acknowledgements

We thank members of the Bukau laboratory for valuable contributions; C. Gläßer for support with data analysis; and H. Bernstein for providing plasmid pHQ4. Sequencing was done at the Genomics & Proteomics Core (DKFZ) facility. I.P. and R.C.W. acknowledge support from the Klaus Tschira Foundation. This work was supported by research grants from the Deutsche Forschungsgemeinschaft (SFB638 and FOR1805) to G.K. and B.B., a Human Frontier Science Program grant to B.B. and a grant from the Swedish Research Council to G.v.H.

Author information

Authors and Affiliations

  1. Center for Molecular Biology of the University of Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg, D-69120, Germany
    Daniela Schibich, Felix Gloge, Rebecca C. Wade, Bernd Bukau & Günter Kramer
  2. German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, D-69120, Germany
    Daniela Schibich, Felix Gloge, Bernd Bukau & Günter Kramer
  3. Heidelberg Institute for Theoretical Studies, (HITS gGmbH), Schloss-Wolfsbrunnenweg 35, Heidelberg, D-69118, Germany
    Ina Pöhner & Rebecca C. Wade
  4. Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Stockholm, 106 91, Sweden
    Patrik Björkholm & Gunnar von Heijne
  5. Science for Life Laboratory, Stockholm University, Box 1031, Solna, 171 21, Sweden
    Patrik Björkholm & Gunnar von Heijne
  6. Department of Molecular Evolution, Cell, and Molecular Biology, Uppsala University, Uppsala, 752 36, Sweden
    Patrik Björkholm
  7. Interdisciplinary Center for Scientific Computing (IWR), University of Heidelberg, Im Neuenheimer Feld 205, Heidelberg, D-69120, Germany
    Rebecca C. Wade

Authors

  1. Daniela Schibich
  2. Felix Gloge
  3. Ina Pöhner
  4. Patrik Björkholm
  5. Rebecca C. Wade
  6. Gunnar von Heijne
  7. Bernd Bukau
  8. Günter Kramer

Contributions

B.B. and G.K. conceived the study. D.S., B.B. and G.K. designed the experiments. D.S. and F.G. performed the experiments. D.S., I.P., R.C.W., P.B., G.vH., B.B. and G.K. analysed the data. B.B. and G.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence toBernd Bukau or Günter Kramer.

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Competing interests

The authors declare no competing financial interests.

Additional information

Data have been deposited in the Figshare database and are accessible from https://dx.doi.org/10.6084/m9.figshare.2058051.

Reviewer Information

Nature thanks N. Stern-Ginossar and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Selective ribosome profiling of E. coli SRP.

a, Experimental scheme of selective ribosome profiling (SeRP) of E. coli SRP-bound RNCs. Cells were harvested in mid-log phase via rapid filtration, frozen in liquid nitrogen and lysed in a frozen state with a cryo mill. After thawing, polysomes were digested with micrococcal nuclease. Monosomes were purified by sucrose cushion centrifugation (translatome). SRP-bound RNCs were immunopurified using an SRP-specific polyclonal rabbit antibody (SRP interactome). b, Bioanalyzer spectra quantifying the amount of co-purified ribosomes in control immunoprecipitation (top) and SRP immunoprecipitation (bottom). The 16S ribosomal RNA (rRNA) of the small ribosomal subunit and the 23S rRNA of the large subunit are indicated. c, Reproducibility of translatome (left) and SRP interactome (right) data sets from biological replicates d, Gene expression levels of translatome and SRP interactome are compared. Only SRP substrates that pass a threshold of twofold enrichment are coloured according to localization (cytoplasm in blue, inner membrane in red, outer membrane, lipoproteins and periplasm in green, no localization known in grey). e, CopA ratio-enrichment profile of SRP interactome and translatome, Pearson correlation coefficient 0.74. Light grey shadows indicate the variation between two biological replicates. f, DsbA ratio-enrichment profile, Pearson correlation coefficient 0.67. Shadows as in e. g, DnaK ratio-enrichment profile of SRP interactome and translatome. Shadows as in e.

Extended Data Figure 2 Selective ribosome profiling of E. coli SRP omitting detergents.

a, Gene expression levels of the translatome and SRP interactome are compared for different experimental setups (with detergents in lysis and wash buffer (n = 2), detergent only in wash buffer (n = 2) and omitting detergents at all (n = 1)). ORFs are coloured according to localization (cytoplasm in blue, inner membrane in red, outer membrane, lipoproteins and periplasm in green, no localization known in grey). b, DsbA ratio-enrichment profile in the absence of detergents (n = 1). c, Metagene SRP interaction profile aligned to the N terminus of the initial TMD that is skipped in the presence of detergents (orange) and in the absence of detergents (black). d, Ratio-enrichment profiles in the absence of detergents of MetI, MsbA and ManZ (n = 1).

Extended Data Figure 4 Heatmap representation of TMD positioning of inner membrane SRP substrates at the time point of first SRP binding.

TMDs are shown in dark red and segments (loops) located outside the membrane bilayer are shown in light grey, dashed lines indicate the area of the ribosomal tunnel exit. Substrates that are bound by SRP without exposing a TMD near the ribosome surface are shown at amino acid resolution. Amino acid colour code: highly hydrophobic amino acids, black; medium hydrophobic amino acids, dark grey; low hydrophobic amino acids, light grey; basic amino acids, blue; acidic amino acids, red; helix breakers, green.

Extended Data Figure 5 NuoA ratio enrichment profile of SRP binding.

Binding events are correlated with topology. SRP binds nascent NuoA when the first TMD is still buried in the tunnel. Light grey shadows indicate the variation between two biological replicates.

Extended Data Figure 6 SRP interaction profiles.

a–c, SRP interaction profile with nascent UraA (a), MetI (b) and SecY (c). SRP-binding peaks are correlated with protein topology. Light grey shading indicates variation between two biological replicates. d, SRP interaction profile with nascent CyoA in wild-type (WT) cells and cells overexpressing SRP and FtsY. Shading as in a–c.

Extended Data Figure 7 SRP interaction with purified RNCs at different salt concentration.

Left, cartoons illustrating the composition of nascent chains. Right, SRP–RNC interaction at indicated salt (ammonium acetate) concentrations analysed by sucrose cushion centrifugation and SDS–PAGE. Proteins are visualized using SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific). I, purified SRP; P, ribosomal pellet fraction; S, supernatant.

Extended Data Figure 8 Quantification of ribosome-exposed nascent-chain binding properties of SRP.

Four categories of binding markers (aliphatic amino acids, FILV motif, helix breakers and aromatic amino acids) quantified in bound TMDs, skipped TMDs and signal sequences.

Extended Data Figure 9 Interaction profile of TF and SRP with nascent MrcA.

TF, blue; SRP, orange. Light grey shading indicates the variation between two biological replicates.

Supplementary information

Supplementary Table 1 (download XLSX )

This file contains additional details about the proteome-wide SeRP data of SRP-nascent chain interactions in E. coli. Read counts of the translatome and the SRP interactome are compared, substrate identification methods are highlighted, quality scores (pearson correlation coefficient) are listed, initial SRP binding sites are given, retargeting and skipping events are indicated and topology information is provided. (XLSX 2035 kb)

Supplementary Table 2 (download XLSX )

This file contains an analysis of the properties of bound and skipped transmembrane domains (TMDs), and signal sequences. (XLSX 10 kb)

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Schibich, D., Gloge, F., Pöhner, I. et al. Global profiling of SRP interaction with nascent polypeptides.Nature 536, 219–223 (2016). https://doi.org/10.1038/nature19070

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