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. 2010 Oct;78(1):203-17.
doi: 10.1111/j.1365-2958.2010.07325.x. Epub 2010 Aug 20.

The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding

Janine H Peterson  1 , Cheryl A Woolhead, Harris D Bernstein
Affiliations

The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding

Janine H Peterson et al. Mol Microbiol. 2010 Oct.

Abstract

In this report, we describe insights into the function of the ribosome tunnel that were obtained through an analysis of an unusual 25 residue N-terminal motif (EspP(1-25) ) associated with the signal peptide of the Escherichia coli EspP protein. It was previously shown that EspP(1-25) inhibits signal peptide recognition by the signal recognition particle, and we now show that fusion of EspP(1-25) to a cytoplasmic protein causes it to aggregate. We obtained two lines of evidence that both of these effects are attributable to the conformation of EspP(1-25) inside the ribosome tunnel. First, we found that mutations in EspP(1-25) that abolished its effects on protein targeting and protein folding altered the cross-linking of short nascent chains to ribosomal components. Second, we found that a mutation in L22 that distorts the tunnel mimicked the effects of the EspP(1-25) mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions and suggest that ribosomal mutations might increase the solubility of at least some aggregation-prone proteins produced in E. coli.

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Figures

Fig. 1
Fig. 1
The signal peptides of a subset of autotransporters and TPS exoproteins contain a conserved sequence motif. A. The EspP signal peptide (EspPSP) consists of typical N, H and C regions plus a ~30 residue N-terminal extension. The derivatives of EspPSP that were used in this study are shown. The OmpA signal peptide (OmpASP) is shown for comparison. B. The first 24 residues of the N-terminal signal peptide extensions of selected serine protease autotransporters of Enterobacteriaceae (SPATEs), other autotransporters, and TPS exoproteins were aligned using Clustal V. The organism that produces each protein is indicated in parentheses. Invariant residues are shown in red and highly conserved residues are shown in black.
Fig. 2
Fig. 2
Mutations in EspP1-25 or L22 suppress the effect of EspP1-25 on protein targeting. A. HDB140 (wild-type) and HDB141 (L22 Δ82-84) cells transformed with a plasmid encoding HA-tagged OmpA or EspP(Cterm)SP-OmpA were grown in M9 at 37°C. Cultures were shifted to 20°C, IPTG was added to induce the expression of the plasmid-borne gene, and cells were subjected to pulse-chase labeling. Sodium azide (NaN3) was added to a portion of one culture to block secretion and to show the position of the precursor. The precursor (p) and mature (m) forms of the OmpA derivatives were immunoprecipitated with an anti-HA antiserum. The percent of the precursor processed to the mature form and the percent of polypeptide chains completed at each time point is shown. B. As in part A, except that HDB140 and HDB141 were transformed with a plasmid encoding EspPSP-OmpA or the indicated derivative. C. As in part A, except that HDB142 (L4 K63E) were transformed with a plasmid encoding EspPSP-OmpA. D. The fraction of EspPSP-OmpA or EspP(E21A)SP-OmpA completed in the indicated strain at each time point is shown.
Fig. 3
Fig. 3
Mutations in EspP1-25 or L22 restore SRP recognition of a highly hydrophobic signal peptide. A. HDB140 and HDB141 cells transformed with plasmid encoding HA-tagged EspP(Cterm/Hydro”)SP-OmpA were grown and treated as described in the legend to Fig. 2A. The precursor (p) and mature (m) forms of the OmpA derivative were immunoprecipitated with an anti-HA antiserum. B. As in part A, except that HDB140 harbored a second plasmid [either pBAD33 or pBAD33-ftsY(G385A)]. Arabinose was added to both cultures before they were shifted to 20°C. C. As in part A, except that HDB140 and HDB141 were transformed with a plasmid encoding EspP(Hydro”)SP-OmpA or EspP(E21A/Hydro”)SP-OmpA. The percent of the precursor processed to the mature form and the percent of EspP(Hydro”)SP-OmpA completed at each time point is shown. D. As in part B, except that HDB140 or HDB141 harbored a plasmid encoding the indicated OmpA derivative.
Fig. 4
Fig. 4
Mutations in EspP1-25 or L22 suppress the effect of EspP1-25 on protein folding. A. HDB140, HDB141 and HDB142 transformed with pTRC99a (vector) or pTRC99a encoding MetE, EspP1-25-MetE, or a derivative of EspP1-25-MetE were grown in LB at 37°C. Expression of the plasmid-borne gene was induced by the addition of 50 μM IPTG, and cell lysates were subjected to high-speed centrifugation. Proteins present in the supernatant and pellet fractions were resolved by SDS-PAGE and visualized by Coomassie Blue staining. MetE-containing polypeptides are indicated with an asterisk. B. HDB140 transformed with pTRC99a or pTRC99a encoding EspP1-25-MetE were grown in LB, and expression of the plasmid-borne gene was induced by the addition of the indicated amount of IPTG. Cell lysates were processed as in part A. (C) The growth of HDB140, HDB141 and HDB142 harboring pTRC99a is shown.
Fig. 5
Fig. 5
Crosslinking of EspP, EspP(I12P) and EspP(E21A) 30-80 residue nascent chains to ribosomal proteins. A. N-terminal fragments containing the indicated number of amino acids (aa) of EspP, EspP(I12P) and EspP(E21A) were synthesized in coupled transcription-translation reactions and radiolabeled. Equal portions of each reaction that were untreated or treated with BS3 were resolved by SDS-PAGE. Prominent crosslinking products are denoted with an asterisk. B. Portions of the reactions shown in part A that were treated with BS3 were subjected to immunoprecipitation with anti-L23 and anti-L24 antisera.
Fig. 6
Fig. 6
Differential crosslinking of EspP, EspP(I12P) and EspP(E21A) 30-50 residue nascent chains to L24. N-terminal fragments containing the indicated number of amino acids (aa) of EspP, EspP(I12P) and EspP(E21A) were synthesized in coupled transcription-translation reactions and radiolabeled. Equal portions of each reaction that were treated with BS3 were subjected to immunoprecipitation with an anti-L24 antisera.
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