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ClpS is an essential component of the N-end rule pathway in Escherichia coli

Nature volume 439, pages 753–756 (2006)Cite this article

Abstract

The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue1. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome1,2. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP)3. Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS–ClpA interface4, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.

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Figure 1: ClpS is essential for ClpAP-mediated degradation of N-end rule substrates in vivo.
Figure 2: ClpS binds to N-end rule peptides in peptide libraries.
Figure 3: ClpS mediates degradation of a N-end rule GFP variant by ClpAP in vitro.
Figure 4: ClpS has a specific binding site for N-end rule substrates.

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Acknowledgements

We thank R. J. Dohmen for pUB23-M, pUB23-R, pUB23-L and pJT184; R. Volkmer for synthesis of peptide libraries; J. M. Weber for technical assistance; R. Nicolay for help with surface plasmon resonance; and laboratory members of the Clp group for discussions. This work was supported by the Deutsche Forschungsgemeinschaft priority program, proteolysis in prokaryotes: protein quality control and regulatory principles, the Fonds der Chemischen Industrie and the Australian Research Council QEII Fellowship to D.A.D.

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Author notes

  1. T. Bornemann

    Present address: Institut für Molekularbiologie, Universität Witten/Herdecke, Stockumer Strasse 10-12, Witten, D-58453, Germany

  2. A. Erbse and R. Schmidt: *These authors contributed equally to this work

Authors and Affiliations

  1. Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, INF 282, D-69120, Heidelberg, Germany

    A. Erbse, R. Schmidt, T. Bornemann, A. Mogk, R. Zahn, D. A. Dougan & B. Bukau

  2. Universitätsklinikum Charite, Humboldt-Universität, Schumannstrasse 20-21, D-10098, Berlin, Germany

    J. Schneider-Mergener

  3. Department of Biochemistry, La Trobe University, 3086, Melbourne, Australia

    D. A. Dougan

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Correspondence to B. Bukau.

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Supplementary information

Supplementary Notes

This file contains Supplementary Figure Legends and Supplementary Methods (PDF 127 kb)

Supplementary Figure 1

N-end rule degradation pathway in prokaryotes and eukaryotes (PDF 629 kb)

Supplementary Figure 2

ClpS binds to N-End rule peptides on C-terminal immobilised peptide library (PDF 46 kb)

Supplementary Figure 3

ClpS inhibits the slow degradation of wt-Linker-GFP (PDF 16 kb)

Supplementary Figure 4

ClpS binds FR-GFP and FR-Linker-GFP (PDF 17 kb)

Supplementary Figure 5

Mutations in the binding side for N-end rule substrates of ClpS do not effect the interaction of ClpS wit ClpA (PDF 731 kb)

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Erbse, A., Schmidt, R., Bornemann, T. et al. ClpS is an essential component of the N-end rule pathway in Escherichia coli. Nature 439, 753–756 (2006). https://doi.org/10.1038/nature04412

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