Charge-driven dynamics of nascent-chain movement through the SecYEG translocon

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Abstract

On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47–49 residues away from the ribosomal P site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential, whereas the second may reflect interactions between charged residues and the periplasmic membrane surface.

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Figure 1: Negatively charged residues experience an electric pulling force during passage through the SecYEG translocon.
Figure 2: The electric force has both PMF-dependent and PMF-independent components and scales with the number of negatively charged residues in the nD stretch.
Figure 3: A positively charged lysine residue placed N terminal to a 4D stretch reduces the electric pulling force.
Figure 4: Physical model for translocation of charged residues (details in Supplementary Note).

References

  1. 1

    Park, E. & Rapoport, T.A. Preserving the membrane barrier for small molecules during bacterial protein translocation. Nature 473, 239–242 (2011).

  2. 2

    Cymer, F. & von Heijne, G. Cotranslational folding of membrane proteins probed by arrest-peptide-mediated force measurements. Proc. Natl. Acad. Sci. USA 110, 14640–14645 (2013).

  3. 3

    Ismail, N., Hedman, R., Schiller, N. & von Heijne, G. A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration. Nat. Struct. Mol. Biol. 19, 1018–1022 (2012).

  4. 4

    Cymer, F., Ismail, N. & von Heijne, G. Weak pulling forces exerted on Nin-orientated transmembrane segments during co-translational insertion into the inner membrane of Escherichia coli. FEBS Lett. 588, 1930–1934 (2014).

  5. 5

    Ito, K., Chiba, S. & Pogliano, K. Divergent stalling sequences sense and control cellular physiology. Biochem. Biophys. Res. Commun. 393, 1–5 (2010).

  6. 6

    Butkus, M.E., Prundeanu, L.B. & Oliver, D.B. Translocon “pulling” of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation. J. Bacteriol. 185, 6719–6722 (2003).

  7. 7

    Gumbart, J., Schreiner, E., Wilson, D.N., Beckmann, R. & Schulten, K. Mechanisms of SecM-mediated stalling in the ribosome. Biophys. J. 103, 331–341 (2012).

  8. 8

    Yap, M.N. & Bernstein, H.D. The plasticity of a translation arrest motif yields insights into nascent polypeptide recognition inside the ribosome tunnel. Mol. Cell 34, 201–211 (2009).

  9. 9

    Yap, M.N. & Bernstein, H.D. The translational regulatory function of SecM requires the precise timing of membrane targeting. Mol. Microbiol. 81, 540–553 (2011).

  10. 10

    Nilsson, I.M. & von Heijne, G. Determination of the distance between the oligosaccharyltransferase active site and the endoplasmic reticulum membrane. J. Biol. Chem. 268, 5798–5801 (1993).

  11. 11

    Nilsson, I. M., Whitley, P. & von Heijne, G. The C-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase. J. Cell Biol. 126, 1127–1132 (1994).

  12. 12

    Stefansson, A., Armulik, A., Nilsson, I.M., von Heijne, G. & Johansson, S. Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits. J. Biol. Chem. 279, 21200–21205 (2004).

  13. 13

    Frauenfeld, J. et al. Cryo-EM structure of the ribosome–SecYE complex in the membrane environment. Nat. Struct. Mol. Biol. 18, 614–621 (2011).

  14. 14

    Voorhees, R.M., Fernández, I.S., Scheres, S.H. & Hegde, R.S. Structure of the mammalian ribosome-Sec61 complex to 3.4 Å resolution. Cell 157, 1632–1643 (2014).

  15. 15

    Fujita, H., Yamagishi, M., Kida, Y. & Sakaguchi, M. Positive charges on the translocating polypeptide chain arrest movement through the translocon. J. Cell Sci. 124, 4184–4193 (2011).

  16. 16

    Park, E. et al. Structure of the SecY channel during initiation of protein translocation. Nature 506, 102–106 (2014).

  17. 17

    Van den Berg, B. et al. X-ray structure of a protein-conducting channel. Nature 427, 36–44 (2004).

  18. 18

    Chimerel, C., Field, C.M., Piñero-Fernandez, S., Keyser, U.F. & Summers, D.K. Indole prevents Escherichia coli cell division by modulating membrane potential. Biochim. Biophys. Acta 1818, 1590–1594 (2012).

  19. 19

    Lu, J., Kobertz, W.R. & Deutsch, C. Mapping the electrostatic potential within the ribosomal exit tunnel. J. Mol. Biol. 371, 1378–1391 (2007).

  20. 20

    Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

  21. 21

    Smith, D.E. et al. The bacteriophage φ29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001).

  22. 22

    Liu, T. et al. Direct measurement of the mechanical work during translocation by the ribosome. eLife 3, e03406 (2014).

  23. 23

    Nakamori, K., Chiba, S. & Ito, K. Identification of a SecM segment required for export-coupled release from elongation arrest. FEBS Lett. 588, 3098–3103 (2014).

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Acknowledgements

This work was supported by grants from the Swedish Foundation for Strategic Research, the European Research Council (ERC-2008-AdG 232648), the Swedish Cancer Foundation, the Swedish Research Council and the Knut and Alice Wallenberg Foundation to G.v.H., and by a grant from the Wenner-Gren Foundation to M.L.

Author information

N.I. contributed to the study design, the experimental work and the writing of the paper. R.H. contributed to the study design, the experimental and modeling work and the writing of the paper. M.L. contributed to the modeling work and the writing of the paper. G.v.H. contributed to the study design, the modeling work and the writing of the paper.

Correspondence to Gunnar von Heijne.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Amino acid sequences of the LepB construct, the test segments and the SecM arrest-peptide variants used in this study.

(a) The amino acid sequence of the full-length LepB construct is shown with the wild type 8-residue M. succiniciproducens SecM arrest peptide (bold) and 5D test segment (bold and underlined). The two natural TM domains of LepB (TM1, TM2) are underlined. The last residue indicated by the left-facing arrow (denoted ‘R’) was fused to the first residue indicated by the right-facing arrows of downstream LepB residues to give constructs with linker lengths, L, indicated by the numbers above the arrows.(b) Sequences of the test segments analyzed in this study. All test segments used have GG...GG flanks, except for the SG constructs which have (SG)5...(SG)5. flanks and the [6L,13A] segment which has GGPG...GPGG flanks.(c) Sequences of SecM arrest peptide variants used. Mutations introduced into the wild type SecM arrest peptide to generate the non-functional variant (mut) and the stronger variant (Sup1) are underlined.

Supplementary Figure 2 Analysis of 10D and (SG)5-5D-(SG)5 constructs containing the SecM(Ms-Sup1) AP.

(a) fFL for the 10D constructs plotted as a function of L+n measured in the absence (black) and presence (blue) of 2 mM indole in the growth medium.(b) Difference plots of fFL values representing the pmf-dependent component obtained by subtracting the 10D (+indole) profile from the 10D (-indole) profile.(c) fFL for the (SG)5-5D-(SG)5 constructs plotted as a function of L+n measured in the absence (purple) and presence (blue) of 2 mM indole in the growth medium.(d) Difference plots of fFL values representing the pmf-dependent component obtained by subtracting the (SG)5-5D-(SG)5 (+indole) profile from the (SG)5-5D-(SG)5 (-indole) profile.

Supplementary Figure 3 Characteristics of the first (i.e., PMF dependent) peak in the nD force profiles obtained with the SecM(Ms-Sup1) arrest peptide.

(a) Maximal value of the pmf-dependent peak as a function of n.(b) Initial slope of the pmf-dependent peak as a function of n.

Supplementary Figure 4 Experimental and calculated fFL profiles for nX-SecM(Ms) and nX-SecM(Ms-Sup1) constructs.

(a) Experimental (blue) and calculated (red) fFL profiles for nX-SecM(Ms) constructs. Note that the experimental nX-SecM(Ms) profiles were not used in the parameter optimization, which only included data for the (SG)5-nX-(SG)5-SecM(Ms) constructs (see Supplementary Note).(b) Experimental (blue) and calculated (red) fFL profiles for nX-SecM(Ms-Sup1) constructs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Note (PDF 2247 kb)

Supplementary Data Set 1

Uncropped version of a gel with the same constructs as in Figure 1b (PDF 372 kb)

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Ismail, N., Hedman, R., Lindén, M. et al. Charge-driven dynamics of nascent-chain movement through the SecYEG translocon. Nat Struct Mol Biol 22, 145–149 (2015) doi:10.1038/nsmb.2940

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