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Stoichiometry and turnover in single, functioning membrane protein complexes

Nature volume 443, pages 355–358 (2006)Cite this article

Abstract

Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media1,2,3. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall4,5. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body6, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP–MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains ∼22 copies of GFP–MotB, consistent with ∼11 stators each containing two MotB molecules. We also observed a membrane pool of ∼200 GFP–MotB molecules diffusing at ∼0.008 µm2 s-1. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP–MotB between the membrane pool and motor with a rate constant of the order of 0.04 s-1: the dwell time of a given stator in the motor is only ∼0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.

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Figure 1: TIRF microscopy of live GFP–MotB cells.
Figure 2: TIRF photobleaching.
Figure 3: Focused laser FRAP and ‘one-shot’ FLIP.

References

  1. Macnab, R. M. in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhardt, F. C.) 123–145 (American Society for Microbiology, Washington DC, 1996)

    Google Scholar 

  2. Berry, R. M. & Armitage, J. P. The bacterial flagella motor. Adv. Microb. Physiol. 41, 291–337 (1999)

    Article  CAS  Google Scholar 

  3. Berg, H. C. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72, 19–54 (2003)

    Article  CAS  Google Scholar 

  4. Block, S. M. & Berg, H. C. Successive incorporation of force-generating units in the bacterial rotary motor. Nature 309, 470–472 (1984)

    Article  ADS  CAS  Google Scholar 

  5. Blair, D. F. & Berg, H. C. Restoration of torque in defective flagellar motors. Science 242, 1678–1681 (1988)

    Article  ADS  CAS  Google Scholar 

  6. Silverman, M. & Simon, M. Flagellar rotation and the mechanism of bacterial motility. Nature 249, 73–74 (1974)

    Article  ADS  CAS  Google Scholar 

  7. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998)

    Article  CAS  Google Scholar 

  8. Mashanov, G. I., Tacon, D., Peckham, M. & Molloy, J. E. The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts. J. Biol. Chem. 279, 15274–15280 (2004)

    Article  CAS  Google Scholar 

  9. Mullineaux, C. W. & Sarcina, M. Probing the dynamics of photosynthetic membranes with fluorescence recovery after photobleaching. Trends Plant Sci. 7, 237–240 (2002)

    Article  CAS  Google Scholar 

  10. Ray, N., Nenninger, A., Mullineaux, C. W. & Robinson, C. Location and mobility of twin-arginine translocase subunits in the Escherichia coli plasma membrane. J. Biol. Chem. 280, 17961–17968 (2005)

    Article  CAS  Google Scholar 

  11. Goodwin, J. S. & Kenworthy, A. K. Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells. Methods 37, 154–164 (2005)

    Article  CAS  Google Scholar 

  12. Mullineaux, C. W., Nenninger, A., Ray, N. & Robinson, C. Diffusion of green fluorescent protein in three cell environments in Escherichia coli. J. Bacteriol. 188, 3442–3448 (2006)

    Article  CAS  Google Scholar 

  13. Berry, R. M., Turner, L. & Berg, H. C. Mechanical limits of bacterial flagellar motors probed by electrorotation. Biophys. J. 69, 280–286 (1995)

    Article  ADS  CAS  Google Scholar 

  14. Khan, S., Dapice, M. & Reese, T. S. Effects of mot gene expression on the structure of the flagellar motor. J. Mol. Biol. 202, 575–584 (1988)

    Article  CAS  Google Scholar 

  15. Reid, S. W. et al. The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11. Proc. Natl Acad. Sci. USA 103, 8066–8071 (2006)

    Article  ADS  CAS  Google Scholar 

  16. Kojima, S. & Blair, D. F. Solubilization and purification of the MotA/MotB complex of Escherichia coli. Biochemistry 43, 26–34 (2004)

    Article  CAS  Google Scholar 

  17. Sowa, Y. et al. Direct observation of steps in rotation of the bacterial flagellar motor. Nature 437, 916–919 (2005)

    Article  ADS  CAS  Google Scholar 

  18. Sourjik, V. & Berg, H. C. Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions. Mol. Microbiol. 37, 740–751 (2000)

    Article  CAS  Google Scholar 

  19. Turner, L., Ryu, W. S. & Berg, H. C. Real-time imaging of fluorescent flagellar filaments. J. Bacteriol. 182, 2793–2801 (2000)

    Article  CAS  Google Scholar 

  20. Dickson, R. M., Cubitt, A. B., Tsien, R. Y. & Moerner, W. E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997)

    Article  ADS  CAS  Google Scholar 

  21. Chu, S. Biology and polymer physics at the single-molecule level. Phil. Trans. R. Soc. Lond. A 361, 689–698 (2003)

    Article  ADS  CAS  Google Scholar 

  22. Leake, M. C., Wilson, D., Gautel, M. & Simmons, R. M. The elasticity of single titin molecules using a two-bead optical tweezers assay. Biophys. J. 87, 1112–1135 (2004)

    Article  CAS  Google Scholar 

  23. Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993)

    Article  ADS  CAS  Google Scholar 

  24. Mashanov, G. I., Tacon, D., Knight, A. E., Peckham, M. & Molloy, J. E. Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. Methods 29, 142–152 (2003)

    Article  CAS  Google Scholar 

  25. Deich, J., Judd, E. M., McAdams, H. H. & Moerner, W. E. Visualization of the movement of single histidine kinase molecules in live Caulobacter cells. Proc. Natl Acad. Sci. USA 101, 15921–15926 (2004)

    Article  ADS  CAS  Google Scholar 

  26. Van Way, S. M., Hosking, E. R., Braun, T. F. & Manson, M. D. Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene. J. Mol. Biol. 297, 7–24 (2000)

    Article  CAS  Google Scholar 

  27. Demot, R. & Vanderleyden, J. The C-terminal sequence conservation between OMPA-related outer membrane proteins and MotB suggests a common function in both gram-positive and gram-negative bacteria, possibly in the interaction of these domains peptidoglycan. Mol. Microbiol. 12, 333–336 (1994)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Blair for antibodies to flagellin and MotB. The research of M.C.L., J.H.C., R.M.B. and J.P.A. was supported by combined UK research councils via an Interdisciplinary Research Collaboration in Bionanotechnology (IRC), that of G.H.W. by the Biotechnology and Biological Sciences Research Council (BBSRC), and that of F.B. by a Clarendon Scholarship. Author Contributions Fluorescence experiments were carried out by M.C.L. and J.H.C. in the laboratory of R.M.B.; strain construction was done by J.H.C. and G.H.W. in the laboratory of J.P.A.; data analysis was done by M.C.L., R.M.B. and G.H.W.; and simulations were carried out by F.B. and M.C.L. The experiment was designed by M.C.L., R.M.B. and J.P.A.

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Author notes

  1. Mark C. Leake and Jennifer H. Chandler: *These authors contributed equally to this work

Authors and Affiliations

  1. Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, OX1 3PU, Oxford, UK

    Mark C. Leake, Fan Bai & Richard M. Berry

  2. Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK

    Jennifer H. Chandler, George H. Wadhams & Judith P. Armitage

Authors
  1. Mark C. Leake
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  6. Judith P. Armitage
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Correspondence to Judith P. Armitage.

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Supplementary information

Supplementary Notes

This file contains Supplementary Methods and Supplementary Figures 1–13. (PDF 1180 kb)

Supplementary Video Legends

This file contains text to accompany the below Supplementary Videos. (PDF 10 kb)

Supplementary Video 1

Brightfield video for a freely rotating tethered GFP-MotB cell and fixed cell in the same field of view. (MPG 141 kb)

Supplementary Video 2

Continuous TIRF bleach of cells in Supplementary Video 1. (MPG 638 kb)

Supplementary Video 3

Continuous TIRF illumination on a different pre-bleached fixed GFP-MotB cells showing diffusing GFP-MotB in the membrane. (AVI 212 kb)

Supplementary Video 4

Continuous TIRF illumination on a different pre-bleached fixed GFP-MotB cells showing diffusing GFP-MotB in the membrane. (AVI 329 kb)

Supplementary Video 5

Brightfield video for a freely rotating tethered GFP-MotB cell. (MPG 232 kb)

Supplementary Video 6

Continuous TIRF bleach of cell in Supplementary Video 5. (MPG 143 kb)

Supplementary Video 7

Continuous TIRF bleach of cell in Supplementary Video 4, 60 min after the end of the initial bleach. (MPG 143 kb)

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Leake, M., Chandler, J., Wadhams, G. et al. Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443, 355–358 (2006). https://doi.org/10.1038/nature05135

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