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Measurement of the Energetics of Protein Transport Across the Chloroplast Thylakoid Membrane

  • Steven M. Theg
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

Protein transport across cellular membranes represents an unknown, possibly significant drain on the total energy pool. Many protein transport systems utilize a mixture of energetic inputs, with contributions from both NTP hydrolysis and transmembrane electrochemical gradients. Both of these parameters will have to be measured before we can know the cost to the cell of its considerable protein transport activities. We describe here methods to evaluate the magnitude of the ΔpH across the thylakoid membrane, which serves as the driving force for protein transport on the cpTat pathway, and to determine how much energy is drained therefrom per protein translocated. The methods derive from spectroscopic techniques, well known in the field of thylakoid energetics, to monitor the light-dependent ΔpH across the membrane and the rate of proton flux through the thylakoid lumen, combined with those to measure the rate of protein transport across the thylakoid membrane.

Key words

Protein transport energetics thylakoid membrane ΔpH proton pump 

Notes

Acknowledgments

This work is supported by US Department of Energy Grant DE-FG02-03ER15405.

References

  1. 1.
    Schatz, G., and Dobberstein, B. (1996) Common principles of protein translocation across membranes. Science 271, 1519–1525.CrossRefPubMedGoogle Scholar
  2. 2.
    Alder, N. N., and Theg, S. M. (2003) Energy use by biological protein transport pathways. Trends Biochem Sci 28, 442–451.CrossRefPubMedGoogle Scholar
  3. 3.
    Mokranjac, D., and Neupert, W. (2008) Energetics of protein translocation into mitochondria. Biochim Biophys Acta 1777, 758–762.CrossRefPubMedGoogle Scholar
  4. 4.
    Wickner, W., and Schekman, R. (2005) Protein translocation across biological membranes. Science 310, 1452–1456.CrossRefPubMedGoogle Scholar
  5. 5.
    Schiebel, E., Driessen, A. M., Hartl, F.-U., and Wickner, W. (1991) Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell 64, 927–939.CrossRefPubMedGoogle Scholar
  6. 6.
    Cline, K., Ettinger, W. F., and Theg, S. M. (1992) Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP. J Biol Chem 267, 2688–2696.PubMedGoogle Scholar
  7. 7.
    Braun, N. A., Davis, A. W., and Theg, S. M. (2007) The chloroplast tat pathway utilizes the transmembrane electric potential as an energy source. Biophys J 93, 1993–1998.CrossRefPubMedGoogle Scholar
  8. 8.
    Bageshwar, U. K., and Musser, S. M. (2007) Two electrical potential-dependent steps are required for transport by the Escherichia coli Tat machinery. J Cell Biol 179, 87–99.CrossRefPubMedGoogle Scholar
  9. 9.
    Diekert, K., Kispal, G., Guiard, B., and Lill, R. (1999) An internal targeting signal directing proteins into the mitochondrial intermembrane space. Proc Natl Acad Sci USA 96, 11752–11757.CrossRefPubMedGoogle Scholar
  10. 10.
    Lill, R., Cunningham, K., Brundage, L. A., Ito, K., Oliver, D., and Wickner, W. (1989) SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli. EMBO J 8, 961–966.PubMedGoogle Scholar
  11. 11.
    Driessen, A. J. M. (1992) Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force. EMBO J 11, 847–853.PubMedGoogle Scholar
  12. 12.
    Tomkiewicz, D., Nouwen, N., van Leeuwen, R., Tans, S., and Driessen, A. J. (2006) SecA supports a constant rate of preprotein translocation. J Biol Chem 281, 15709–15713.CrossRefPubMedGoogle Scholar
  13. 13.
    Alder, N. N., and Theg, S. M. (2003) Energetics of protein transport across biological membranes: A study of the thylakoid DeltapH-dependent/cpTat pathway. Cell 112, 231–242.CrossRefPubMedGoogle Scholar
  14. 14.
    Hulford, A., Hazell, L., Mould, R. M., and Robinson, C. (1994) Two distinct mechanisms for the translocation of proteins across the thylakoid membrane, one requiring the presence of a stromal protein factor and nucleotide triphosphates. J Biol Chem 269, 3251–3256.PubMedGoogle Scholar
  15. 15.
    Bolter, B., and Soll, J. (2007) Import of plastid precursor proteins into pea chloroplasts. Methods Mol Biol 390, 195–206.CrossRefPubMedGoogle Scholar
  16. 16.
    Berry, S., and Rumberg, B. (1996) H+/ATP coupling ratio at the unmodulated CF0CF1-ATP synthase determined by proton flux measurements. Biochim Biophys Acta 1276, 51–56.CrossRefGoogle Scholar
  17. 17.
    Schuldiner, S., Rottenberg, H., and Avron, M. (1972) Determination of delta pH in chloroplasts: 2. Fluorescent amines as a probe for the determination of delta pH in chloroplasts. Eur J Biochem 25, 64–70.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Steven M. Theg
    • 1
  1. 1.Department of Plant BiologyUniversity of CaliforniaDavisUSA

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