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Transient Expression and Analysis of Chloroplast Proteins in Arabidopsis Protoplasts

  • Dong Wook Lee
  • Inhwan Hwang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 774)

Abstract

Although chloroplasts have their own genome, most chloroplast proteins are encoded in the nuclear genome and are targeted to chloroplasts posttranslationally. In vitro import studies with isolated chloroplasts have been widely used and have helped to elucidate the complex mechanisms involved in protein targeting to chloroplasts. Recently, an in vivo targeting method using protoplasts emerged as an alternative method to investigate protein targeting into chloroplasts. The present study describes a set of principles and methods, including polyethylene glycol-mediated reporter plasmid transformation, fluorescence microscopy, immunocytochemistry, and Western blotting, for studying chloroplast interior and envelope membrane protein targeting using protoplasts isolated from Arabidopsis thaliana leaf tissues.

Key words

Chloroplast Protoplast Transit peptide Green fluorescent protein Polyethylene glycol 

Notes

Acknowledgments

This work was supported in part by grants from the National Research Foundation of Korea (NRF) (20100000737), World Class University Program (Project No. R31-2008-000-10105-0) of Ministry of Education, Science and Technology, and Technology Development Program (609004-05-1-SB210) for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (Republic of Korea).

References

  1. 1.
    Li, H. M., and Chiu, C. C. (2010) Protein transport into chloroplasts. Annu. Rev. Plant Biol. 61, 157–180.PubMedCrossRefGoogle Scholar
  2. 2.
    Lee, D. W., Lee, S., Lee, G. J., Lee, K. H., Kim, S., Cheong, G. W., and Hwang, I. (2006) Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco. Plant Physiol. 140, 466–483.PubMedCrossRefGoogle Scholar
  3. 3.
    Lee, D. W., Kim, J. K., Lee, S., Choi, S., Kim, S., and Hwang, I. (2008) Arabidopsis nuclear-encoded plastid transit peptides contain multiple sequence subgroups with distinctive chloroplast-targeting sequence motifs. Plant Cell 20, 1603–1622.PubMedCrossRefGoogle Scholar
  4. 4.
    Zhang, X. P., and Glaser, E. (2002) Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone. Trends Plant Sci. 7, 14–21.PubMedCrossRefGoogle Scholar
  5. 5.
    Perry, S. E., Li, H. M., and Keegstra, K. (1991) In vitro reconstitution of protein transport into chloroplasts. Methods Cell Biol. 34, 327–344.PubMedCrossRefGoogle Scholar
  6. 6.
    Smith, M. D., Schnell, D. J., Fitzpatrick, L., and Keegstra, K. (2003) In vitro analysis of chloroplast protein import. Curr. Protoc. Cell Biol. Chapter 11, Unit 11.16.Google Scholar
  7. 7.
    Jin, J. B., Kim, Y. A., Kim, S. J., Lee, S. H., Kim, D. H., Cheong, G. W., and Hwang, I. (2001) A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis. Plant Cell 13, 1511–1526.PubMedCrossRefGoogle Scholar
  8. 8.
    Lee, D. W., Lee, S., Oh, Y. J., and Hwang, I. (2009) Multiple sequence motifs in the rubisco small subunit transit peptide independently contribute to Toc159-dependent import of proteins into chloroplasts. Plant Physiol. 151, 129–141.PubMedCrossRefGoogle Scholar
  9. 9.
    Lee, S., Lee, D. W., Lee, Y., Mayer, U., Stierhof, Y. D., Lee, S., Jürgens, G., and Hwang, I. (2009) Heat shock protein cognate 70–4 and an E3 ubiquitin ligase, CHIP, mediate plastid-destined precursor degradation through the ubiquitin-26S proteasome system in Arabidopsis. Plant Cell 21, 3984–4001.PubMedCrossRefGoogle Scholar
  10. 10.
    Kikuchi, S., Oishi, M., Hirabayashi, Y., Lee, D. W., Hwang, I., and Nakai, M. (2009) A 1-megadalton translocation complex containing Tic20 and Tic21 mediates chloroplast protein import at the inner envelope membrane. Plant Cell 21, 1781–1797.PubMedCrossRefGoogle Scholar
  11. 11.
    Kim, D. H., Eu, Y. J., Yoo, C. M., Kim, Y. W., Pih, K. T., Jin, J. B., Kim, S. J., Stenmark, H., and Hwang, I. (2001) Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells. Plant Cell 13, 287–301.PubMedCrossRefGoogle Scholar
  12. 12.
    Lee, Y. J., Kim, D. H., Kim, Y. W., and Hwang, I. (2001) Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo. Plant Cell 13, 2175–2190.PubMedCrossRefGoogle Scholar
  13. 13.
    Lee, Y. J., Sohn, E. J., Lee, K. H., Lee, D. W., and Hwang, I. (2004) The transmembrane domain of AtToc64 and its C-terminal lysine-rich flanking region are targeting signals to the chloroplast outer envelope membrane. Mol. Cells 17, 281–291.PubMedGoogle Scholar
  14. 14.
    Bae, W., Lee, Y. J., Kim, D. H., Lee, J., Kim, S., Sohn, E. J., and Hwang, I. (2008) AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis. Nat. Cell Biol. 10, 220–227.PubMedCrossRefGoogle Scholar
  15. 15.
    Boyes, D. C., Zayed, A. M., Ascenzi, R., McCaskill, A. J., Hoffman, N. E., Davis, K. R., and Görlach, J. (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13, 1499–1510.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Life SciencePohang University of Science and TechnologyPohangRepublic of Korea
  2. 2.Division of Integrative Bioscience and BiotechnologyPohang University of Sciences and TechnologyPohangRepublic of Korea

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