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Studying Arabidopsis Envelope Protein Localization and Topology Using Thermolysin and Trypsin Proteases

  • John Froehlich
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 774)

Abstract

Chloroplasts are metabolically important organelles that perform many essential functions within plant cells. The chloroplasts can be subdivided into six distinct sub-compartments to which a protein may be ultimately targeted. These sub-compartments are defined as the outer envelope membrane (OEM), the inner envelope membrane (IEM), the thylakoid membrane, and three aqueous sub-compartments – the intermembrane space (IMS), the stroma, and the thylakoid lumen. The process by which proteins are targeted to the chloroplastic envelope membrane remains a challenging question in cell biology. Our understanding of protein targeting to the OEM is very limited, whereas targeting of membrane proteins to the IEM appears to utilize at least two targeting pathways called the stop-transfer and the conservative sorting (or post-import) pathways. Furthermore, once a membrane protein arrives at the envelope membrane, our understanding of how it achieves its final topology remains limited. One method that can be used to determine the topology of an envelope membrane protein is to apply the “dual protease” strategy. This approach involves several steps: first, performing an in vitro import assay; second, applying a “dual protease” protection assay using thermolysin and trypsin; and finally, isolating and analyzing chloroplastic subcellular fractionations (i.e., total membrane and soluble fractions). By using this multistep approach, one can gain critical information regarding the final topology of an OEM or IEM protein. Likewise, the “dual protease” approach may help in elucidating the possible targeting pathway that a membrane protein utilizes prior to its insertion into the envelope membrane.

Key words

Envelope membrane Protein targeting Transmembrane domain Protease Topology Thermolysin Trypsin 

Notes

Acknowledgments

I wish to thank Drs. Jon Glynn and Kathy Osteryoung for the generous gift of the prARC6/pBluescript plasmid and Dr. Christoph Benning for the gift of the prTGD2/pGEM-TEasy plasmid. The author is funded by DOE Grant no. DE-FG02-91ER20021 to Ken Keegstra.

References

  1. 1.
    Gutensohn, M., Fan, E., Frielingsdorf, S., Hanner, P., Hou, B., Hust, B., and Klosgen, R. B. (2006) Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts. J. Plant Physiol. 163, 333347.Google Scholar
  2. 2.
    Jarvis, P., and Robinson, C. (2004) Mechanisms of protein import and routing in chloroplasts. Curr. Biol. 14, R1064-R1077.PubMedCrossRefGoogle Scholar
  3. 3.
    Schwacke, R., Schneider, A., van der Graaff, E., Fischer, K., Catoni, E., Desimone, M., Frommer, W. B., Flugge, U. I., and Kunze, R. (2003) ARAMEMNON, a novel database for Arabidopsis integral membrane proteins. Plant Physiol. 131, 1626.PubMedCrossRefGoogle Scholar
  4. 4.
    van Wijk, K.J. (2004) Plastid proteomics. Plant Physiol. Biochem. 42, 963977.CrossRefGoogle Scholar
  5. 5.
    Marmagne, A., Salvi, D., Rolland, N., Ephritikhine, G., Joyard, J., and Barbier-Brygoo, H. (2006) Purification and fractionation of membranes for proteomic analyses. Methods Mol. Biol. 323, 403420.PubMedGoogle Scholar
  6. 6.
    Froehlich, J. E., Wilkerson, C. G., Ray, W. K., McAndrew, R. S., Osteryoung, K. W., Gage, D. A., and Phinney, B. S. (2003) Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis. J. Proteome Res. 2, 413–425.PubMedCrossRefGoogle Scholar
  7. 7.
    Ferro, M., Salvi, D., Brugiere, S., Miras, S., Kowalski, S., Louwagie, M., Garin, J., Joyard, J., and Rolland, N. (2003) Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana. Mol. Cell. Proteomics 2, 325–345.PubMedGoogle Scholar
  8. 8.
    Rolland, N., Ferro, M., Seigneurin-Berny, D., Garin, J., Douce, R., and Joyard, J. (2003) Proteomics of chloroplast envelope membranes. Photosynth. Res. 78, 205–230.PubMedCrossRefGoogle Scholar
  9. 9.
    Sveshnikova, N., Grimm, R., Soll J., and Schleiff, E. (2000) Topology studies of the chloroplast protein import channel Toc75. Biol. Chem. 381, 687693.PubMedCrossRefGoogle Scholar
  10. 10.
    Chen, D., and Schnell, D. J. (1997) Insertion of the 34-kDa chloroplast protein import ­component, IAP34, into the chloroplast outer membrane is dependent on its intrinsic GTP-binding capacity. J. Biol. Chem. 272, 6614–6620.PubMedCrossRefGoogle Scholar
  11. 11.
    Li, H.-M., Moore, T., and Keegstra, K. (1991) Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts. Plant Cell 3, 709–717.PubMedCrossRefGoogle Scholar
  12. 12.
    Froehlich, J. E., Itoh, A., and Howe, G. A. (2001) Tomato allene oxide synthase and fatty acid hydroperoxide lyase, two cytochrome P450s involved in oxylipin metabolism, are targeted to different membranes of chloroplast envelope. Plant Physiol. 125, 306–317.PubMedCrossRefGoogle Scholar
  13. 13.
    Froehlich, J. E., Benning, C., and Dörmann, P. (2001) The digalactosyldiacylglycerol (DGDG) synthase DGD1 is inserted into the outer envelope membrane of chloroplasts in a manner independent of the general import pathway and does not depend on direct interaction with monogalactosyldiacylglycerol synthase for DGDG biosynthesis. J. Biol. Chem. 276, 31806–31812.PubMedCrossRefGoogle Scholar
  14. 14.
    Vitha, S., Froehlich, J. E., Koksharova, O., Pyke, K. A., van Erp, H., and Osteryoung, K. W. (2003) ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2. Plant Cell 15, 1918–1933.PubMedCrossRefGoogle Scholar
  15. 15.
    Awai, K., Xu, C., Tamot. B., and Benning, C. (2006) A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proc. Natl. Acad. Sci. USA 103, 10817–10822.PubMedCrossRefGoogle Scholar
  16. 16.
    Viana, A. A., Li, M., and Schnell, D.J. (2010) Determinants for stop-transfer and post-import pathways for protein targeting to the chloroplast inner envelope membrane. J. Biol. Chem. 285, 12948–12960.PubMedCrossRefGoogle Scholar
  17. 17.
    Dreses-Werringloer, U., Fischer, K., Wachter, E., Link, T. A., and Flugge, U. I. (1991) cDNA sequence and deduced amino acid sequence of the precursor of the 37-kDa inner envelope membrane polypeptide from spinach chloroplasts. Its transit peptide contains an amphiphilic alpha-helix as the only detectable structural element. Eur. J. Biochem. 195, 361–368.Google Scholar
  18. 18.
    Tripp, J., Inoue, K., Keegstra, K., and Froehlich, J. E. (2007) A novel serine/proline-rich domain in combination with a transmembrane domain is required for the insertion of AtTic40 into the inner envelope membrane of chloroplasts. Plant J. 52, 824–838.PubMedCrossRefGoogle Scholar
  19. 19.
    Li, M., and Schnell, D. J. (2006) Reconstitution of protein targeting to the inner envelope membrane of chloroplasts. J. Cell Biol. 175, 249259.PubMedCrossRefGoogle Scholar
  20. 20.
    Chou, M.-L., Fitzpatrick, L. M., Tu, S.-L., Budziszewski, G., Potter-Lewis, S., Akita, M., Levin, J. Z., Keegstra, K., and Li, H.-M. (2003) Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon. EMBO J. 22, 2970–2980.PubMedCrossRefGoogle Scholar
  21. 21.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.Google Scholar
  22. 22.
    Cline, K., Werner-Washburne, M., Andrews, J., and Keegstra K. (1984) Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol. 75, 675–678.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.MSU-DOE Plant Research LaboratoryMichigan State UniversityEast LansingUSA

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