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self-assembling GFP: A Versatile Tool for Plant (Membrane) Protein Analyses

  • Katharina Wiesemann
  • Lucia E. Groß
  • Manuel Sommer
  • Enrico Schleiff
  • Maik S. Sommer
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1033)

Abstract

The investigation of cellular processes on the molecular level is important to understand the functional network within plant cells. self-assembling GFP has evolved to be a versatile tool for (membrane) protein analyses. Based on the autocatalytical reassembling property of the nonfluorescent strands 1–10 and 11, protein distribution and membrane protein topology can be analyzed in vivo. Here, we provide basic protocols to determine membrane protein topology in Arabidopsis thaliana protoplasts.

Key words

Membrane proteins Topology self-assembling GFP Plant protoplasts Arabidopsis thaliana 

Notes

Acknowledgments

We thank Geoffrey S. Waldo (Los Alamos National Laboratory, Los Alamos, NM) for providing templates for the self-assembling GFP. This work was supported by Deutsche Forschungsgemeinschaft (SFB807 P17).

References

  1. 1.
    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239PubMedCrossRefGoogle Scholar
  2. 2.
    Remington SJ (2011) Green fluorescent protein: a perspective. Protein Sci 20:1509–1519PubMedCrossRefGoogle Scholar
  3. 3.
    Chalfie M, Tu Y, Euskirchen G et al (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805PubMedCrossRefGoogle Scholar
  4. 4.
    Thomas JD, Daniel RA, Errington J et al (2001) Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Mol Microbiol 39:47–53PubMedCrossRefGoogle Scholar
  5. 5.
    Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877PubMedCrossRefGoogle Scholar
  6. 6.
    van Dooren GG, Tomova C, Agrawal S et al (2008) Toxoplasma gondii Tic20 is essential for apicoplast protein import. Proc Natl Acad Sci USA 105:13574–13579PubMedCrossRefGoogle Scholar
  7. 7.
    Fiebiger E, Story C, Ploegh HL et al (2002) Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein. EMBO J 21:1041–1053PubMedCrossRefGoogle Scholar
  8. 8.
    Ciruela F (2008) Fluorescence-based methods in the study of protein-protein interactions in living cells. Curr Opin Biotechnol 19:338–343PubMedCrossRefGoogle Scholar
  9. 9.
    Ormo M, Cubitt AB, Kallio K et al (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395PubMedCrossRefGoogle Scholar
  10. 10.
    Cody CW, Prasher DC, Westler WM et al (1993) Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry 32: 1212–1218PubMedCrossRefGoogle Scholar
  11. 11.
    Cabantous S, Terwilliger TC, Waldo GS (2005) Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol 23:102–107PubMedCrossRefGoogle Scholar
  12. 12.
    Machettira AB, Gross LE, Sommer MS et al (2011) The localization of Tic20 proteins in Arabidopsis thaliana is not restricted to the inner envelope membrane of chloroplasts. Plant Mol Biol 77:381–390PubMedCrossRefGoogle Scholar
  13. 13.
    Sommer MS, Daum B, Gross LE et al (2011) Chloroplast Omp85 proteins change orientation during evolution. Proc Natl Acad Sci USA 108:13841–13846PubMedCrossRefGoogle Scholar
  14. 14.
    Ulrich T, Gross LE, Sommer MS et al (2012) Chloroplast beta-barrel proteins are assembled into the mitochondrial outer membrane in a process that depends on the TOM and TOB complexes. J Biol Chem 287:27467–27479PubMedCrossRefGoogle Scholar
  15. 15.
    Kaddoum L, Magdeleine E, Waldo GS et al (2010) One-step split GFP staining for sensitive protein detection and localization in mammalian cells. Biotechniques 49:727–728, 730, 732 passimGoogle Scholar
  16. 16.
    Hempel F, Bullmann L, Lau J et al (2009) ERAD-derived preprotein transport across the second outermost plastid membrane of diatoms. Mol Biol Evol 26:1781–1790PubMedCrossRefGoogle Scholar
  17. 17.
    Bullmann L, Haarmann R, Mirus O et al (2010) Filling the gap, evolutionarily conserved Omp85 in plastids of chromalveolates. J Biol Chem 285:6848–6856PubMedCrossRefGoogle Scholar
  18. 18.
    Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475PubMedCrossRefGoogle Scholar
  19. 19.
    Ferrara F, Listwan P, Waldo GS et al (2011) Fluorescent labeling of antibody fragments using split GFP. PLoS One 6:e25727PubMedCrossRefGoogle Scholar
  20. 20.
    Chaudhary A, Ganguly K, Cabantous S et al (2012) The Brucella TIR-like protein TcpB interacts with the death domain of MyD88. Biochem Biophys Res Commun 417:299–304PubMedCrossRefGoogle Scholar
  21. 21.
    Chun W, Waldo GS, Johnson GV (2011) Split GFP complementation assay for quantitative measurement of tau aggregation in situ. Methods Mol Biol 670:109–123PubMedCrossRefGoogle Scholar
  22. 22.
    von Arnim AG, Deng XW, Stacey MG (1998) Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene 221:35–43CrossRefGoogle Scholar
  23. 23.
    Gross LE, Machettira AB, Rudolf M et al (2011) GFP-based in vivo protein topology determination in plant protoplasts. J Endocytobiosis Cell Res 21:89–97Google Scholar
  24. 24.
    Kobayashi K, Nakamura Y, Ohta H (2009) Type A and type B monogalactosyldiacylglycerol synthases are spatially and functionally separated in the plastids of higher plants. Plant Physiol Biochem 47:518–525PubMedCrossRefGoogle Scholar
  25. 25.
    Sun Q, Zybailov B, Majeran W et al (2009) PPDB, the plant proteomics database at Cornell. Nucleic Acids Res 37:D969–D974PubMedCrossRefGoogle Scholar
  26. 26.
    Johnson TL, Olsen LJ (2003) Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes. Plant Physiol 133:1991–1999PubMedCrossRefGoogle Scholar
  27. 27.
    Bionda T, Tillmann B, Simm S et al (2010) Chloroplast import signals: the length requirement for translocation in vitro and in vivo. J Mol Biol 402:510–523PubMedCrossRefGoogle Scholar
  28. 28.
    Clausen C, Ilkavets I, Thomson R et al (2004) Intracellular localization of VDAC proteins in plants. Planta 220:30–37PubMedCrossRefGoogle Scholar
  29. 29.
    Geissler A, Chacinska A, Truscott KN et al (2002) The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel. Cell 111:507–518PubMedCrossRefGoogle Scholar
  30. 30.
    Jansch L, Kruft V, Schmitz UK et al (1996) New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria. Plant J 9: 357–368PubMedCrossRefGoogle Scholar
  31. 31.
    Salomon M, Fischer K, Flugge UI et al (1990) Sequence analysis and protein import studies of an outer chloroplast envelope polypeptide. Proc Natl Acad Sci USA 87: 5778–5782PubMedCrossRefGoogle Scholar
  32. 32.
    Schleiff E, Tien R, Salomon M et al (2001) Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7. Mol Biol Cell 12:4090–4102PubMedCrossRefGoogle Scholar
  33. 33.
    Goetze TA, Philippar K, Ilkavets I et al (2006) OEP37 is a new member of the chloroplast outer membrane ion channels. J Biol Chem 281:17989–17998PubMedCrossRefGoogle Scholar
  34. 34.
    Schleiff E, Eichacker LA, Eckart K et al (2003) Prediction of the plant beta-barrel proteome: a case study of the chloroplast outer envelope. Protein Sci 12:748–759PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Katharina Wiesemann
    • 1
  • Lucia E. Groß
    • 1
  • Manuel Sommer
    • 1
  • Enrico Schleiff
    • 1
  • Maik S. Sommer
    • 1
  1. 1.Cluster of Excellence Frankfurt, Center for Membrane Proteomics, Department of Biosciences, Molecular Cell BiologyGoethe UniversityFrankfurtGermany

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