GFP tagging sheds light on protein translocation: implications for key methods in cell biology

Cellular and Molecular Life Sciences volume 69, pages1025–1033(2012)Cite this article

Abstract

Green fluorescent protein (GFP) is a powerful tool for studying gene expression, protein localization, protein–protein interactions, calcium concentrations, and redox potentials owing to its intrinsic fluorescence. However, GFP not only contains a chromophore but is also tightly folded in a temperature-dependent manner. The latter property of GFP has recently been exploited (1) to characterize the translocase of the outer mitochondrial membrane and (2) to discriminate between protein transport across and into biomembranes in vivo. I therefore suggest that GFP could be a valuable tool for the general analysis of protein transport machineries and pathways in a variety of organisms. Moreover, results from such studies could be important for the interpretation and optimization of classical experiments using GFP tagging.

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References

  1. 1.

    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Zimmer M (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102(3):759–781

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312(5771):217–224

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA 95(12):6803–6808

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Matsuyama S, Llopis J, Deveraux QL, Tsien RY, Reed JC (2000) Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2(6):318–325

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Bjornberg O, Ostergaard H, Winther JR (2006) Measuring intracellular redox conditions using GFP-based sensors. Antioxid Redox Signal 8(3–4):354–361

    PubMed  Article  Google Scholar 

  7. 7.

    Gutscher M, Pauleau AL, Marty L, Brach T, Wabnitz GH, Samstag Y, Meyer AJ, Dick TP (2008) Real-time imaging of the intracellular glutathione redox potential. Nat Methods 5(6):553–559

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Meyer AJ, Dick TP (2010) Fluorescent protein-based redox probes. Antioxid Redox Signal 13(5):621–650

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Ostergaard H, Henriksen A, Hansen FG, Winther JR (2001) Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J 20(21):5853–5862

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Craggs TD (2009) Green fluorescent protein: structure, folding and chromophore maturation. Chem Soc Rev 38(10):2865–2875

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Reid BG, Flynn GC (1997) Chromophore formation in green fluorescent protein. Biochemistry 36(22):6786–6791

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Fukuda H, Arai M, Kuwajima K (2000) Folding of green fluorescent protein and the cycle3 mutant. Biochemistry 39(39):12025–12032

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Hsu ST, Blaser G, Jackson SE (2009) The folding, stability and conformational dynamics of beta-barrel fluorescent proteins. Chem Soc Rev 38(10):2951–2965

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Harner M, Neupert W, Deponte M (2011) Lateral release of proteins from the TOM complex into the outer membrane of mitochondria. EMBO J 30(16):3232–3241

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Dalbey RE, Wang P, Kuhn A (2011) Assembly of bacterial inner membrane proteins. Annu Rev Biochem 80:161–187

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Zimmermann R, Eyrisch S, Ahmad M, Helms V (2011) Protein translocation across the ER membrane. Biochim Biophys Acta 1808(3):912–924

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Robinson C, Matos CF, Beck D, Ren C, Lawrence J, Vasisht N, Mendel S (2011) Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria. Biochim Biophys Acta 1808(3):876–884

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Funes S, Kauff F, van der Sluis EO, Ott M, Herrmann JM (2011) Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts. Biol Chem 392(1–2):13–19

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Hagan CL, Silhavy TJ, Kahne D (2011) β-Barrel membrane protein assembly by the Bam complex. Annu Rev Biochem 80:189–210

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Dukanovic J, Rapaport D (2011) Multiple pathways in the integration of proteins into the mitochondrial outer membrane. Biochim Biophys Acta 1808(3):971–980

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Sommer MS, Daum B, Gross LE, Weis BL, Mirus O, Abram L, Maier UG, Kuhlbrandt W, Schleiff E (2011) Chloroplast Omp85 proteins change orientation during evolution. Proc Natl Acad Sci USA 108(33):13841–13846

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N (2009) Importing mitochondrial proteins: machineries and mechanisms. Cell 138(4):628–644

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Endo T, Yamano K (2009) Multiple pathways for mitochondrial protein traffic. Biol Chem 390(8):723–730

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Schleiff E, Becker T (2011) Common ground for protein translocation: access control for mitochondria and chloroplasts. Natl Rev Mol Cell Biol 12(1):48–59

    Article  CAS  Google Scholar 

  25. 25.

    Bagola K, Mehnert M, Jarosch E, Sommer T (2011) Protein dislocation from the ER. Biochim Biophys Acta 1808(3):925–936

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Meinecke M, Cizmowski C, Schliebs W, Kruger V, Beck S, Wagner R, Erdmann R (2010) The peroxisomal importomer constitutes a large and highly dynamic pore. Nat Cell Biol 12(3):273–277

    PubMed  CAS  Google Scholar 

  27. 27.

    Bleves S, Viarre V, Salacha R, Michel GP, Filloux A, Voulhoux R (2010) Protein secretion systems in Pseudomonas aeruginosa: a wealth of pathogenic weapons. Int J Med Microbiol 300(8):534–543

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Simeone R, Bottai D, Brosch R (2009) ESX/type VII secretion systems and their role in host-pathogen interaction. Curr Opin Microbiol 12(1):4–10

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    de Koning-Ward TF, Gilson PR, Boddey JA, Rug M, Smith BJ, Papenfuss AT, Sanders PR, Lundie RJ, Maier AG, Cowman AF, Crabb BS (2009) A newly discovered protein export machine in malaria parasites. Nature 459(7249):945–949

    PubMed  Article  Google Scholar 

  30. 30.

    Spork S, Hiss JA, Mandel K, Sommer M, Kooij TW, Chu T, Schneider G, Maier UG, Przyborski JM (2009) An unusual ERAD-like complex is targeted to the apicoplast of Plasmodium falciparum. Eukaryot Cell 8(8):1134–1145

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Vogel F, Bornhovd C, Neupert W, Reichert AS (2006) Dynamic subcompartmentalization of the mitochondrial inner membrane. J Cell Biol 175(2):237–247

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Rapaport D (2005) How does the TOM complex mediate insertion of precursor proteins into the mitochondrial outer membrane? J Cell Biol 171(3):419–423

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Karbowski M, Youle RJ (2011) Regulating mitochondrial outer membrane proteins by ubiquitination and proteasomal degradation. Curr Opin Cell Biol 23(4):476–482

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Hearn EM, Patel DR, Lepore BW, Indic M, van den Berg B (2009) Transmembrane passage of hydrophobic compounds through a protein channel wall. Nature 458(7236):367–370

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Popov-Celeketic D, Mapa K, Neupert W, Mokranjac D (2008) Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria. EMBO J 27(10):1469–1480

    PubMed  CAS  Google Scholar 

  36. 36.

    Chacinska A, Rehling P, Guiard B, Frazier AE, Schulze-Specking A, Pfanner N, Voos W, Meisinger C (2003) Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex. EMBO J 22(20):5370–5381

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Donzeau M, Kaldi K, Adam A, Paschen S, Wanner G, Guiard B, Bauer MF, Neupert W, Brunner M (2000) Tim23 links the inner and outer mitochondrial membranes. Cell 101(4):401–412

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Ishihara N, Mihara K (1998) Identification of the protein import components of the rat mitochondrial inner membrane, rTIM17, rTIM23, and rTIM44. J Biochem 123(4):722–732

    PubMed  CAS  Google Scholar 

  39. 39.

    Bauer MF, Gempel K, Reichert AS, Rappold GA, Lichtner P, Gerbitz KD, Neupert W, Brunner M, Hofmann S (1999) Genetic and structural characterization of the human mitochondrial inner membrane translocase. J Mol Biol 289(1):69–82

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Murcha MW, Lister R, Ho AY, Whelan J (2003) Identification, expression, and import of components 17 and 23 of the inner mitochondrial membrane translocase from Arabidopsis. Plant Physiol 131(4):1737–1747

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Eilers M, Schatz G (1986) Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature 322(6076):228–232. doi:10.1038/322228a0

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    Eilers M, Hwang S, Schatz G (1988) Unfolding and refolding of a purified precursor protein during import into isolated mitochondria. EMBO J 7(4):1139–1145

    PubMed  CAS  Google Scholar 

  43. 43.

    Rassow J, Guiard B, Wienhues U, Herzog V, Hartl FU, Neupert W (1989) Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites. J Cell Biol 109(4 Pt 1):1421–1428

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    Chen WJ, Douglas MG (1987) The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation. J Biol Chem 262(32):15605–15609

    PubMed  CAS  Google Scholar 

  45. 45.

    Endo T, Nakayama Y, Nakai M (1995) Avidin fusion protein as a tool to generate a stable translocation intermediate spanning the mitochondrial membranes. J Biochem 118(4):753–759

    PubMed  CAS  Google Scholar 

  46. 46.

    Schulke N, Sepuri NB, Gordon DM, Saxena S, Dancis A, Pain D (1999) A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates. J Biol Chem 274(32):22847–22854

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    Schulke N, Sepuri NB, Pain D (1997) In vivo zippering of inner and outer mitochondrial membranes by a stable translocation intermediate. Proc Natl Acad Sci USA 94(14):7314–7319

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    Vestweber D, Schatz G (1988) A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites. J Cell Biol 107(6 Pt 1):2037–2043

    PubMed  Article  CAS  Google Scholar 

  49. 49.

    Kobayashi T, Morone N, Kashiyama T, Oyamada H, Kurebayashi N, Murayama T (2008) Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research. PLoS One 3(12):e3822

    PubMed  Article  Google Scholar 

  50. 50.

    Zhuang R, Zhang Y, Zhang R, Song C, Yang K, Yang A, Jin B (2008) Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography. Protein Expr Purif 59(1):138–143

    PubMed  Article  CAS  Google Scholar 

  51. 51.

    Kumar A, Agarwal S, Heyman JA, Matson S, Heidtman M, Piccirillo S, Umansky L, Drawid A, Jansen R, Liu Y, Cheung KH, Miller P, Gerstein M, Roeder GS, Snyder M (2002) Subcellular localization of the yeast proteome. Genes Dev 16(6):707–719

    PubMed  Article  CAS  Google Scholar 

  52. 52.

    Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O’Shea EK (2003) Global analysis of protein localization in budding yeast. Nature 425(6959):686–691

    PubMed  Article  CAS  Google Scholar 

  53. 53.

    Prokisch H, Scharfe C, Camp DG 2nd, Xiao W, David L, Andreoli C, Monroe ME, Moore RJ, Gritsenko MA, Kozany C, Hixson KK, Mottaz HM, Zischka H, Ueffing M, Herman ZS, Davis RW, Meitinger T, Oefner PJ, Smith RD, Steinmetz LM (2004) Integrative analysis of the mitochondrial proteome in yeast. PLoS Biol 2(6):e160

    PubMed  Article  Google Scholar 

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Acknowledgments

I thank the Deutsche Forschungsgemeinschaft (DFG) for financial support of my work on mitochondrial protein transport (Grant DE 1431/2). I would also like to thank Max Harner for his extraordinary work on the TOM complex.

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  1. Department of Parasitology, Ruprecht-Karls University, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany

    Marcel Deponte

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Correspondence to Marcel Deponte.

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Deponte, M. GFP tagging sheds light on protein translocation: implications for key methods in cell biology. Cell. Mol. Life Sci. 69, 1025–1033 (2012). https://doi.org/10.1007/s00018-012-0932-6

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Keywords

  • Green fluorescent protein
  • Protein translocase
  • Protein transport machineries
  • Protein localization
  • GFP tagging