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Blue Native PAGE Analysis of Bacterial Secretion Complexes

  • Susann Zilkenat
  • Tobias Dietsche
  • Julia V. Monjarás Feria
  • Claudia E. Torres-Vargas
  • Mehari Tesfazgi Mebrhatu
  • Samuel Wagner
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.

Key words

Bacterial secretion systems Membrane proteins Blue native polyacrylamide gel electrophoresis Two-dimensional polyacrylamide gel electrophoresis Sucrose gradients Bacterial cell fractionation Type III secretion systems Salmonella typhimurium Escherichia coli 

Notes

Acknowledgments

Work performed in the laboratory of SW was supported by the Alexander von Humboldt Foundation in the framework of the Sofja Kovalevskaja Award endowed by the Federal Ministry of Education and Research (BMBF) and in the framework of the Georg Forster Research Fellowships (to J.V.M.F.), and by the Deutsche Forschungsgemeinschaft (DFG) as part of the Collaborative Research Center (SFB) 766 Bacterial cell envelope, project B14.

References

  1. 1.
    Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231CrossRefPubMedGoogle Scholar
  2. 2.
    Bessonneau P, Besson V, Collinson I, Duong F (2002) The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure. EMBO J 21:995–1003CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Oates J, Barrett CML, Barnett JP, Byrne KG, Bolhuis A, Robinson C (2005) The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex. J Mol Biol 346:295–305CrossRefPubMedGoogle Scholar
  4. 4.
    Wiedemann N, Kozjak V, Chacinska A, Schönfisch B, Rospert S, Ryan MT, Pfanner N, Meisinger C (2003) Machinery for protein sorting and assembly in the mitochondrial outer membrane. Nature 424:565–571CrossRefPubMedGoogle Scholar
  5. 5.
    Kikuchi S, Hirohashi T, Nakai M (2006) Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE. Plant Cell Physiol 47:363–371CrossRefPubMedGoogle Scholar
  6. 6.
    Wagner S, Baars L, Ytterberg AJ, Klussmeier A, Wagner CS, Nord O, Nygren PA, van Wijk KJ, de Gier JW (2007) Consequences of membrane protein overexpression in Escherichia coli. Mol Cell Proteomics 6:1527–1550CrossRefPubMedGoogle Scholar
  7. 7.
    Baars L, Wagner S, Wickström D, Klepsch M, Ytterberg AJ, van Wijk KJ, de Gier JW (2008) Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli. J Bacteriol 190:3505–3525CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wagner S, Klepsch MM, Schlegel S, Appel A, Draheim R, Tarry M, Högbom M, van Wijk KJ, Slotboom DJ, Persson JO, de Gier JW (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105:14371–14376CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Wickström D, Wagner S, Baars L, Ytterberg AJ, Klepsch M, van Wijk KJ, Luirink J, de Gier JW (2011) Consequences of depletion of the signal recognition particle in Escherichia coli. J Biol Chem 286:4598–4609CrossRefPubMedGoogle Scholar
  10. 10.
    Wickström D, Wagner S, Simonsson P, Pop O, Baars L, Ytterberg AJ, van Wijk KJ, Luirink J, de Gier JWL (2011) Characterization of the consequences of YidC depletion on the inner membrane proteome of Escherichia coli using 2D blue native/SDS-PAGE. J Mol Biol 409:124–135CrossRefPubMedGoogle Scholar
  11. 11.
    Krall L, Wiedemann U, Unsin G, Weiss S, Domke N, Baron C (2002) Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens. Proc Natl Acad Sci U S A 99:11405–11410CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kuroda T, Kubori T, Thanh Bui X, Hyakutake A, Uchida Y, Imada K, Nagai H (2015) Molecular and structural analysis of Legionella DotI gives insights into an inner membrane complex essential for type IV secretion. Sci Rep 5:10912CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wagner S, Königsmaier L, Lara-Tejero M, Lefebre M, Marlovits TC, Galán JE (2010) Organization and coordinated assembly of the type III secretion export apparatus. Proc Natl Acad Sci U S A 107:17745–17750CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lara-Tejero M, Kato J, Wagner S, Liu X, Galán JE (2011) A sorting platform determines the order of protein secretion in bacterial type III systems. Science 331:1188–1191CrossRefPubMedGoogle Scholar
  15. 15.
    Fischer M, Zilkenat S, Gerlach RG, Wagner S, Renard BY (2014) Pre- and post-processing workflow for affinity purification mass spectrometry data. J Proteome Res 13:2239–2249CrossRefPubMedGoogle Scholar
  16. 16.
    Monjarás Feria JV, Lefebre MD, Stierhof YD, Galán JE, Wagner S (2015) Role of autocleavage in the function of a type III secretion specificity switch protein in Salmonella enterica serovar Typhimurium. MBio 6:e01459–e01415CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zilkenat S, Franz-Wachtel M, Stierhof YD, Galán JE, Macek B, Wagner S (2016) Determination of the stoichiometry of the complete bacterial type III secretion needle complex using a combined quantitative proteomic approach. Mol Cell ProteomicsGoogle Scholar
  18. 18.
    Houben ENG, Bestebroer J, Ummels R, Wilson L, Piersma SR, Jiménez CR, Ottenhoff THM, Luirink J, Bitter W (2012) Composition of the type VII secretion system membrane complex. Mol Microbiol 86:472–484CrossRefPubMedGoogle Scholar
  19. 19.
    Wittig I, Braun HP, Schägger H (2006) Blue native PAGE. Nat Protoc 1:418–428CrossRefPubMedGoogle Scholar
  20. 20.
    Schraidt O, Marlovits TC (2011) Three-dimensional model of Salmonella’s needle complex at subnanometer resolution. Science 331:1192–1195CrossRefPubMedGoogle Scholar
  21. 21.
    Loquet A, Sgourakis NG, Gupta R, Giller K, Riedel D, Goosmann C, Griesinger C, Kolbe M, Baker D, Becker S, Lange A (2012) Atomic model of the type III secretion system needle. Nature 486:276–279CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Klepsch M, Schlegel S, Wickström D, Friso G, van Wijk KJ, Persson JO, de Gier JW, Wagner S (2008) Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes. Methods 46:48–53CrossRefPubMedGoogle Scholar
  23. 23.
    Schlegel S, Klepsch M, Wickström D, Wagner S, de Gier JW (2010) Comparative analysis of cytoplasmic membrane proteomes of Escherichia coli using 2D blue native/SDS-PAGE. Methods Mol Biol 619:257–269CrossRefPubMedGoogle Scholar
  24. 24.
    Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262CrossRefPubMedGoogle Scholar
  25. 25.
    Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858CrossRefGoogle Scholar
  26. 26.
    Sessler N, Krug K, Nordheim A, Mordmüller B, Macek B (2012) Analysis of the Plasmodium falciparum proteasome using blue native PAGE and label-free quantitative mass spectrometry. Amino Acids 43:1119–1129CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Susann Zilkenat
    • 1
  • Tobias Dietsche
    • 1
  • Julia V. Monjarás Feria
    • 1
  • Claudia E. Torres-Vargas
    • 1
  • Mehari Tesfazgi Mebrhatu
    • 1
  • Samuel Wagner
    • 1
    • 2
  1. 1.Section of Cellular and Molecular Microbiology, Interfaculty Institute of Microbiology and Infection Medicine (IMIT)University of TübingenTübingenGermany
  2. 2.German Center for Infection Research (DZIF)TübingenGermany

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