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Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAM™)

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Bacterial Protein Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1615))

Abstract

A described simple and advanced protocol for the substituted-cysteine accessibility method as applied to transmembrane (TM) orientation (SCAM™) permits a topology analysis of proteins in their native state and can be universally adapted to any membrane system to either systematically map an uniform topology or identify and quantify the degree of mixed topology. In this approach, noncritical individual amino acids that are thought to reside in the putative extracellular or intracellular loops of a membrane protein are replaced one at a time by cysteine residue, and the orientation with respect to the membrane is evaluated using a pair of membrane-impermeable nondetectable and detectable thiol-reactive labeling reagents.

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References

  1. von Heijne G (2006) Membrane-protein topology. Nat Rev Mol Cell Biol 7:909–918

    Article  CAS  Google Scholar 

  2. Bogdanov M, Xie J, Dowhan W (2009) Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive inside rule. J Biol Chem 284:9637–9641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dowhan W, Bogdanov M (2009) Lipid-dependent membrane protein topogenesis. Annu Rev Biochem 78:515–540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bogdanov M, Dowhan W, Vitrac H (2014) Lipids and topological rules governing membrane protein assembly. Biochim Biophys Acta 1843:1475–1488

    Article  CAS  PubMed  Google Scholar 

  5. Bogdanov M, Zhang W, Xie J, Dowhan W (2005) Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM™): application to lipid-specific membrane protein topogenesis. Methods 36:148–171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fleishman SJ, Unger VM, Ben-Tal N (2006) Transmembrane protein structures without X-rays. Trends Biochem Sci 31:106–113

    Article  CAS  PubMed  Google Scholar 

  7. Lacapere JJ, Pebay-Peyroula E, Neumann JM, Etchebest C (2007) Determining membrane protein structures: still a challenge! Trends Biochem Sci 32:259–270

    Article  CAS  PubMed  Google Scholar 

  8. Bochud A, Ramachandra N, Conzelmann A (2013) Adaptation of low-resolution methods for the study of yeast microsomal polytopic membrane proteins: a methodological review. Biochem Soc Trans 41:35–42

    Article  CAS  PubMed  Google Scholar 

  9. Bogdanov M, Heacock PN, Dowhan W (2010) Study of polytopic membrane protein topological organization as a function of membrane lipid composition. Methods Mol Biol 619:79–101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bogdanov M, Xie J, Heacock P, Dowhan W (2008) To flip or not to flip: lipid-protein charge interactions are a determinant of final membrane protein topology. J Cell Biol 182:925–935

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nasie I, Steiner-Mordoch S, Gold A, Schuldiner S (2010) Topologically random insertion of EmrE supports a pathway for evolution of inverted repeats in ion-coupled transporters. J Biol Chem 285:15234–15244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhu Q, Casey JR (2007) Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology. Methods 41:439–450

    Article  CAS  PubMed  Google Scholar 

  13. Islam ST, Lam JS (2013) Topological mapping methods for alpha-helical bacterial membrane proteins--an update and a guide. Microbiology 2:350–364

    CAS  Google Scholar 

  14. Lee H, Kim H (2014) Membrane topology of transmembrane proteins: determinants and experimental tools. Biochem Biophys Res Commun 453:268–276

    Article  CAS  PubMed  Google Scholar 

  15. Liapakis G (2014) Obtaining structural and functional information for GPCRs using the substituted-cysteine accessibility method (SCAM). Curr Pharm Biotechnol 15:980–986

    Article  CAS  PubMed  Google Scholar 

  16. van Geest M, Lolkema JS (2000) Membrane topology and insertion of membrane proteins: search for topogenic signals. Microbiol Mol Biol Rev 64:13–33

    Article  PubMed  PubMed Central  Google Scholar 

  17. van Geest M, Lolkema JS (1999) Transmembrane segment (TMS) VIII of the Na(+)/citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane. J Biol Chem 274:29705–29711

    Article  PubMed  Google Scholar 

  18. Karlin A, Akabas MH (1998) Substituted-cysteine accessibility method. Methods Enzymol 293:123–145

    Article  CAS  PubMed  Google Scholar 

  19. Bogdanov M, Heacock PN, Dowhan W (2002) A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition. EMBO J 21:2107–2116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bogdanov M, Heacock P, Guan Z, Dowhan W (2010) Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli. Proc Natl Acad Sci U S A 107:15057–15062

    Article  PubMed  PubMed Central  Google Scholar 

  21. Bogdanov M, Dowhan W (2012) Lipid-dependent generation of a dual topology for a membrane protein. J Biol Chem 287:37939–37948

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Vitrac H, Bogdanov M, Heacock P, Dowhan W (2011) Lipids and topological rules of membrane protein assembly: balance between long- and short-range lipid-protein interactions. J Biol Chem 286:15182–15194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vitrac H, Bogdanov M, Dowhan W (2013) In vitro reconstitution of lipid-dependent dual topology and postassembly topological switching of a membrane protein. Proc Natl Acad Sci U S A 110:9338–9343

    Article  PubMed  PubMed Central  Google Scholar 

  24. Vitrac H, Bogdanov M, Dowhan W (2013) Proper fatty acid composition rather than an ionizable lipid amine is required for full transport function of lactose permease from Escherichia coli. J Biol Chem 288:5873–5885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tang XB, Casey JR (1999) Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1. Biochemistry 38:14565–14572

    Article  CAS  PubMed  Google Scholar 

  26. Hu YK, Kaplan JH (2000) Site-directed chemical labeling of extracellular loops in a membrane protein. The topology of the Na,K-ATPase alpha-subunit. J Biol Chem 275:19185–19191

    Article  CAS  PubMed  Google Scholar 

  27. Nagamori S, Nishiyama K, Tokuda H (2002) Membrane topology inversion of SecG detected by labeling with a membrane-impermeable sulfhydryl reagent that causes a close association of SecG with SecA. J Biochem 132:629–634

    Article  CAS  PubMed  Google Scholar 

  28. Dale H, Angevine CM, Krebs MP (2000) Ordered membrane insertion of an archaeal opsin in vivo. Proc Natl Acad Sci U S A 97:7847–7852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kerr JE, Christie PJ (2010) Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the agrobacterium VirB/VirD4 type IV secretion system. J Bacteriol 192:4923–4934

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Xie J, Bogdanov M, Heacock P, Dowhan W (2006) Phosphatidylethanolamine and monoglucosyldiacylglycerol are interchangeable in supporting topogenesis and function of the polytopic membrane protein lactose permease. J Biol Chem 281:19172–19178

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang W, Bogdanov M, Pi J, Pittard AJ, Dowhan W (2003) Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition. J Biol Chem 278:50128–50135

    Article  CAS  PubMed  Google Scholar 

  32. Wang X, Bogdanov M, Dowhan W (2002) Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition. EMBO J 21:5673–5681

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bernsel A, Viklund H, Falk J, Lindahl E, von Heijne G, Elofsson A (2008) Prediction of membrane-protein topology from first principles. Proc Natl Acad Sci U S A 105:7177–7181

    Article  PubMed  PubMed Central  Google Scholar 

  34. Zhao G, London E (2006) An amino acid “transmembrane tendency” scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: relationship to biological hydrophobicity. Protein Sci 15:1987–2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Dobson L, Remenyi I, Tusnady GE (2015) CCTOP: a consensus constrained TOPology prediction web server. Nucleic Acids Res 43:W408–W412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bayer EA, Zalis MG, Wilchek M (1985) 3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent. Anal Biochem 149:529–536

    Article  CAS  PubMed  Google Scholar 

  37. Berezuk AM, Goodyear M, Khursigara CM (2014) Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK. J Biol Chem 289:23287–23301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Moss K, Helm A, Lu Y, Bragin A, Skach WR (1998) Coupled translocation events generate topological heterogeneity at the endoplasmic reticulum membrane. Mol Biol Cell 9:2681–2697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Woodall NB, Yin Y, Bowie JU (2015) Dual-topology insertion of a dual-topology membrane protein. Nat Commun 6:8099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gafvelin G, von Heijne G (1994) Topological “frustration” in multispanning E. coli inner membrane proteins. Cell 77:401–412

    Article  CAS  PubMed  Google Scholar 

  41. Nasie I, Steiner-Mordoch S, Schuldiner S (2013) Topology determination of untagged membrane proteins. Methods Mol Biol 1033:121–130

    Article  CAS  PubMed  Google Scholar 

  42. Gelis-Jeanvoine S, Lory S, Oberto J, Buddelmeijer N (2015) Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction. Mol Microbiol 95:692–705

    Article  CAS  PubMed  Google Scholar 

  43. Liu Y, Basu A, Li X, Fliegel L (2015) Topological analysis of the Na+/H+ exchanger. Biochim Biophys Acta 1848:2385–2393

    Article  CAS  PubMed  Google Scholar 

  44. Abramson J, Smirnova I, Kasho V, Verner G, Kaback HR, Iwata S (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science 301:610–615

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, University of Texas Health Science Center at Houston, McGovern Medical School, UT-GSBS, P.O. Box 20334, Houston, TX, 77030, USA

    Mikhail Bogdanov

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  1. Mikhail Bogdanov
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Correspondence to Mikhail Bogdanov .

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Editors and Affiliations

  1. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Laure Journet

  2. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Eric Cascales

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Bogdanov, M. (2017). Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAM™). In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7033-9_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7031-5

  • Online ISBN: 978-1-4939-7033-9

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