Flip-Flopping Membrane Proteins: How the Charge Balance Rule Governs Dynamic Membrane Protein Topology

  • Mikhail BogdanovEmail author
  • Heidi Vitrac
  • William Dowhan
Living reference work entry
Part of the Handbook of Hydrocarbon and Lipid Microbiology book series (HHLM)


Transmembrane and lateral phospholipid asymmetries are not absolute as is the case for integral membrane proteins where asymmetry does not have to be actively maintained due to the enormous energy required to flip across the hydrophobic barrier of the membrane. Although the lipid bilayer is widely considered as a non-flipping zone for most proteins, some integral membrane proteins possess the capacity to reversibly reorient themselves during or after insertion if membrane phospholipid composition is changed, the membrane is depolarized or components of the translocon interact with each other during ATP-driven protein substrate translocation. Membrane proteins can be also engineered to flip after assembly if a strong topological retention signal is introduced at the very end of the polypeptide and then removed post-insertionally. Phosphorylation of an extramembrane domain, which alters its charge nature, could also induce post-insertional topological changes. A structural approach for dynamic membrane protein organization is not achievable by X-ray crystallography. Therefore, a set of unique in vivo and in vitro approaches should be used to establish a detailed mechanistic understanding for how lipid-protein interactions govern dynamic membrane protein structure and function. Novel approaches and concepts have been developed to analyze dynamic lipid-protein interactions and mechanisms of membrane protein folding and topogenesis. Such methods have the advantage of probing the dynamics of biological membrane organization, membrane protein structure, and lipid-protein interactions both in vitro and in vivo.


  1. Awe K, Lambert C, Prange R (2008) Mammalian BiP controls posttranslational ER translocation of the hepatitis B virus large envelope protein. FEBS Lett 582(21–22):3179–3184CrossRefPubMedGoogle Scholar
  2. 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(1):35–42CrossRefPubMedGoogle Scholar
  3. Bogdanov M (2017) Mapping of membrane protein topology by substituted cysteine accessibility method (SCAM™). In: Bacterial protein secretion systems: methods and protocols, pp 105–128CrossRefGoogle Scholar
  4. Bogdanov M, Dowhan W (2012) Lipid-dependent generation of a dual topology for a membrane protein. J Biol Chem 287:37939–37948CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bogdanov M, Heacock PN, Dowhan W (2002) A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition. EMBO J 21(9):2107–2116CrossRefPubMedPubMedCentralGoogle Scholar
  6. 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(2):148–171CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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(5):925–935CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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(15):9637–9641CrossRefPubMedPubMedCentralGoogle Scholar
  9. Bogdanov M, Heacock PN, Dowhan W (2010a) Study of polytopic membrane protein topological organization as a function of membrane lipid composition. Methods Mol Biol 619:79–101CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bogdanov M, Heacock P, Guan Z, Dowhan W (2010b) 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(34):15057–15062CrossRefPubMedPubMedCentralGoogle Scholar
  11. Bogdanov M, Dowhan W, Vitrac H (2014) Lipids and topological rules governing membrane protein assembly. Biochim Biophys Acta 1843(8):1475–1488CrossRefPubMedGoogle Scholar
  12. Bowie JU (2006) Flip-flopping membrane proteins. Nat Struct Mol Biol 13(2):94–96CrossRefPubMedGoogle Scholar
  13. Cheng H-T, London E (2009) Preparation and properties of asymmetric vesicles that mimic cell membranes effect upon lipid raft formation and transmembrane helix orientation. J Biol Chem 284(10):6079–6092CrossRefPubMedPubMedCentralGoogle Scholar
  14. Cymer F, von Heijne G, White SH (2015) Mechanisms of integral membrane protein insertion and folding. J Mol Biol 427(5):999–1022CrossRefPubMedGoogle Scholar
  15. Dale H, Angevine CM, Krebs MP (2000) Ordered membrane insertion of an archaeal opsin in vivo. Proc Natl Acad Sci USA 97(14):7847–7852CrossRefPubMedGoogle Scholar
  16. Dorobantu C, Macovei A, Lazar C, Dwek RA, Zitzmann N, Branza-Nichita N (2011) Cholesterol depletion of hepatoma cells impairs hepatitis B virus envelopment by altering the topology of the large envelope protein. J Virol 85(24):13373–13383CrossRefPubMedPubMedCentralGoogle Scholar
  17. Dowhan W, Bogdanov M (2009) Lipid-dependent membrane protein topogenesis. Annu Rev Biochem 78:515–540CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fleishman SJ, Unger VM, Ben-Tal N (2006) Transmembrane protein structures without X-rays. Trends Biochem Sci 31(2):106–113CrossRefPubMedGoogle Scholar
  19. Gafvelin G, von Heijne G (1994) Topological “frustration” in multispanning E. coli inner membrane proteins. Cell 77(3):401–412CrossRefPubMedGoogle Scholar
  20. Goder V, Bieri C, Spiess M (1999) Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon. J Cell Biol 147(2):257–266CrossRefPubMedPubMedCentralGoogle Scholar
  21. Herate C, Ramdani G, Grant NJ, Marion S, Gasman S, Niedergang F, Benichou S, Bouchet J (2016) Phospholipid scramblase 1 modulates FcR-mediated phagocytosis in differentiated macrophages. PLoS One 11(1):e0145617CrossRefPubMedPubMedCentralGoogle Scholar
  22. Huggett J, Vaughan-Thomas A, Mason D (2000) The open reading frame of the Na+-dependent glutamate transporter GLAST-1 is expressed in bone and a splice variant of this molecule is expressed in bone and brain. FEBS Lett 485(1):13–18CrossRefPubMedGoogle Scholar
  23. Islam ST, Lam JS (2013) Topological mapping methods for alpha-helical bacterial membrane proteins–an update and a guide. Microbiology 2(2):350–364Google Scholar
  24. Jakes KS, Kienker PK, Slatin SL, Finkelstein A (1998) Translocation of inserted foreign epitopes by a channelforming protein. Proc Natl Acad Sci USA 95(8):4321–4326CrossRefPubMedGoogle Scholar
  25. Karlin A, Akabas MH (1998) Substituted-cysteine accessibility method. Methods Enzymol 293:123–145CrossRefPubMedGoogle Scholar
  26. 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(19):4923–4934CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kienker P, Qiu X-Q, Slatin S, Finkelstein A, Jakes K (1997) Transmembrane insertion of the colicin Ia hydrophobic hairpin. J Membrane Biol 157(1):27–37CrossRefGoogle Scholar
  28. Kim SJ, Hegde RS (2002) Cotranslational partitioning of nascent prion protein into multiple populations at the translocation channel. Mol Biol Cell 13(11):3775–3786CrossRefPubMedPubMedCentralGoogle Scholar
  29. Lambert C, Prange R (2003) Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: implications for translocational regulation. Proc Natl Acad Sci 100(9):5199–5204CrossRefPubMedGoogle Scholar
  30. Lee H, Kim H (2014) Membrane topology of transmembrane proteins: determinants and experimental tools. Biochem Biophys Res Commun 453(2):268–276CrossRefPubMedGoogle Scholar
  31. Levy D (1996) Membrane proteins which exhibit multiple topological orientations. Essays Biochem 31:49–60PubMedGoogle Scholar
  32. Liapakis G (2014) Obtaining structural and functional information for GPCRs using the substituted-cysteine accessibility method (SCAM). Curr Pharm Biotechnol 15(10):980–986CrossRefPubMedGoogle Scholar
  33. Lu Y, Turnbull IR, Bragin A, Carveth K, Verkman AS, Skach WR (2000) Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum. Mol Biol Cell 11(9):2973–2985CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lundin M, Monne M, Widell A, Von Heijne G, Persson MA (2003) Topology of the membrane-associated hepatitis C virus protein NS4B. J Virol 77(9):5428–5438CrossRefPubMedPubMedCentralGoogle Scholar
  35. McIlwain BC, Vandenberg RJ, Ryan RM (2015) Transport rates of a glutamate transporter homologue are influenced by the lipid bilayer. J Biol Chem 290(15):9780–9788CrossRefPubMedPubMedCentralGoogle Scholar
  36. 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(4):629–634CrossRefPubMedGoogle Scholar
  37. Nilsson I, von Heijne G (1990) Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids. Cell 62(6):1135–1141CrossRefPubMedGoogle Scholar
  38. Nishiyama K-i, Suzuki T, Tokuda H (1996) Inversion of the membrane topology of SecG coupled with SecA-dependent preprotein translocation. Cell 85(1):71–81CrossRefPubMedGoogle Scholar
  39. Rapp M, Granseth E, Seppälä S, Von Heijne G (2006) Identification and evolution of dual-topology membrane proteins. Nat Struct Mol Biol 13(2):112CrossRefPubMedGoogle Scholar
  40. Rutz C, Rosenthal W, Schulein R (1999) A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain. J Biol Chem 274(47):33757–33763CrossRefPubMedGoogle Scholar
  41. Schlebach JP, Sanders CR (2015) Influence of pathogenic mutations on the energetics of translocon-mediated bilayer integration of transmembrane helices. J Membr Biol 248(3):371–381CrossRefPubMedGoogle Scholar
  42. Schuldiner S (2007) Controversy over EmrE structure. Science 317(5839):748–751CrossRefPubMedGoogle Scholar
  43. Slatin SL, Nardi A, Jakes KS, Baty D, Duché D (2002) Translocation of a functional protein by a voltage-dependent ion channel. Proc Natl Acad Sci 99(3):1286–1291CrossRefPubMedGoogle Scholar
  44. van Klompenburg W, Nilsson I, von Heijne G, de Kruijff B (1997) Anionic phospholipids are determinants of membrane protein topology. EMBO J 16(14):4261–4266CrossRefPubMedPubMedCentralGoogle Scholar
  45. 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 286:15182–15194CrossRefPubMedPubMedCentralGoogle Scholar
  46. 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 USA 110(23):9338–9343CrossRefPubMedGoogle Scholar
  47. Vitrac H, MacLean DM, Jayaraman V, Bogdanov M, Dowhan W (2015) Dynamic membrane protein topological switching upon changes in phospholipid environment. Proc Natl Acad Sci 112(45):13874–13879CrossRefPubMedGoogle Scholar
  48. Vitrac H, Dowhan W, Bogdanov M (2017a) Effects of mixed proximal and distal topogenic signals on the topological sensitivity of a membrane protein to the lipid environment. Biochim Biophys Acta 1859(7):1291–1300CrossRefPubMedPubMedCentralGoogle Scholar
  49. Vitrac H, MacLean DM, Karlstaedt A, Taegtmeyer H, Jayaraman V, Bogdanov M, Dowhan W (2017b) Dynamic lipid-dependent modulation of protein topology by post-translational phosphorylation. J Biol Chem 292(5):1613–1624CrossRefPubMedGoogle Scholar
  50. von Heijne G (2006) Membrane-protein topology. Nat Rev Mol Cell Biol 7(12):909–918CrossRefGoogle Scholar
  51. Wang X, Bogdanov M, Dowhan W (2002) Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition. EMBO J 21(21):5673–5681CrossRefPubMedPubMedCentralGoogle Scholar
  52. Wikstrom M, Xie J, Bogdanov M, Mileykovskaya E, Heacock P, Wieslander A, Dowhan W (2004) Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli. J Biol Chem 279(11):10484–10493CrossRefPubMedGoogle Scholar
  53. Wikstrom M, Kelly AA, Georgiev A, Eriksson HM, Klement MR, Bogdanov M, Dowhan W, Wieslander A (2009) Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function. J Biol Chem 284(2):954–965CrossRefPubMedPubMedCentralGoogle Scholar
  54. Woodall NB, Yin Y, Bowie JU (2015) Dual-topology insertion of a dual-topology membrane protein. Nat Commun 6:8099CrossRefPubMedPubMedCentralGoogle Scholar
  55. Woodall NB, Hadley S, Yin Y, Bowie JU (2017) Complete topology inversion can be part of normal membrane protein biogenesis. Protein Sci 26(4):824–833CrossRefPubMedPubMedCentralGoogle Scholar
  56. 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(28):19172–19178CrossRefPubMedPubMedCentralGoogle Scholar
  57. Yao X, Hong M (2006) Effects of anionic lipid and ion concentrations on the topology and segmental mobility of colicin Ia channel domain from solid-state NMR. Biochemistry 45(1):289–295CrossRefPubMedGoogle Scholar
  58. Zakharov SD, Kotova EA, Antonenko YN, Cramer WA (2004) On the role of lipid in colicin pore formation. Biochim Biophys Acta 1666(1):239–249CrossRefPubMedGoogle Scholar
  59. 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(50):50128–50135CrossRefPubMedGoogle Scholar
  60. Zhang Y, Ren Y, Li S, Hayes JD (2014) Transcription factor Nrf1 is topologically repartitioned across membranes to enable target gene transactivation through its acidic glucose-responsive domains. PLoS One 9(4):e93458CrossRefPubMedPubMedCentralGoogle Scholar
  61. Zhao YJ, Lam CMC, Lee HC (2012) The membrane-bound enzyme CD38 exists in two opposing orientations. Sci Signal 5(241):ra67CrossRefPubMedGoogle Scholar
  62. Zhao YJ, Zhu WJ, Wang XW, Zhang L-H, Lee HC (2015) Determinants of the membrane orientation of a calcium signaling enzyme CD38. Biochim Biophys Acta Mol Cell Res 1853(9):2095–2103CrossRefGoogle Scholar
  63. Zhu Q, Casey JR (2007) Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology. Methods 41(4):439–450CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Mikhail Bogdanov
    • 1
    Email author
  • Heidi Vitrac
    • 1
  • William Dowhan
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Texas Health Science Center, McGovern Medical SchoolHoustonUSA

Personalised recommendations