Article | Published:

How curved membranes recruit amphipathic helices and protein anchoring motifs

Nature Chemical Biology volume 5, pages 835841 (2009) | Download Citation

Abstract

Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50–700 nm in diameter. Our results revealed that sensing is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity. We proposed a model based on curvature-induced defects in lipid packing that related these findings to lipid sorting and accurately predicted the existence of a new ubiquitous class of curvature sensors: membrane-anchored proteins. The fact that unrelated structural motifs such as α-helices and alkyl chains sense MC led us to propose that MC sensing is a generic property of curved membranes rather than a property of the anchoring molecules. We therefore anticipate that MC will promote the redistribution of proteins that are anchored in membranes through other types of hydrophobic moieties.

  • Compound

    1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate

  • Compound

    5-Hexadecanoylaminofluorescein

  • Compound

    5-Dodecanoylaminofluorescein

  • Compound

    5-Octadecanoylaminofluorescein

  • Compound

    N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt

  • Compound

    3,3'-Dioctadecyloxacarbocyanine perchlorate

  • Compound

    1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)

  • Compound

    1,2-Dioleoyl-sn-glycero-3-phosphocholine

  • Compound

    1,2-Dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)

  • Compound

    1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt

  • Compound

    N-((4-Maleimidylmethyl)cyclohexane-1-carbonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine, triethylammonium salt

  • Compound

    3-Amino-9-(2-carboxy-4-(2,3,5,6-tetrafluorobenzoyl)-6-iminoxanthene-4,5-disulfonate, tetraethylammonium salt

  • Compound

    Palmitic acid N-hydroxysuccinimide ester

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell Biol. 7, 9–19 (2006).

  2. 2.

    & Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005).

  3. 3.

    & Sheets, ribbons and tubules - how organelles get their shape. Nat. Rev. Mol. Cell Biol. 8, 258–264 (2007).

  4. 4.

    & Mechanisms of membrane deformation. Curr. Opin. Cell Biol. 15, 372–381 (2003).

  5. 5.

    & Curvature and spatial organization in biological membranes. Soft Matter 3, 24–33 (2007).

  6. 6.

    Membranes are more mosaic than fluid. Nature 438, 578–580 (2005).

  7. 7.

    et al. BAR domains as sensors of membrane curvature: The amphiphysin BAR structure. Science 303, 495–499 (2004).

  8. 8.

    et al. A general amphipathic alpha-helical motif for sensing membrane curvature. Nat. Struct. Mol. Biol. 14, 138–146 (2007).

  9. 9.

    & Amphipathic helices as mediators of the membrane interaction of amphitropic proteins, and as modulators of bilayer physical properties. Curr. Protein Pept. Sci. 7, 539–552 (2006).

  10. 10.

    et al. Role of curvature and phase transition in lipid sorting and fission of membrane tubules. EMBO J. 24, 1537–1545 (2005).

  11. 11.

    & Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132 (2004).

  12. 12.

    , & Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425, 821–824 (2003).

  13. 13.

    , , & Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Nature 426, 563–566 (2003).

  14. 14.

    & Effect of cholesterol, fatty acyl-chain composition, and bilayer curvature on the interaction of cytochrome B(5) with liposomes of phosphatidylcholines. Biochemistry 34, 3841–3850 (1995).

  15. 15.

    , & Thermodynamics of the coil-alpha-helix transition of amphipathic peptides in a membrane environment: the role of vesicle curvature. Biophys. Chem. 96, 191–201 (2002).

  16. 16.

    Thermodynamics of lipid-peptide interactions. Biochim. Biophys. Acta 1666, 40–50 (2004).

  17. 17.

    , , & A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles. Biophys. J. 95, 1176–1188 (2008).

  18. 18.

    et al. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell 122, 605–617 (2005).

  19. 19.

    , , , & Asymmetric tethering of flat and curved lipid membranes by a golgin. Science 320, 670–673 (2008).

  20. 20.

    et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002).

  21. 21.

    α-synuclein has a high affinity for packing defects in a bilayer membrane—a thermodynamics study. J. Biol. Chem. 279, 21966–21975 (2004).

  22. 22.

    et al. Mechanism of endophilin N-BAR domain-mediated membrane curvature. EMBO J. 25, 2898–2910 (2006).

  23. 23.

    , , & Self-assembled microarrays of attoliter molecular vessels. Angew. Chem. Int. Ed. 42, 5580–5583 (2003).

  24. 24.

    , , , & Multiple intermediates in SNARE-induced membrane fusion. Proc. Natl. Acad. Sci. USA 103, 19731–19736 (2006).

  25. 25.

    , & Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers. Proc. Natl. Acad. Sci. USA 104, 18913–18918 (2007).

  26. 26.

    & Surface-based lipid vesicle reactor systems: fabrication and applications. Soft Matter 3, 828–836 (2007).

  27. 27.

    , , & Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous. J. Am. Chem. Soc. 128, 3233–3240 (2006).

  28. 28.

    , & Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 106, 12341–12346 (2009).

  29. 29.

    et al. Amphipathic motifs in BAR domains are essential for membrane curvature sensing. EMBO J. (in the press).

  30. 30.

    et al. Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature. Biochemistry 46, 1779–1790 (2007).

  31. 31.

    et al. Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains. J. Biol. Chem. 278, 28993–28999 (2003).

  32. 32.

    & Geometric packing constraints in egg phosphatidylcholine vesicles. Proc. Natl. Acad. Sci. USA 75, 308–310 (1978).

  33. 33.

    et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210–213 (2008).

  34. 34.

    & Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, 605–611 (2005).

  35. 35.

    & Palmitoylation: policing protein stability and traffic. Nat. Rev. Mol. Cell Biol. 8, 74–84 (2007).

  36. 36.

    et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752 (2005).

  37. 37.

    et al. COPI coat assembly occurs on liquid-disordered domains and the associated membrane deformations are limited by membrane tension. Proc. Natl. Acad. Sci. USA 105, 16946–16951 (2008).

  38. 38.

    Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell Biol. 9, 99–111 (2008).

  39. 39.

    Trafficking and signaling by fatty-acylated and prenylated proteins. Nat. Chem. Biol. 2, 584–590 (2006).

  40. 40.

    et al. Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation. Nature 456, 904–909 (2008).

  41. 41.

    & Binding of acylated peptides and fatty-acids to phospholipid-vesicles—pertinence to myristoylated proteins. Biochemistry 32, 10436–10443 (1993).

  42. 42.

    & Fluorometric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry 33, 3014–3022 (1994).

  43. 43.

    Purification of recombinant G protein α and βγ subunits from Sf9 cells. in G-protein Techniques of Analysis 1st edn, vol. 1 (ed. Manning, D.R.) 23–38 (CRC Press, Washington, D.C., 1999).

  44. 44.

    et al. Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins. Proc. Natl. Acad. Sci. USA 106, 5622–5626 (2009).

  45. 45.

    & Sorting of lipids and proteins in membrane curvature gradients. Biophys. J. 96, 2676–2688 (2009).

  46. 46.

    , , & Physiological membrane tension causes an increase in lipid diffusion: a single molecule fluorescence study. Biophys. J. 96 (suppl. 1): 197a–198a (2009).

  47. 47.

    et al. Effect of vesicle size on their interaction with class A amphipathic helical peptides. J. Lipid Res. 38, 2147–2154 (1997).

  48. 48.

    , , & Geometric cue for protein localization in a bacterium. Science 323, 1354–1357 (2009).

  49. 49.

    , & How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).

  50. 50.

    & Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane. EMBO J. 27, 27–37 (2008).

Download references

Acknowledgements

We thank H. Wennerstrom, T. Heimburg and T. Bjornholm for critically reading the manuscript; J.-B. Perez and K.L. Martinez for help with data treatment; N. Kirkby (University of Copenhagen Hospital, Rigshospitalet, Department of Clinical Microbiology) for providing palmitoylated ovalbumin; J.L. Baneres (Institut des Biomolécules Max Mousseron, University of Montpellier) for the generous contribution of Gβ1γ2 and H. McMahon (MRC Laboratory of Molecular Biology) for kindly providing the plasmid for rat endophilin A1. This work was supported by the Danish Councils for Scientific and Strategic Research and partly by the European Union FP6–2004–IST–4 program NEMOSLAB.

Author information

Author notes

    • Pierre-Yves Bolinger
    •  & John Castillo

    Present addresses: Credit Suisse, Zürich, Switzerland (P.-Y.B.); School of Chemistry, Industrial University of Santander, Bucaramanga, Colombia (J.C.).

    • Nikos S Hatzakis
    •  & Vikram K Bhatia

    These authors contributed equally to this work.

Affiliations

  1. Bio-Nanotechnology Laboratory, Department of Neuroscience and Pharmacology & Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.

    • Nikos S Hatzakis
    • , Vikram K Bhatia
    • , Jannik Larsen
    • , Pierre-Yves Bolinger
    • , Andreas H Kunding
    • , John Castillo
    •  & Dimitrios Stamou
  2. Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.

    • Kenneth L Madsen
    •  & Ulrik Gether
  3. Nano-Science Center, Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark.

    • Per Hedegård

Authors

  1. Search for Nikos S Hatzakis in:

  2. Search for Vikram K Bhatia in:

  3. Search for Jannik Larsen in:

  4. Search for Kenneth L Madsen in:

  5. Search for Pierre-Yves Bolinger in:

  6. Search for Andreas H Kunding in:

  7. Search for John Castillo in:

  8. Search for Ulrik Gether in:

  9. Search for Per Hedegård in:

  10. Search for Dimitrios Stamou in:

Contributions

V.K.B. developed the assay; N.S.H. designed most experiments and recorded and treated most data with help from J.L., V.K.B., P.-Y.B. and J.C.; A.H.K. developed the image treatment; K.L.M. purified and labeled the AH and GST constructs; P.H. helped formulate the model; D.S. designed and supervised the project and wrote the main text together with N.S.H. and V.K.B. The manuscript was discussed and corrected by all co-authors.

Corresponding author

Correspondence to Dimitrios Stamou.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Methods and Supplementary Results

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchembio.213

Further reading