Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

  • B. V. Belugin
  • I. M. Zhestkova
  • M. S. Trofimova


The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm3 and in the DRM fraction (1.146 g/cm3). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm3 had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm3. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.


Pisum sativum L. plasma membrane buoyant density detergent-resistant membranes sterols PIP-aquaporins laurdan 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Verkman A.S. 1992. Water channels in cell membranes. Annu. Rev. Physiol. 54, 97–108.PubMedCrossRefGoogle Scholar
  2. 2.
    Agre P., Sasaki S., Chrispeels M.J. 1993. Aquaporins: A family of water channel proteins. Am. J. Physiol. 265, F641–F677.Google Scholar
  3. 3.
    Maurel C. 1997. Aquaporins and water permeability of plant membranes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 399–429.PubMedCrossRefGoogle Scholar
  4. 4.
    Tyerman S.D., Bohnert H.J., Maurel C., Steule E., Smith J.A.C. 1999. Plant aquaporins: Their molecular biology, biophysics and significance for plant water relations. J. Exp. Bot. 50, 1055–1071.CrossRefGoogle Scholar
  5. 5.
    Verkman A.S., Mitra A.K. 2000. Structure and function of aquaporin water channels. Am. J. Physiol. Renal. Physiol. 278, F13–F28.PubMedGoogle Scholar
  6. 6.
    Zeidel M.L., Ambudkar S.V., Smith B. L., Agre P. 1992. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry. 25, 7436–7440.CrossRefGoogle Scholar
  7. 7.
    Preston G.M., Agre P. 1991. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: Member of an ancient channel family. Proc. Natl. Acad. Sci. USA. 88, 11110–11114.PubMedCrossRefGoogle Scholar
  8. 8.
    Zardoya R., Villalba S. 2001. A phylogenetic framework for the aquaporin family in eukaryotes. J. Mol. Evol. 52, 391–404.PubMedGoogle Scholar
  9. 9.
    Hachez C., Zelazny E., Chaumont F. 2006. Modulating the expression of aquaporin genes in planta: A key to understand their physiological functions? Biochim. Biophys. Acta. 1758, 1142–1156.PubMedCrossRefGoogle Scholar
  10. 10.
    Maurel C., Verdoucq L., Luu D.-T., Santoni V. 2008. Plant aquaporins: Membrane channels with multiple integrated functions. Annu. Rev. Plant Biol. 59, 595–624.PubMedCrossRefGoogle Scholar
  11. 11.
    Lande M.B., Donovan J.M., Zeidel M.L. 1995. The relationship between membrane fluidity and permeabilities to water, solutes, ammonia, and protons. J. Gen. Physiol. 106, 67–84.PubMedCrossRefGoogle Scholar
  12. 12.
    Olbrich K., Rawicz W., Needman D., Evans E. 2000. Water permeability and mechanical strength of polyunsaturated lipid bilayers. Biophys. J. 79, 321–327.PubMedCrossRefGoogle Scholar
  13. 13.
    Mathai J.C., Tristram-Nagle S., Nagle J.F., Zeidel M.L. 2008. Structural determinants of water permeability through the lipid membrane. J. Gen. Physiol. 31, 69–76.Google Scholar
  14. 14.
    Simons K., Ikonen E. 1997. Functional rafts in cell membranes. Nature. 387, 569–572.PubMedCrossRefGoogle Scholar
  15. 15.
    Brown D.A., London E. 2000. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224.PubMedCrossRefGoogle Scholar
  16. 16.
    Pike L.J. 2003. Lipid rafts: Bringing order to chaos. J. Lipid Res. 44, 655–667.PubMedCrossRefGoogle Scholar
  17. 17.
    Lingwood D., Simons K. 2010. Lipid rafts as a membrane organizing principle. Science. 327, 46–50.PubMedCrossRefGoogle Scholar
  18. 18.
    Simons K., Vaz W.L.C. 2004. Model systems, lipid rafts, and cell membranes. Annu. Rev. Biophys. Biomol. Struct. 33, 269–295.PubMedCrossRefGoogle Scholar
  19. 19.
    Brown D.A. 2006. Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology. 21, 430–439.PubMedCrossRefGoogle Scholar
  20. 20.
    Zappel N.F., Panstruga R. 2008. Heterogeneity and lateral compartmentalization of plant plasma membranes. Curr. Opin. Plant Biol. 11, 632–640.PubMedCrossRefGoogle Scholar
  21. 21.
    Kusumi A., Suzuki K. 2005. Toward understanding the dynamics of membrane-raft-based molecular interactions. Biochim. Biophys. Acta. 1746, 234–251.PubMedCrossRefGoogle Scholar
  22. 22.
    Lichtenberg D., Gonñi F.M. Heerklotz H. 2005. Detergent-resistant membranes should not be identified with membrane rafts. Trends Biochem. Sci. 30, 430–436.PubMedCrossRefGoogle Scholar
  23. 23.
    Lingwood D., Simons K. 2007. Detergent resistance as a tool in membrane research. Nat. Prot. 2, 2159–2165.CrossRefGoogle Scholar
  24. 24.
    Mongrand S., Morel J., Laroche J., Claverol S., Carde J.-P., Hartmann M.-A., Bonneu M., Simon-Plas F., Lessire R., Bessoule J.-J. 2004. Lipid rafts in higher plant cells. J. Biol. Chem. 279, 36277–36286.PubMedCrossRefGoogle Scholar
  25. 25.
    Borner G.H.H., Sherrier D.J., Weimar T., Michaelson L.V., Hawkins N.D., MacAskill A., Napier J.A., Beale M.H., Lilley K.S., Dupree P. 2005. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiol. 137, 104–116.PubMedCrossRefGoogle Scholar
  26. 26.
    Morel J., Claverol S., Mongrand S., Furt F., Fromentin J., Bessoule J.-J., Blein J.-P., Simon-Plas F. 2006. Proteomics of plant detergent-resistant membranes. Mol. Cell. Proteomics. 5, 1396–1411.PubMedCrossRefGoogle Scholar
  27. 27.
    Marmagne A., Ferro M., Meinnel T., Bruley C., Kuhn L., Garin J., Barbier-Brygoo H., Ephritikhine G. 2007. A high content in lipid-modified peripheral proteins and integral receptor kinases features in the Arabidopsis plasma membrane proteome. Mol. Cell. Proteomics. 6, 1980–1996.PubMedCrossRefGoogle Scholar
  28. 28.
    Larsson C., Sommarin M., Widell S. 1994. Isolation of highly purified plasma membranes and the separation of inside-out and right-side-out vesicles. Methods Enzymol. 228, 451–469.CrossRefGoogle Scholar
  29. 29.
    Ampilogova Ya.N., Zhestkova I.M., Trofimova M.S. 2006. Redox modulation of osmotic water permeability in plasma membranes isolated from roots and shoots of pea seedlings. Fiziologia rastenii (Rus.) 53, 622–628.Google Scholar
  30. 30.
    Briskin D.P., Leonard R.T., Hodges T.K. 1987. Isolation of the plasma membrane: Membrane markers and general principles. Methods Enzymol. 148, 542–558.CrossRefGoogle Scholar
  31. 31.
    Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685.PubMedCrossRefGoogle Scholar
  32. 32.
    Parasassi T., Gratton E. 1995. Membrane lipid domains and dynamics as detected by laurdan fluorescence. J. Fluoresc. 5, 59–68.CrossRefGoogle Scholar
  33. 33.
    Peskan T., Westermann M., Oelmüller R. 2000. Identification of low density Triton X-100-insoluble plasma membrane microdomains in higher plants. Eur. J. Biochem. 267, 6989–6995.PubMedCrossRefGoogle Scholar
  34. 34.
    Poole R.J., Briskin D.P., Kratky Z., Johnstone R.M. 1984. Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet. Plant Physiol. 74, 549–556.PubMedCrossRefGoogle Scholar
  35. 35.
    Beck J.G., Mathieu D., Loudet C., Buchoux S., Dufourc E.J. 2007. Plant sterols in “rafts”: A better way to regulate membrane thermal shocks. FASEB J. 21, 1714–1723.PubMedCrossRefGoogle Scholar
  36. 36.
    Roche Y., Gerbeau-Pissot P., Buhot B., Thomas D., Bonneau L., Gresti J., Mongrand S., Perrier-Cornet J.-M., Simon-Plas F. 2008. Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts. FASEB J. 22, 3980–3991.PubMedCrossRefGoogle Scholar
  37. 37.
    Minami A., Fujiwara M., Furuto A., Fukao Y., Yamashita T., Kamo M., Kawamura Y., Uemura M. 2009. Alterations in detergent-resistant plasma membrane microdomains in Arabidopsis thaliana during cold acclimation. Plant Cell. Physiol. 50, 341–359.PubMedCrossRefGoogle Scholar
  38. 38.
    Uemura M., Joseph R.A., Steponkus P.L. 1995. Cold acclimation of Arabidopsis thaliana: Effect on plasma membrane lipid composition and freeze-induced lesions. Plant Physiol. 109, 15–30.PubMedGoogle Scholar
  39. 39.
    Uemura M., Tominaga Y., Nakagawara C., Shigematsu S., Minami A., Kawamura Y. 2006. Responses of the plasma membrane to low temperatures. Physiol. Plantarum. 126, 81–89.CrossRefGoogle Scholar
  40. 40.
    Wang X., Li W., Li M., Welti R. 2006. Profiling lipid changes in plant response to low temperatures. Physiol. Plantarum. 126, 90–96.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2011

Authors and Affiliations

  • B. V. Belugin
    • 1
  • I. M. Zhestkova
    • 1
  • M. S. Trofimova
    • 1
  1. 1.Timiryazev Institute of Plant PhysiologyRussian Academy of SciencesMoscowRussia

Personalised recommendations