Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels

• Autorzy: Rajasekharan A. , Gummadi S. N.
• Języki publikacji: EN

Abstrakty

EN

Biogenic membranes or self-synthesizing membranes are the site of synthesis of new lipids such as the endoplasmic reticulum (ER) in eukaryotes. Newly synthesized phospholipids (PLs) at the cytosolic leaflet of ER need to be translocated to the lumen side for membrane biogenesis and this is facilitated by a special class of lipid translocators called biogenic membrane flippase. Even though ER is the major site of cholesterol synthesis, it contains very low amounts of cholesterol, since newly synthesized cholesterol in ER is rapidly transported to other organelles and is highly enriched in plasma membrane. Thus, only low levels of cholesterol are present at the biosynthetic compartment (ER), which results in loose packing of ER lipids. We hypothesize that the prevalence of cholesterol in biogenic membranes might affect the rapid flip-flop. To validate our hypothesis, detergent solubilized ER membranes from both bovine liver and spinach leaves were reconstituted into proteoliposomes with varying mol% of cholesterol. Our results show that (i) with increase in the cholesterol/PL ratio, the half-life time of PL translocation increased, suggesting that cholesterol affects the kinetics of flipping, (ii) flipping activity was completely inhibited in proteoliposomes reconstituted with 1 mol% cholesterol, and (iii) FRAP and DSC experiments revealed that 1 mol% cholesterol did not alter the bilayer properties significantly and that flippase activity inhibition is probably mediated by interaction of cholesterol with the protein.

Słowa kluczowe

EN

inhibition; biogenic membrane; flippase activity; proteoliposome; low cholesterol level; cholesterol level; flipase zob.flippase; endoplasmic reticulum; new lipid; lipid; phospholipid; phosphatidylcholine

• Wydawca: -
• Czasopismo: Cellular and Molecular Biology Letters
• Rocznik: 2012
• Tom: 17
• Numer: 1
• Opis fizyczny: p.136-152,fig.,ref.

Twórcy

autor

Rajasekharan A.

• Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India

autor

Gummadi S. N.

Bibliografia

• 1. Albert, A.D. and Boesze-Battaglia, K. The role of cholesterol in rod outer segment membranes. Prog. Lipid Res. 44 (2005) 99-124.
• 2. Yeagle, P.L. Modulation of membrane function by cholesterol. Biochemie 73 (1991) 1303-1310.
• 3. López-Revuelta, A., Sánchez-Gallego, J.I., García-Montero, C.A., HernándezHernández, A.,Sánchez-Yagüe, J. and Llanillo, M. Membrane cholesterol in the regulation of aminophospholipid asymmetry and phagocytosis in oxidized erythrocytes. Free. Radic. Biol. Med. 42 (2007) 1106-1118.
• 4. Lange, Y., Echevarria, F. and Steck, T.L. Movement of zymosterol, a precursor of cholesterol, among three membranes in human fibroblast. J. Biol. Chem. 266 (1991) 21439-21443.
• 5. Ridsdale, A., Denis, M., Gougeon, P.Y., Ngsee, J.K., Presley, J.F. and Zha, X. Cholesterol is required for efficient endoplasmic reticulum-to-Golgi transport of secretory membrane proteins. Mol. Biol. Cell. 17 (2006) 1593-1605.
• 6. DeGrella, R.F. and Simonia, R.D. Intracellular transport of cholesterol to the plasma membrane. J. Biol. Chem. 257 (1982) 14256-14262.
• 7. Kornberg, R.D. and McConnell, H.M. Inside-outside transitions of phospholipids in vesicle membranes. Biochemistry 10 (1971) 1111-1120.
• 8. Gummadi, S.N. and Kumar, K.S. The mystery of phospholipid flip-flop in biogenic membranes. Cell. Mol. Biol. Lett. 10 (2005) 101-121.
• 9. Menon, A.K. Flippases. Trends Cell. Biol. 5 (1995) 355-360.
• 10. Hrafnsdo′ttir, S. and Menon, A.K. Reconstitution and partial characterization of phospholipid flippase activity from detergent extracts of Bacillus subtilis cell membrane. J. Bacteriol. 182 (2000) 4198-4206.
• 11. Gummadi, S.N. and Menon, A.K. Transbilayer movement of dipalmitoylphosphatidylcholine in proteoliposomes reconstituted from detergent extracts of endoplasmic reticulum: kinetics of transbilayer transport mediated by a single flippase and identification of protein fractions enriched in flippase activity. J. Biol. Chem. 277 (2002) 25337-25343.
• 12. Sahu, S.K. and Gummadi, S.N. Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants. Biochemistry 47 (2008) 10481-10490.
• 13. Lord, J.M., Kiagawva, T. and Beevers, H. Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm. Proc. Nat. Acad. Sci. U.S.A. 69 (1972) 2429-2432.
• 14. Backer, J.M. and Dawidowicz, E.A. Reconstitution of a phospholipid flippase from rat liver microsomes. Nature 327 (1987) 341-343.
• 15. Menon, A.K., Watkins, W.E. and Hrafnsdo´ttir, S. Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum. Curr Biol. 10 (2000) 241-252.
• 16. Lèvy, D., Bluzat, A., Seigneuret, M. and Rigaud, J.L. A systematic study of liposome and proteoliposomes reconstitution involving Bio-Bead mediated Triton X-100 removal. Biochim. Biophys. Acta 1025 (1990) 179-190.
• 17. Bligh, E.G. and Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Med. Sci. 37 (1959) 911-917.
• 18. Kaplan, R.S. and Pedersen, P.L. Sensitive protein assay in the presence of high levels of lipid. Methods Enzymol. 172 (1989) 393-399.
• 19. Akashi, K., Miyata, H., Itoh, H. and Kinosita, K. Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. Biophys. J. 71 (1996) 3242-3250.
• 20. Dimova1, R., Aranda, S., Bezlyepkina, N., Nikolov, V., Riske, K.A. and Lipowsky, R. A practical guide to giant vesicles - Probing the membrane nanoregime via optical microscopy. J. Phys. Condens. Matter. 18 (2006) S1151-S1176.
• 21. Taylor, K.M.G. and Morris, R.M. Thermal analysis of phase transition behaviour in liposomes. Thermochim. Acta 248 (1994) 289-301.
• 22. Yancey, P.G., Rodrigueza, W.V., Kilsdonk, E.P.C., Stoudt, G.W., Johnson, W.J., Phillips, M.C. and Rothblat, G.H. Cellular cholesterol efflux mediated by cyclodextrins. J. Biol. Chem. 271 (1996) 16026-16034.
• 23. Eckford, P.D.W. and Sharom, F.J. Interaction of the P-glycoprotein multidrug efflux pump with cholesterol: effects on ATPase activity, drug binding and transport. Biochemistry 47 (2008) 13686-13698.
• 24. Chang, Q., Gummadi, S.N. and Menon, A.K. Chemical modification identifies two population of glycerophospholipid flippase in rat liver ER. Biochemistry 43 (2004) 10710-10718.
• 25. Bishop, R.W. and Bell, R.M. Assembly of the endoplasmic reticulum phospholipid bilayer: the phosphatidylcholine transporter. Cell 42 (1985) 51-60.
• 26. Vigh, L., Escriba, P.V., Sonnleitner, A., Sonnleitner, M., Piotto, S., Marseca, M., Horvath, I. and Harwood J.L. The significance of lipid composition for membrane activity: New concepts and ways of assessing function. Prog. Lipid Res. 44 (2005) 303-344.
• 27. Betts, H. and Moore, I. Plant cell polarity: The Ins-and-outs of sterol transport. Curr. Biol. 13 (2003) 781-783.
• 28. Moreau, P., Hartmann, M.A., Perret, A.M., Sturbois-Balcerzak, B. and Cassagne C. Transport of sterols to the plasma membrane of leek seedlings. Plant. Physiol. 117 (1998) 931-937.
• 29. Grebe, M., Xu, J., Möbius, W., Ueda, T., Nakano, A., Geuze, H.J., Rook, M.B. and Scheres, B. Arabidopsis sterol endocytosis involves actin mediated trafficking via ARA6-positive early endosomes. Curr. Biol. 13 (2003) 1378-1387.
• 30. Boutte, Y. And Grebe, M. Cellular processes relying on sterol function in plants. Curr. Opin. Plant Biol. 12 (2009) 705-713.
• 31. Meer, G.V., Voelker, D.R. and Feigenson, G.W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell. Biol. 9 (2008) 112-124.
• 32. Smith, M.L. and McConnell, H.M. Pattern photobleaching of fluorescent lipid vesicles using polarized laser light. Biophys. J. 3 (1981) 139-146.
• 33. McMullen, T.P.W., Vilcheze, C., McElhaney, R.N. and Bittman, R. Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behaviour. Biophys. J. 69 (1995) 169-176.
• 34. Zidovetzki, R. and Levitan, I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim. Biophys. Acta 1768 (2007) 1311-1324.
• 35. Tieleman, D.P. and Marrink, S.J. Lipids out of equilibrium: energetics of desorption and pore mediated flip-flop. J. Am. Chem. Soc. 128 (2006) 12462-12467.
• 36. Gurtovenko, A.A. and Vattulainen, I. Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide. J. Phys. Chem. B. 111 (2007) 13554-13559.
• 37. Schroeder, F., Frolov, A., Schoer, J., Gallegos, A., Atshaves, B.P., Stolowich, N.J., Scott, A.I. and Kier, A.B. Intracellular sterol binding proteins, cholesterol transport and membrane domains, in intracellular cholesterol trafficking (Chang, T.Y. and Freeman, D.A., Eds), 1998, Kluwer Academic Publishers, Boston.
• 38. Stolowich, N., Frolov, A., Petrescu, A.D., Scott, A.I., Billheimeri, J.T. and Schroeder, F. Holo-sterol carrier protein-2. J. Biol. Chem. 274 (1999) 35425-35433.
• 39. Schroeder, F., Frolov, A., Starodub, O., Atshaves, B.B., Russelli, W., Petrescu, A., Huang, H., Gallegos, A.M., McIntosh, A., Tahotna, D., Russelli, D.H., Billheimer, J.T., Baum, C.L. and Kier, A.B. Pro-sterol carrier protein-2. J. Biol. Chem. 275 (2000) 25547-25555.
• 40. Avdulov, N.A., Chochina, S.V., Igbavboa, U., Warden, C.S., Schroeder, F. and Wood, W.G. Lipid binding to sterol carrier protein-2 is inhibited by ethanol. Biochim. Biophys. Acta. 1437 (1999) 37-45.
• 41. Serrero, G., Frolov, A., Schroeder, F., Tanaka, K. and Gelhaar, L. Adipose differentiation related protein: expression, purification of recombinant protein in Escherichia coli and characterization of its fatty acid binding properties. Biochim. Biophys. Acta. 1488 (2000) 245-254.
• 42. Atshaves, B.P., Starodub, O., McIntosh, A., Petrescu, A., Roths, J.B., Kier, A.B. and Schroeder, F. Sterol carrier protein-2 alters high density lipoproteinmediated cholesterol efflux. J. Biol. Chem. 275 (2000) 36852-36861