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Cells have 500-1000 different lipids; Why? How? Lipidomics

Cholesterol trafficking machinery Gerrit van Meer; g.vanmeer@uu.nl Membrane Enzymology Bijvoet Center / Institute of Biomembranes. Cells have 500-1000 different lipids; Why? How? Lipidomics 30% of the cellular proteins are membrane proteins Proteomics

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Cells have 500-1000 different lipids; Why? How? Lipidomics

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  1. Cholesterol trafficking machinery Gerrit van Meer; g.vanmeer@uu.nl Membrane Enzymology Bijvoet Center / Institute of Biomembranes Cells have 500-1000 different lipids; Why? How? Lipidomics 30% of the cellular proteins are membrane proteins Proteomics How do cells use proteins and lipids for their vital functions? Systems Biology In many cases there must be an interplay between the lipids and the proteins! They co-evolved in evolution.

  2. Glycerolipids Sphingolipids Sterols glycerophospholipids PC, PE, PS, PI sphingomyelin glycosphingolipids P G glucose O O sphingosine cholesterol glycerol O O O OH O N OH OH O oleic acid fatty acid fatty acid 65 mol% 10% 25%

  3. P G = phosphate = choline, ethanolamine, serine, inositol A = glucose Glycerolipids Sphingolipids Sterols PC, PE, PS, PI Sphingomyelin Glycosphingolipids Cholesterol A A A P P P G O O OH O O O O O O O O N OH O O N OH OH O O 65 mol% 10% 25%

  4. The hydrophobic effect Nonpolar molecules break the organization of the water; caged water Aggregation increases the entropy of the water more than the decrease in entropy of the nonpolar molecule Lipid molecules will not dissolve in water

  5. Membranes consisting of unsaturated phospholipids are in the liquid phase; fluid Cholesterol reduces fluidity Membranes consisting of saturated phospholipids are in the gel phase; solid Cholesterol fluidizes A second fluid state exists Solid-fluid immiscibility Fluid-fluid immiscibility Fluidity does not increase linearly with cholesterol content: “phase transitions”

  6. OH Cholesterol Phase diagram of the ternary mixture palmitoyl-oleoyl phosphatidylcholine, palmitoyl sphingomyelin, cholesterol de Almeida et al. (2003) Biophys. J. 85, 2406 Follow any line from the bottom (chol = 0) to the top (chol = 100) and you cross phase boundaries: Phase transitions. lo C C P P O O O O O O N O OH ld + lo lo + so ld + lo + so ld ld + so so POPC PSM

  7. Proteins are sorted by lateral segregation into different coated pits This must also occur for lipids. How does a certain lipid composition recoggnize and capture a certain SNARE required for targeting?

  8. Segregation of transferrin Tfn (recycling) and epidermal growth factor EGF (to late endosomes) after endocytosis Sharma et al (2003) JBC 278, 7564

  9. Insertion of Bodipy LacCer on surface Endocytosis Removal of Bodipy-LacCer from surface Concentration of fluorescent (Bodipy-) glycosphingolipid LacCer in endosome subdomains (green: low concentration; red high concentration) Sharma et al (2003) JBC 278, 7564

  10. Lipids spontaneously aggregated during endocytosis This must have involved lateral segregation Does this only occur during endocytotic recycling?

  11. PC SM GlcCer cholesterol Plasma membrane Golgi PE PS ER PC PE Cellular membranes differ in lipid composition: because ER and plasma membrane are connected by vesicular transport in both directions sorting must take place

  12. O O N OH Lipid sorting must occur at the Golgi OH C Sphingolipids and cholesterol P TGN E G C P O L O ER O O O UnsaturatedPC Lipid raft microscopy: Eggeling, C., Ringemann, C., Medda, R., Schwarzmann, G., Sandhoff, K., Polyakova, S., Belov, V.N., Hein, B., von Middendorff, C., Schonle, A., et al. 2009. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457:1159-1162.

  13. Phospholipid The lipoprotein LDL P Triacylglycerol 20 nm Apo-A1 Cholesterol ester HDL

  14. Lipid structure predicts flip rate in model membrane 10-1 s seconds 10 h >10 h DAG Cer Chol PC LPC ganglioside

  15. Lipid structure predicts “off rate” in model membrane Bodipy 102h 10 min 10 min Native 60 h < 102s 103h DAG Cer PC LPC ganglioside Cholesterol < 2 hours

  16. 1 Transport mechanisms of lipids Outside 3 2 4 Cytosol Lumen cholesterol glucosylceramide 1. Lateral mobility + + Vesicular traffic + + Flip-flop + – Monomolecular transfer + –

  17. Cholesterol moves rapidly across membranes Cholesterol moves rapidly between membranes Thus its localization must be determined by affinity for other lipids or proteins

  18. Cholesterol binds to specific proteins Thiele et al. (2000) Nature Cell Biol 2, 42-49

  19. Interactions of proteins with membranes

  20. Cholesterol has an increased affinity for some types of lipids (saturated glycerophospholipids and sphingolipids), and for some sorts of proteins. The high affinity of cholesterol for a certain protein may make this a raft protein just like the effect of palmitoylation Still, cholesterol moves quickly between and across membranes. What is the problem? Well there are a number of cholesterol transport diseases that are caused by mutations in what may be cholesterol transport proteins. What do they do?

  21. ABCG5/G8 NPC1L1 G E NPC1 NPC2 MLN64 StAR ABCA1 SCP L ER N M Proteins of cholesterol transport

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