1. 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. Cholesterol trafficking machinery Gerrit van Meer; g.vanmeer@uu.nl Membrane Enzymology Bijvoet Center / Institute of Biomembranes

2. P G OH Glycerolipids Sterols sphingomyelin glycosphingolipids glycerophospholipids PC, PE, PS, PI OH N O O O O O O O Sphingolipids 65 mol% 10% 25% glycerol sphingosinecholesterol fattyacid fattyacid glucose oleicacid

3. A P G OH GlycerolipidsGlycerolipidsSterolsSterols SphingomyelinGlycosphingolipids PC, PE, PS, PI Cholesterol OH OH O O OH O O SphingolipidsSphingolipids 65 mol% 10% 25% A P O O O O O A P O O O O O N N P = phosphate A = choline, ethanolamine, serine, inositol G = glucose

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

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

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

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

12. ER G TGNE L Sphingolipids and cholesterol Unsaturated PC P O O O O O C OH P O O N C Lipid sorting must occur at the Golgi 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. P 20 nm HDL Apo-A1 The lipoprotein LDL Phospholipid Triacylglycerol Cholesterol ester

14. DAGCerCholLPCPCganglioside 10 -1 s seconds 10 h >10 h Lipid structure predicts flip rate in model membrane

15. DAGCerLPCPCganglioside Bodipy 10 2 h 10 min 10 min Native 60 h < 10 2 s 10 3 h Lipid structure predicts “off rate” in model membrane Cholesterol < 2 hours

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

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. ER G L E N ABCA1 ABCG5/G8 NPC1L1 NPC1NPC2MLN64StAR M SCP Proteins of cholesterol transport