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

30% of the cellular proteins are membrane proteins30% of the cellular proteins are membrane proteinsProteomicsProteomics

How do cells use proteins and lipids for their vital functions?How do cells use proteins and lipids for their vital functions?Systems BiologySystems Biology

In many cases there must be an interplay between the lipids In many cases there must be an interplay between the lipids and the proteins! They co-evolved in evolution.and the proteins! They co-evolved in evolution.

Cholesterol trafficking machineryCholesterol trafficking machineryGerrit van Meer; g.vanmeer@uu.nlGerrit van Meer; g.vanmeer@uu.nl

Membrane EnzymologyMembrane EnzymologyBijvoet Center / Institute of BiomembranesBijvoet Center / Institute of Biomembranes

PG

OH

GlycerolipidsGlycerolipids SterolsSterols

sphingomyelinglycosphingolipids

glycerophospholipidsPC, PE, PS, PI

OHOH N

O O

OO

O

O

O

SphingolipidsSphingolipids

65 mol% 10% 25%

glycerolglycerol sphingosinesphingosine cholesterolcholesterol

fattyfattyacidacid

fattyfattyacidacid

glucose

oleicoleicacidacid

AA

PPGG

OHOH

GlycerolipidsGlycerolipidsGlycerolipidsGlycerolipids SterolsSterolsSterolsSterols

SphingomyelinSphingomyelin GlycosphingolipidsGlycosphingolipidsPC, PE, PS, PIPC, PE, PS, PI CholesterolCholesterol

OHOH

OHOH

OO OOOHOH

OO OO

SphingolipidsSphingolipidsSphingolipidsSphingolipids

65 mol%65 mol% 10% 10% 25% 25%

AA

PP

OO

OO

OO

OO

OO

AA

PP

OO

OO

OO

OO

OO

NNNN

PP = phosphate= phosphate AA = choline, ethanolamine, serine, inositol= choline, ethanolamine, serine, inositol GG = glucose= glucose

The hydrophobic effectThe hydrophobic effectNonpolar molecules break the organizationNonpolar molecules break the organization

of the water; caged waterof the water; caged waterAggregation increases the entropy of the Aggregation increases the entropy of the

water more than the decrease in water more than the decrease in entropy of the nonpolar moleculeentropy of the nonpolar molecule

Lipid molecules will not dissolve in waterLipid molecules will not dissolve in water

Membranes consisting of unsaturated phospholipids

are in the liquid phase; fluidCholesterol reduces fluidityMembranes consisting

of saturated phospholipids are in the gel phase; solidCholesterol fluidizes

A second fluid state exists

Solid-fluid immiscibilityFluid-fluid immiscibility

Fluidity does not increase linearly with cholesterol content:“phase transitions”

lo

ld + lo

CholesterolCholesterol

ldld + lo + so

ld + so

POPCPOPC PSMPSM

OH

C

P

OH NO O

C

P

OO

O

O

O

Phase diagram of the ternary mixturePhase diagram of the ternary mixturepalmitoyl-oleoyl phosphatidylcholine, palmitoyl-oleoyl phosphatidylcholine, palmitoyl sphingomyelin,palmitoyl sphingomyelin,cholesterolcholesterol

de Almeida et al. (2003) de Almeida et al. (2003) Biophys. J. 85, 2406Biophys. J. 85, 2406

lo + solo + so

soso

Follow any line from the bottomFollow any line from the bottom(chol = 0) to the top (chol = 100)(chol = 0) to the top (chol = 100)and you cross phase boundaries:and you cross phase boundaries:Phase transitions.Phase transitions.

Proteins are sorted by lateral segregation into different coated pitsProteins are sorted by lateral segregation into different coated pitsThis must also occur for lipids. How does a certain lipid compositionThis must also occur for lipids. How does a certain lipid compositionrecoggnize and capture a certain SNARE required for targeting?recoggnize and capture a certain SNARE required for targeting?

Segregation of transferrin Segregation of transferrin Tfn Tfn (recycling) and epidermal (recycling) and epidermal growth factor growth factor EGFEGF (to late endosomes) after endocytosis (to late endosomes) after endocytosis

Sharma et al (2003) JBC 278, 7564Sharma et al (2003) JBC 278, 7564

Concentration of fluorescent (Bodipy-) glycosphingolipid LacCer in Concentration of fluorescent (Bodipy-) glycosphingolipid LacCer in endosome subdomainsendosome subdomains

(green: low concentration; red high concentration)(green: low concentration; red high concentration)Sharma et al (2003) JBC 278, 7564Sharma et al (2003) JBC 278, 7564

EndocytosisEndocytosis

Removal of Bodipy-LacCerRemoval of Bodipy-LacCerfrom surfacefrom surface

Insertion of BodipyInsertion of BodipyLacCer on surfaceLacCer on surface

Lipids spontaneously aggregated during endocytosisLipids spontaneously aggregated during endocytosisThis must have involved lateral segregationThis must have involved lateral segregation

Does this only occur during endocytotic recycling?Does this only occur during endocytotic recycling?

PSPS PEPE

SMSM PCPC GlcCerGlcCer

PCPC PEPE

cholesterolcholesterol

Plasma membrane

ER

Golgi

Cellular membranes differ in lipid composition:Cellular membranes differ in lipid composition:because ER and plasma membrane are connected bybecause ER and plasma membrane are connected byvesicular transport in both directions sorting must take placevesicular transport in both directions sorting must take place

ER

G

TGN E

L

Sphingolipids and cholesterolSphingolipids and cholesterol

UnsaturatedUnsaturated PCPC

P

OO

O

O

O

C

OH

PO

OH

O

N

CLipid sorting must occur at the GolgiLipid sorting must occur at the Golgi

Lipid raft microscopy: Eggeling, C., Ringemann, C., Medda, R., Lipid raft microscopy: Eggeling, C., Ringemann, C., Medda, R., Schwarzmann, G., Sandhoff, K., Polyakova, S., Belov, V.N., Hein, B., Schwarzmann, G., Sandhoff, K., Polyakova, S., Belov, V.N., Hein, B.,

von Middendorff, C., Schonle, A., et al. 2009. Direct observation of the von Middendorff, C., Schonle, A., et al. 2009. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. nanoscale dynamics of membrane lipids in a living cell. Nature 457:1159-1162.Nature 457:1159-1162.

P

20 nm

HDL

Apo-A1

The lipoprotein LDLThe lipoprotein LDLPhospholipidPhospholipid

TriacylglycerolTriacylglycerol

Cholesterol esterCholesterol ester

DAGDAG CerCerCholChol

LPCLPCPCPC gangliosideganglioside

1010-1 -1 s seconds 10 h >10 hs seconds 10 h >10 h

Lipid structure predicts flip rate in model membraneLipid structure predicts flip rate in model membrane

DAGDAG CerCer LPCLPCPCPC gangliosideganglioside

Bodipy 10Bodipy 1022 h 10 min 10 minh 10 min 10 min

NativeNative 60 h < 10 60 h < 1022 s 10s 1033

hh

Lipid structure predicts “off rate” in model membraneLipid structure predicts “off rate” in model membrane

CholesterolCholesterol< 2 hours< 2 hours

Outside

Cytosol

Lumen

cholesterol cholesterol glucosylceramideglucosylceramide

1.1. Lateral mobilityLateral mobility ++ ++2.2. Vesicular trafficVesicular traffic ++ ++3.3. Flip-flop Flip-flop ++ ––4.4. Monomolecular transfer Monomolecular transfer ++ ––

2

1

4

3

Transport mechanisms of lipidsTransport mechanisms of lipids

Cholesterol moves rapidly across membranes Cholesterol moves rapidly across membranes Cholesterol moves rapidly between membranesCholesterol moves rapidly between membranes

Thus its localization must be determined Thus its localization must be determined by affinity for other lipids or proteinsby affinity for other lipids or proteins

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

Interactions of proteins with membranesInteractions of proteins with membranes

Cholesterol has an increased affinity for some types of lipids (saturated Cholesterol has an increased affinity for some types of lipids (saturated glycerophospholipids and sphingolipids), and for some sorts of proteins. glycerophospholipids and sphingolipids), and for some sorts of proteins. The high affinity of cholesterol for a certain protein may make this The high affinity of cholesterol for a certain protein may make this a raft protein just like the effect of palmitoylationa raft protein just like the effect of palmitoylation

Still, cholesterol moves quickly between and across membranes. What isStill, cholesterol moves quickly between and across membranes. What isthe problem? the problem?

Well there are a number of cholesterol transport diseases that are causedWell there are a number of cholesterol transport diseases that are causedby mutations in what may be cholesterol transport proteins. by mutations in what may be cholesterol transport proteins. What do they do?What do they do?

ERER

GG

LL

EE

NN

ABCA1ABCA1

ABCG5/G8 ABCG5/G8 NPC1L1NPC1L1

NPC1NPC1NPC2NPC2MLN64MLN64

StARStAR MM

SCPSCP

Proteins of cholesterol transportProteins of cholesterol transport