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Cholesterol trafficking machinery Gerrit van Meer; [email protected] 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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Cholesterol trafficking machinery

Gerrit van Meer; [email protected]

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.


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%


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%


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


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”


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


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?


Segregation of transferrin coated pitsTfn (recycling) and epidermal growth factor EGF (to late endosomes) after endocytosis

Sharma et al (2003) JBC 278, 7564


Insertion of Bodipy coated pits

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


Lipids spontaneously aggregated during endocytosis coated pits

This must have involved lateral segregation

Does this only occur during endocytotic recycling?


PC coated pits

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


O coated pits

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.


Phospholipid coated pits

The lipoprotein LDL

P

Triacylglycerol

20 nm

Apo-A1

Cholesterol ester

HDL


Lipid structure predicts flip rate in model membrane coated pits

10-1 s seconds 10 h >10 h

DAG

Cer

Chol

PC

LPC

ganglioside


Lipid structure predicts “off rate” in model membrane coated pits

Bodipy 102h 10 min 10 min

Native 60 h < 102s 103h

DAG

Cer

PC

LPC

ganglioside

Cholesterol

< 2 hours


1 coated pits

Transport mechanisms of lipids

Outside

3

2

4

Cytosol

Lumen

cholesterol glucosylceramide

1. Lateral mobility + +

Vesicular traffic + +

Flip-flop + –

Monomolecular transfer + –


Cholesterol moves rapidly across membranes coated pits

Cholesterol moves rapidly between membranes

Thus its localization must be determined

by affinity for other lipids or proteins


Cholesterol binds to specific proteins coated pits

Thiele et al. (2000)

Nature Cell Biol 2, 42-49



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?


ABCG5/G8 lipids (saturated NPC1L1

G

E

NPC1

NPC2

MLN64

StAR

ABCA1

SCP

L

ER

N

M

Proteins of cholesterol transport


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