3. Biological membranes and cell
compartments





Structure of lipid bilayers
Structure of membrane proteins
Membrane dynamics
Purification of intracellular compartments
Visualization of intracellular compartments and proteins
1
Self-assembly of amphiphilic molecules
Amphipathic (amphiphilic) molecules
hydrophobe hydrophile
spontaneously form
micelles
or bilayers
depending on the relative size of the hydrophilic and hydrophobic parts
2
Biological membranes are made of lipids and proteins
The fluid mosaic model of cell membranes
S. J. Singer and G. L. Nicolson. Science 175(1972):723
 The main biological lipids are phospholipides, sphingolipides, glycolipides and
cholesterol
 Biological membranes contain between 25% and 75% proteins (w/w)
 In cells, 20% (w/w) of proteins are membrane-bound
 70% of all eukaryote proteins interact with membranes
3
Fatty acids
palmitate
oleate
acyl chain
carboxylic
acid group
Natural fatty acids contain an even number of carbon atoms: C14->C24
Some bonds may be unsaturated and induce a bend in the structure
4
Phospholipids
example of phosphoglycerides
ALCOHOL
PHOSPHATE
G
L
Y
C
E
R
O
L
FATTY ACID
FATTY ACID
5
Phosphoglycerides contain a glycerol, two fatty acids, a
phosphate and an alcohol group linked by ester bonds
O
H3C
H2C
O
C
(CH2)14
HC
O
C
(CH2)7
O
H3C
N+
CH2
H3C
CH2
O
P
O
palmitate
CH2
CH3
C
H
C
H
(CH2)7
CH3
O
oleate
Oalcohol
phosphate
glycerol
fatty acids
 phosphatidylcholine
6
Phospholipid diversity derives from many different alcohol
moieties...
NH3+
H3N+
CH2
CH2
-OOC
OH
ethanolamine
C
H
CH2
CH2
OH
OH
CH2
C
H
H3C
choline
OH
OH
H
H
H
OH H
HO
OH
N+
OH
OH
serine
H3C
H3C
CH2
CH2
H
H
OH
glycerol
OH
inositol
proportion in %
Plasma
membrane
Mitochondria
Endoplasmic
reticulum
Phosphatidylethanolamine
Phosphatidylserine
Phosphatidylcholine
Phosphatidylinositol
Sphingomyelin
Glycolipids
Cholesterol
Others
7
4
24
<1
19
7
17
22
35
2
39
0
0
0
3
21
17
5
40
0
5
0
6
30
7
... associated with many different fatty acids
Number of
carbon atoms
Number of double
bonds
Laurate
Myristate
Palmitate
Stearate
Arachidate
Behenate
Lignocerate
12
14
16
18
20
22
24
0
0
0
0
0
0
0
Palmitoleate
Oleate
Linoleate
Linolenate
Arachidonate
16
18
18
18
20
1
1
2
3
4
 hundreds of different phospholipids !
8
Sphingolipids
oleate
O
C
HC
CH
O
H3C
N+
H3C
HN
CH2
H3C
alcohol (choline)
CH2
O
P
O
CH2
(CH2)7
C
H
C
H
C
H
C
H
(CH2)12
(CH2)7
CH3
CH3
OH
Ophosphate
sphingosine
 sphingomyelin
9
Glycolipids
oleate
O
H
HO
O H
CH2OH
H
OH
O
HN
C
HC
CH
CH2
(CH2)7
C
H
C
H
C
H
C
H
(CH2)12
(CH2)7
CH3
CH3
OH
H
OH H
sphingosine
polysaccharide (glucose)
Glucosylcerebroside and gangliosides
For more details concerning ganglioside nomenclature, see
http://www.chups.jussieu.fr/polys/biochimie/STbioch/POLY.Chp.10.12.html
10
In biological membranes, the lipid distribution is asymmetric
example : plasma membrane
Phosphatidylserine
Phosphatidylethanolamine
Phosphatidylcholine
Glycolipids
outside
inside
0
10
90
100
100
90
10
0
11
Polysaccharides are exposed in the outer leaflet of the plasma
membrane
12
Most eukaryote membranes contain cholesterol or ergosterol
HO
HO
Wayne W. LaMorte,
Boston University
School of Public Health
cholesterol
ergosterol
animal cells
fungi cells
13
Cholesterol is essential for the growth and viability of cells in
higher organisms
The presence of cholesterol and phospholipid diversity are required for
the function of many membrane proteins
Glutamate transporter 
GABA transporter

Shouffani & Kanner (1990)
J Biol Chem 265 : 6002
Steroid hormones derive from cholesterol
14
Lipid mixtures can form microdomains in biological membranes
Demixion of lipid mixtures
 Some microdomains of the plasma
membrane called ‘lipid rafts’ seem to be
enriched in specific proteins (caveolins),
sphingolipids and cholesterol (K. Simons,
G van Meer 1988).
 The existence and physiological
function of these structures is still
discussed
DPPC/Cholesterol
2/1
DPPC : di-palmitoyl
phosphatidyl choline
GM1 : a ganglioside
containing 4 sugars
and 1 sialic acid
DPPC/Cholesterol/GM1
68/30/2
Lipid monolayer on mica
Yuan et Johnston (2000) Biophys. J 79 : 2768
15
Biological membranes and cell
compartments





Structure of lipid bilayers
Structure of membrane proteins
Membrane dynamics
Purification of intracellular compartments
Visualization of intracellular compartments and proteins
16
Interaction of proteins with membranes
Integral membrane
proteins
Solubilized only by using detergents
(membrane disruption)
Peripheral membrane
proteins
Solubilized by adding salts or changing the pH (no
disruption of the membrane)
17
Membrane protein solubilization by detergents
non-ionic
detergent
ionic
detergent
18
The membrane-spanning region of integral membrane
proteins is often made of hydrophobic a-helices
example : glycophorin A
outside
21 hydrophobic amino acids
example :
b-adrenergic
receptor
inside
4 nm
19
Transmembrane a-helix can be predicted from the primary
sequence
Polarity scale
Amino acid
Phe
Met
Ile
Leu
Val
Cys
Trp
Ala
Thr
Gly
Ser
Pro
Tyr
His
Gln
Asn
Glu
Lys
Asp
Arg
F
M
I
L
V
C
W
A
T
G
S
P
Y
H
Q
N
E
K
D
R
Transfert energy
(kcal/mole)
+ 3,7
+ 3,4
+ 3,1
+ 2,8
+ 2,6
+ 2,0
+ 1,9
+ 1,6
+ 1,2
+ 1,0
+ 0,6
- 0,2
- 0,7
- 3,0
- 4,1
- 4,8
- 8,2
- 8,8
- 9,2
- 12,3
LSTTEVAMHTTTSSSVSKSYISSQTNDTHK...
Score
4,6
2,4
2,4
-2,9
20
Some integral membrane proteins contain several transmembrane
segments arranged as a rigid β-barrel
Receptor
8 b-strands
Lipase
12 b-strands
H20 transport
16 b-strands
iron transport
22 b-strands
In bacteria, mitochondria, chloroplasts
21
In pore-forming toxins, several polypeptides associate to form
a large transmembrane domain
perfringolysin O (1PFO),
a bacterial toxin
Plu-MAC/PF ( 2QP2), the core component of the
mammalian membrane attack complex (MAC)
and perforin (PF) (cf immunology : complement,
cytotoxic T cells, natural killer cells)
Pore-forming toxins have the ability to switch from a
water-soluble form to a membrane-inserted pore
22
form
Biological membranes and cell
compartments





Structure of lipid bilayers
Structure of membrane proteins
Membrane dynamics
Purification of intracellular compartments
Visualization of intracellular compartments and proteins
23
Lipids and membrane proteins diffuse laterally in the plane of the
membrane
Fluorescence Recovery After Photobleaching (FRAP)
t
r
Lateral diffusion coefficient:
Lipids
Peripheral membrane proteins
Free integral membrane proteins
Cytoskeleton anchored proteins
1-2 mm2/s
1 mm2/s
0.1-0.5 mm2/s
10-4 mm2/s
t = r2/D
24
Membrane dynamics : transport across the lipid bilayer
Lipid bilayers are impermeable to ions and polar molecules, but permeable
to hydrophobic molecules
(moles/sec)
Flux
permeability =
(cm.sec-1)
Concentration . Area
gradient
(cm2)
(moles/cm3)
Specific membrane
proteins carry out
the transport of
polar molecules
ions
25
Facilitated transport
Diffusive transport
Flux = Permeability . Concentration gradient . Area
Facilitated diffusion
(Michaelis-Menten kinetics)
Flux = Carrier density . Carrier activity .
Concentration gradient . Area
 Carried-mediated diffusion is saturable and mediated by transmembrane
proteins (e.g. ion channels)
 Passive transport carries ions in the direction of the concentration gradient
 Active transport used ATP hydrolysis energy or another favorable
concentration gradient to carry ions against a concentration gradient
26
Ionic channels and transmembrane carriers
Binding pockets
Selectivity pore
Ionic channels and transmembrane carriers transport ions or small polar
molecules across membranes in the direction of the concentration gradient
27
Pumps
Example : the Na+/K+ ATPase
 The motive force is provided by ATP hydrolysis
28
Coupled transport (exchangers)
Transported molecule
 The motive force is provided by the favorable concentration gradient of
the co-transported ion
29
Example : active glucose transport
glucose
2 Na+
120 mM
X?
Na+/glucose
symporter
Transmembrane
potential
- 60 mV
30 mM
carrier
pump
30
Conclusion : membrane structure
 Biological membranes are made of
lipids and proteins
 The lipid bilayer is asymmetrical
 Membrane proteins are intrinsically
or peripherally associated with the
lipid bilayer
 Most intrinsic membrane proteins
have transmembrane helices
 The phospholipids, the
sphingolipids, the glycolipids and the
cholesterol are the major lipids
 Lipid mixtures can form subdomains in the biological membranes
(lipid rafts)
 Biological membranes are two-dimensional fluids
 Biological membranes are dynamic, molecules can cross it thanks to specific
proteins
 Biological membranes are often associated to the cell cytoskeletons
 The plasma membrane is anchored to the extracellular matrix by adhesion proteins
31
Biological membranes and cell
compartments





Structure of lipid bilayers
Structure of membrane proteins
Membrane dynamics
Purification of intracellular compartments
Visualization of intracellular compartments and proteins
32
Subcellular compartment preparation
Cells in culture
Cells in tissue
Trypsin-EDTA treatment
Mechanical shear stress
Cells in suspension (106)
• Rupture of the plasma membrane
Mechanical rupture or plasma membrane solubilization by non-ionic
detergents
• Separation of the nucleus from the cytoplasm by centrifugation
 Post-nuclear supernatant
• Organelle purification by centrifugation
• Separation of organelle membranes and soluble components
• Solubilization of organelle membranes using detergents
33
Sedimentation velocity
Steady state sedimentation :
Friction force + Centrifugal force = 0
f.u
DrV0 w2 r
f.u = DrV0 w2 r
u : particle velocity
V0 : particle volume
Dr : difference between the density of the particle and that of the
surrounding fluid
w2 r : relative centrifugal force (often expressed in g); w angular velocity of
the rotor ; r distance to the rotor axis
S = u/w2r = DrV0/f is the sedimentation coefficient
(unit S, svedberg, 1 Svedberg (S) = 10-13 s)
For a spherical particle or radius r0,
f = 6phr0 (Stocke’s law)
h: fluid dynamic viscosity (Pa.s)  S = (Drh).(r02/9)
Otherwise, f can be determined by measuring D (for instance in DLS
experiments), thanks to Einstein formula : f = kBT/D = RT/NAD
34
Sedimentation equilibrium
D.dC/dr
Steady state :
Diffusion flow + Centrifugal flow = 0
C.u(r)
D.dC/dr = C.u = C.DrV0 w2 r/f
C : particle concentration
D : diffusion coefficient
V0 : particle volume
Dr : difference between the density of the particle and that of the
surrounding fluid
w2 r : relative centrifugal force (often expressed in g); w angular velocity of
bottom
the rotor ; r distance to the rotor axis
2
Igor-Bricker sample
Absorbance
Integrating the differential equation gives
C = C0exp[(r2-r02).(DrV0 w2 /2RT)]
T=20.0 oC
24,000 RPM v 2=0.73 mL/g
meniscus
1
This allows for a precise determination
of DrV0  molecular weight of proteins
or protein complexes
0
40
45
2
2
r /cm
50
35
Sedimentation equilibrium in a gradient
The particles are centrifuged
in a density gradient (sucrose,
glycerol, Percoll)
DrV0 w2 r
Buoyancy
DrV0 w2 r
At steady state : Dr = 0
The position of the particle matches its density
This allows for the separation of particles according to their density
Step gradients, linear or exponential gradients are used
Percoll is a self-forming density gradient particle mix
36
Subcellular compartment fractionation
Differential centrifugation
sedimentation coefficient (S) =
speed (v)
acceleration (Dmw2r)
proteins ≈ 2-20 S
protein complexes ≈ 20 S
rRNA ≈ 13, 26, 50 S
ribosomes : 50S + 30 S  70S
vesicles & virus ≈ 130 S
1 Svedberg (S) = 10-13 s
w
pelleting factor k = sedimentation coefficient (S) x time (h)
ln (rmax/rmin)
=
w2
X 2,5 1011
rmax
Gradient centrifugation
rmin
sucrose
density & osmosis
subcellular compartments
glycerol
density
protein complexes
density
nucleic acids
Cesium chloride
Percoll®
density
cells
subcellular compartments
37
Gradient centrifugation
Velocity sedimentation (separation according to sedimentation velocity S)
Fraction collector
Equilibrium sedimentation (separation
according to buoyant density r)
38
Differential centrifugation
Post-nuclear
supernatant
1000 x g,
10 min
20 000 x g,
20 min
80 000 x g,
1 hr
150 000 x g,
3 hr
Endoplasmic
reticulum
Golgi apparatus
39
Bovine pulmonary artery endothelial cell labeled with
probes to visualize mitochondria, peroxisomes, and the
nucleus. Mitochondria were stained with the MitoTracker®
Red CMXRos reagent. Peroxisomal labeling was
achieved with a primary antibody directed against PMP70,
visualized using green-fluorescent Alexa Fluor® 488 goat
anti–rabbit IgG. The nucleus was stained with bluefluorescent DAPI. Molecular Probes
40
Immunofluorescence
0. Staining living cells with permeable reagents
1. Cell fixation and permabilization
2. Binding of primary antibodies to specific antigens
3. Visualization using fluorescent secondary antibodies
4. Nucleus visualization using DNA binding fluorophores
Thermodynamical balance
(1 glucose and 2 Na+ enter together)
ln(10)*RT = 1,41 kcal.mole-1
Ne = F = 23 kcal.mole-1.V-1
DG = DGglucose + 2DGNa+
= RT( ln[glucose]int - ln[glucose]ext ) + 2Ne(Vint - Vext) + 2RT( ln[Na+]int - ln[Na+]ext )
= 1.41*( log[glucose]int - log[glucose]ext ) – 2*23*0.06 +
2*1.41*( log[Na+]int - log[Na+]ext )
Transport is possible when DG ≤ 0 and stops when DG = 0
log([glucose]int /[glucose]ext ) ≤ 2*23*0.06/1.41 – 2*log([Na+]int/[Na+]ext )
≤ 1.96 – 2*log(30/120)
≤ 1.96 + 1.20 = 3.16
Therefore, [glucose]int /[glucose]ext ≤ 1450
42