Lipid/Membrane Methods
Course Overview:
1) Introduction, Kusumi Video
2) Lipids rafts, Mayor iBioSeminar
3) Lipid rafts affect protein transport/stability
4) Questions to Videos and Seminars
5) Lipid analysis by mass spectrometry
How to study lipids
1.  Biophysical methods, # protein free symmetrical
membrane of simple lipid composition
2.  Optical methods, # lipids are modified
3.  Genetics, i.e. synthetic lethal interactions
between mutations in enzymes that synthesize
sterols and enzymes that synthesize
sphingolipids, # indirect ?
4.  Biochemical, i.e. radiolabeled precursors
- In vivo, combined with genetics
- In vitro, defining minimal system, i.e. vesicle budding
- Analytical, mass spectrometry etc.
1. Kusumi video
Concepts:
o 
The Singer-Nicholson model of the “Fluid Mosaic”
must be modified to accommodate lipid and protein
inhomogeneity within the plane of the membrane.
o 
Single molecule microscopy, FRET/FRAP
o 
Lipid/membrane protein diffusion and raft formation,
diffusion barrier in neuronal cells
Ref. A. Kusumi et al. Paradigm shift of the plasma membrane concept from the twodimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of
membrane molecules. Annu. Rev. Biophys. Biomol. Struct. 34, 351-378 (2005)
Kusumi video
Key Words:
Methods:
Biology:
• 
EM Tomography
• 
Fence model
• 
FRET
• 
SCF signaling
• 
Laser Tweezers
• 
Diffusion barrier
• 
Rafts
• 
RAS/RAF Signaling
Proximity: < 10nm
Proper orientation
Mit der FRET-Technik erhalten Sie quantitative zeitliche
und räumliche Informationen über die Bindung und
Interaktion zwischen Proteinen, Lipiden, Enzymen,
DNS und RNS in vivo.
Laser Tweezers / Optical
Tweezers
Lipid Raft
Lateral domain in the membrane with special lipid and protein
composition, operational definition: material that resists
extraction by non-ionic detergent
1 Non-raft membrane; 2 Lipid raft; 3 Lipid raft associated transmembrane
protein; 4 Non-raft membrane protein; 5 Glycosylation modifications (on
glycoproteins and glycolipids); 6 GPI-anchored protein; 7 Cholesterol; 8
Glycolipid
2. iBioSeminars
Membrane Rafts:
Satyajit 'Jitu' Mayor, Bangalor, (iBioSeminars.org)
o 
Historical Perspectives: What are Membrane Rafts
(39 min)
o 
Looking for functional Rafts in Cell Membranes (42
min)
o 
Making Rafts in Living Cell Membranes (21 min)
3. Lipid rafts, protein
transport and protein stability
1.  Introduction
A. 
B. 
C. 
D. 
Lipid rafts
Pma1 biogenesis
Protein sorting
Missorting of Pma1 in lipid mutants
2.  Methods
A. 
B. 
C. 
D. 
E. 
Pulse-chase analysis
Triton-X100 extraction
Optiprep gradient
Localization of GFP fusion
Protein complex analysis by BN-PAGE
The plasma membrane H+-ATPase Pma1
ER
Lipids
?
1. Pma1 synthesis, 10TMD
2. Oligomerization, 8-12mer
2. Raft association, 80-120 TMDs
3. ER export
6. Regulated activity
H+
4. Surface delivery
•  Essential
7. Turnover
5. Stabilization, T1/2~11h
•  Very abundant protein, 1/4 of plasma membrane proteins
•  Major cargo protein of the secretory pathway
•  Model to study plasma membrane biogenesis
Domains in the
yeast plasma
membrane
Can1-GFP
(Arg permease)
Pma1-RFP
(proton pump)
Pulse-chase analysis
o  Principle: radio-label newly synthesized
proteins and follow their maturation/
disappearance over time
o  Can be done to see all proteins or
selectively only one protein which can be
isolated by immunoprecipitation
o  Alternative: - Cycloheximide Western –[?]
- Promoter shut-off
o  Restriction: Protein abundance
Pulse-Chase
Insulin transport in beta cells
Protocol Overview
Cells
Pulse [35S]Met/Cys, 5min
Chase, cold Met/Cys
Samples after 0, 15, 30min
On ice, NaN3, NaF
Break cells, glass beads
Load total proteins on gel
or go on with IP
Log phase, grown in SC-Met,
5 OD per time point, spin, up
at 5 OD/ml in fresh SC-Met,
pre-incubate 15 min at Exp. T
100µCi/time point, T1/2= 87.4d,
Ca 85pmol
0.3% met, cys; 25mM in 300mM
(NH4)2SO4
20mM each
Centrifuge cells, up in TEPI
(50mM Tris, pH 7.5, 5mM EDTA
Protease inhibitors
Immunoprecipitation
Immunoprecipitation
Poisoned Homogenate
+SDS, 45°C 10min
+TNET, mix,
centrifuge
Sup to 3ml TNET
Protein A sepharose
1h, 4°C, head over end rotation
Centrifuge, sup to new tube
Add primary Ab (titrated)
1h, 4°C, head over end rotation
Protein A sepharose
o/n, 4°C, head over end rotation
Containing glass beads
25µl, 20%
0.8ml, vortex, 13krpm, 10min,
no glass beads !!!
In 15ml falcon
(30mM Tris 7.5, 150mM NaCl,
5mM EDTA, 1% Triton X100)
100µl, 7% slurry in TNET
100µl, 15% slurry equilibrated
in TNET for 1h
Immunoprecipitation (2)
Pellet sepharose beads
Wash beads with TNET
Resuspend beads
in sample buffer
Incubate 45°C 10min
centrifuge
Load on SDS PAGE
Stain coomassie, destain,
dry gel
Expose to phosphor imager o/n
2krpm, 5min, store sup at -20°C
4times, 1ml, centrifuge 6krpm
1min, after last wash pellet again
Immunoprecipitation (3)
Result
0
15 30
60 min
WT
Pma1
elo3Δ
Pma1
Immunoprecipitation (4)
protein maturation
CPY
Gas1
Elo3 is required for the synthesis of sphingolipids
with C26 very long-chain fatty acid
Mannosyldiinositolphosphoryl
HEAD
GROUP
elongases
Elo2
Lcb1
Elo3
Long chain base
Very long-chain
fatty acid
C26
CERAMIDE
M(IP)2C
ER
How is Pma1 degraded in
elo3∆ ?
Two degradative pathways:
1) In the vacuole, can be blocked by mutations that
prevent proper transport of the substrate to the
or by inhibiting vacuolar proteases (sec, end, vps,
pep4)
2) By the proteasome, can be blocked by proteosomal
mutations, i.e. cin5ts, by drugs (MG132) or by
preventing ubiquitination of the substrate (doa4∆)
The C26 fatty acid is required for Pma1 stability
0 15 30 60 min
0 15 30 60 min
WT
Pma1
elo3Δ end4Δ
Pma1
elo3Δ
Pma1
elo3Δ pep4Δ
Pma1
pep4∆
end4∆
end4
Vacuolar degradation
of newly synthesized
Pma1, in elo3∆ at 37°C
Eisenkolb et al. MBC 13, 4414 (2002)
Lipid Raft
Lateral domain in the membrane with special lipid and protein
composition, operational definition: material that resists
extraction by non-ionic detergent
1 Non-raft membrane; 2 Lipid raft; 3 Lipid raft associated transmembrane
protein; 4 Non-raft membrane protein; 5 Glycosylation modifications (on
glycoproteins and glycolipids); 6 GPI-anchored protein; 7 Cholesterol; 8
Glycolipid
Triton-X100 extraction
Background:
Membrane domains = lipid rafts are thought to
be clusters of certain lipids and proteins in
the plane of the membrane. Formation of
these clusters or platforms are functionally
important for efficient signal transduction
from the plasma membrane, i.e. formation of
the immunological synapse, and for sorting of
membrane proteins in the late exocytotic
pathway and in endocytotic recycling
Biochemically, these domains are operationally
defined by proteins and lipids that resist
extraction by 1% Triton-X100 (DRMs,
DIGs), a non-ionic detergent, at 4°C during
30 min.
Raft isolation
Principle:
1) Break open cells, glass beads, pellet = total
membranes
2) Incubate membranes in 1% Triton-X100 on
ice for 30 min
3) Flotate membranes that have not been
detergent solubilized by density
centrifugation (sucrose or optiprep)
4) Take equal volume (# equal protein)
fractions from top, TCA precipitate
proteins and run Western
Optiprep gradient
Gas1, is a GPI-anchored proteins, that is glycosylated in the
Golgi, 105 kDa (ER form) to 125 kDa (mature form)
Gas1
Pma1
Pma1 acquires detergent
resistance during biogenesis
Couple pulse-chase analysis with TritonX100 extraction
Separate detergent treated material in
soluble and insoluble fraction, no flotation
gradient, but only centrifugation after
detergent treatment
C26 is required for raft association of Pma1
Pulse chase
15min
T
sample
WT
TX100
Total
Pellet
detergent
Resistant
=
Supernatant
detergent
extractable
elo3Δ
P
S
Pma1
Pma1
Lipid raft
In the absence of C26,
newly synthesized Pma1 does not
associate with lipid rafts
Eisenkolb et al. MBC 13, 4414 (2002)
Localization of
GFP fusion
•  238 Aa, 23 kDa, from A. victoria (Nobel Prize 2008)
•  N- or C-terminal fusion
•  If N-terminal, promoter replacement ?
•  Genomic integration or plasmid-borne (high/low copy...)
•  Live cell imaging !!!
•  Microscopic pulse-chase by shut-off promoter, i.e. GAL1
or cycloheximid addition
•  Formation of GFP chromophore is slow (10 min)
requires O2
•  Absorption ca 488nm emission 509nm (S65T mutation)
•  Strong secondary structure, 11 stranded beta-barrel,
resists proteolytic degradation in vacuole -> vacuolar
staining
•  Div. color versions, pH- or Ca-sensitive
C26 is required for Pma1p stability
WT
Pma1p-GFP
elo3Δ
Working Hypothesis
C26 may affect membrane thickness
Hydrophobic mismatch may induce degradation
of newly synthesized Pma1
Destabilisation of
the protein
structure
C26
C22
Test hypothesis - how ?
The plasma membrane H+-ATPase Pma1
ER
Lipids
?
1. Pma1 synthesis, 10TMD
2. Oligomerization, 8-12mer
2. Raft association, 80-120 TMDs
3. ER export
6. Regulated activity
H+
4. Surface delivery
•  Essential
7. Turnover
5. Stabilization, T1/2~11h
•  Very abundant protein, 1/4 of plasma membrane proteins
•  Major cargo protein of the secretory pathway
•  Model to study plasma membrane biogenesis
Is oligomerization of Pma1 in the ER
affected by lipids ?
How do you determine the oligomeric state of a protein ?
Of a protein in transit through an organelle ?
•  Co-IP of differentially tagged versions
•  Ultracentrifugation of the purified complex
•  Gel-filtration chromatography of the purified complex
•  Chemical crosslinking
•  Two-hybrid
•  Blue-native electrophoresis
Blue-native electrophoresis
Specialized version of native electrophoresis for
membrane proteins
•  Membrane proteins must first be solubilized be detergents
•  Are then incubated with coomassie blue which provides
a negative charge but does not denature (# SDS)
•  Amount of dye bound to complex is proportional to
complex size -> constant size/charge ratio as in
SDS-PAGE -> separation acc. to size = pore size of gel
•  First dimension can be blotted and Western probed, or
denatured with SDS and used for second dimension
Coomassie brilliant blue
BN-PAGE
Example: nucleotide transporter IMM
3. Lipid turnover
Questions:
1. Are lipid degraded ?
Phospholipids, sphingolipids, sterols, neutral lipids
2. Lipid turnover could be important for maintaining
the specific lipid composition of a certain membrane,
i.e. the wrong lipid in a membrane would be degraded
-> how do you test this hypothesis ?
3. Upon energy demand, fat is degraded and the
liberated fatty acids are beta-oxidized to yield ATP
-> how is this achieved and regulated ?
Fat
= neutral lipids
are composed of triacylglycerol (TAG)
and steryl esters (STE)
are stored together in intracellular lipid droplets
There must be a signal for degradation -> probably a
kinase -> test hypothesis, find and characterize kinase
There must be a lipase that cleaves the fatty acid from
TAG and STE -> find and characterize this lipase
Identification of steryl ester
hydrolases
Candidate gene approach:
1. Make a list of potential lipase encoding genes in
the yeast genome
2. Unfreeze the corresponding mutants - any
essential ?
3. Test these mutants for defects in STE hydrolysis
- how ?
- label fat with [3H]palmitate o/n
- dilute cells in fresh medium with terbinafine
- take samples at 0, 2, 4, 6h
- extract lipids
- separate STE on TLC
-> result !?!
Steryl ester hydrolases
in vivo mobilization assay:
Candidate lipase mutants
STE>
TAG>
[3H]-Palmitic acid
0
Terbinafine
2
4
WT
6h
0
2
4
Triple
6h
yeh1 yeh2 tgl1
Samples
Lipid analysis by TLC
STE [%]
140
100
60
20
0
2
4
6h
Localization and topology of steryl
ester hydrolases
Periphery
Yeh2
Yeh1-GFP Erg6-RFP Nomarski
C
Lumenal
N
Tgl1-GFP
N
N
GFP-Yeh2
DAPI
C
C
Yeh1
Tgl1
LD
Cytosolic
LD
Köffel et al., Mol. Cell Biol. 25, 1655 (2005)
Who is controlling the activity
of these lipases ??
Hypothesis: it is kinase controlled
Test hypothesis:
Yeast has 116 kinases, 13 are essential
test 103 kinase mutants for defects in TAG and/or
STE mobilization with a two point assay: 0, 6h
Repeat 2-3 times !! To obtain “trustable” results
-> find many ?
-> find one ?
-> find none ?
5. Lipid analysis by
mass spectrometry
Mass spectrometry is the only practicable method to
determine the lipid molecular species composition of a
cellular membrane.
Comparison of lipid composition of subcellular membranes:
-  Isolate membranes (ER, PM, Golgi, IMM, OMM, Vac)
-  Western for marker enrichment
-  EM of membranes, rel. purity/homogeneity, thickness
-  Extract lipids, ESI-MS/MS analysis
-  Try to make sense out of data
- > hypothesis
- > test hypothesis
-  publish (Schneiter et al. JCB 1999)
Nano ESI-MS/MS
+/
-
CAPILLARY
QUADRUPOLE
COLLISION
CHAMBER
RF
Scan Modes:
QUADRUPOLE
MASS
DETECTOR
single-stage MS: negative ion (PS, PE, PI)
positive ion (PC)
tandem MS:
product ion (daughter scan)
precursor ion (parental scan)
neutral loss scan
Example of a phospholipid molecular
species: 1-Stearoyl-2-Oleoyl-3Phosphatidylcholine
Lipid specific scans
•  Fatty Acids: parental scan for m/z= 253 (C16:1)
255 (C16:0)
281 (C18:1)
283 (C18:0)
•  Lipid Headgroups: parental scan for m/z= 241 (PI)
parental scan for m/z= 184 (PC)
neutral loss of m/z= 141 (PE)
185 (PS)
40k Microsomes
835
100
714
807
%
686
678
760
742
863
952
968
0
500
550
600
650
700
750
800
850
900
950
Da/e
1000
The acyl chain composition of
yeast lipids
de novo
Kennedy
ER
Plasma membrane
PS
10%
PS
34%
34:1, 37%
34:1, 64%
LP
PS
5%
34:1, 22%