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 two-dimensional 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: • EM Tomography • FRET • Laser Tweezers • Rafts
Biology: • Fence model
• SCF signaling
• Diffusion barrier
• RAS/RAF Signaling
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.
Proximity: < 10nm Proper orientation
Laser Tweezers / Optical Tweezers
Lipid Raft
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
Lateral domain in the membrane with special lipid and protein composition, operational definition: material that resists extraction by non-ionic detergent
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. Lipid rafts B. Pma1 biogenesis C. Protein sorting D. Missorting of Pma1 in lipid mutants
2. Methods A. Pulse-chase analysis B. Triton-X100 extraction C. Optiprep gradient D. Localization of GFP fusion E. Protein complex analysis by BN-PAGE
ER 1. Pma1 synthesis, 10TMD
3. ER export
4. Surface delivery
Lipids ?
5. Stabilization, T1/2~11h
6. Regulated activity
H+
2. Raft association, 80-120 TMDs 2. Oligomerization, 8-12mer
The plasma membrane H+-ATPase Pma1
• Very abundant protein, 1/4 of plasma membrane proteins
• Essential
• Major cargo protein of the secretory pathway
7. Turnover
• 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
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
Containing glass beads
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
4times, 1ml, centrifuge 6krpm 1min, after last wash pellet again
2krpm, 5min, store sup at -20°C
Immunoprecipitation (3) Result
WT Pma1
0 15 30 60 min
elo3Δ Pma1
Immunoprecipitation (4) protein maturation
CPY
Gas1
Mannosyl-diinositolphosphoryl
CERAMIDE
HEAD GROUP
Long chain base
Very long-chain fatty acid
C26
Elo3 is required for the synthesis of sphingolipids with C26 very long-chain fatty acid
elongases Elo2
Lcb1 Elo3
ER M(IP)2C
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∆)
Vacuolar degradation of newly synthesized
Pma1, in elo3∆ at 37°C
Eisenkolb et al. MBC 13, 4414 (2002)
The C26 fatty acid is required for Pma1 stability
WT Pma1
0 15 30 60 min
elo3Δ end4Δ Pma1
end4
0 15 30 60 min
elo3Δ pep4Δ Pma1
pep4∆
elo3Δ Pma1
end4∆
Lipid Raft
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
Lateral domain in the membrane with special lipid and protein composition, operational definition: material that resists extraction by non-ionic detergent
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
Pma1
Gas1, is a GPI-anchored proteins, that is glycosylated in the Golgi, 105 kDa (ER form) to 125 kDa (mature form)
Pma1 acquires detergent resistance during biogenesis
Couple pulse-chase analysis with Triton-X100 extraction
Separate detergent treated material in soluble and insoluble fraction, no flotation gradient, but only centrifugation after detergent treatment
Pulse chase
sample
Total TX100
Pellet detergent Resistant
=
Lipid raft
Supernatant detergent
extractable
C26 is required for raft association of Pma1
Eisenkolb et al. MBC 13, 4414 (2002)
In the absence of C26, newly synthesized Pma1 does not
associate with lipid rafts
elo3Δ Pma1
WT
T P S
15min
Pma1
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
WT elo3Δ Pma1p-GFP
C26 is required for Pma1p stability
C26 may affect membrane thickness
Working Hypothesis
C26
Destabilisation of the protein structure
C22
Hydrophobic mismatch may induce degradation of newly synthesized Pma1
Test hypothesis - how ?
ER 1. Pma1 synthesis, 10TMD
3. ER export
4. Surface delivery
Lipids ?
5. Stabilization, T1/2~11h
6. Regulated activity
H+
2. Raft association, 80-120 TMDs 2. Oligomerization, 8-12mer
The plasma membrane H+-ATPase Pma1
• Very abundant protein, 1/4 of plasma membrane proteins
• Essential
• Major cargo protein of the secretory pathway
7. Turnover
• 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 !?!
Triple yeh1 yeh2 tgl1
0 2 4 6h
STE>
TAG>
WT 0 2 4 6h
Steryl ester hydrolases in vivo mobilization assay: Candidate lipase mutants
[3H]-Palmitic acid
Terbinafine
Samples
Lipid analysis by TLC 0 2 4 6h
STE
[%]
100 60 20
140
N
C
Periphery Yeh2
GFP-Yeh2 DAPI
Localization and topology of steryl ester hydrolases
Köffel et al., Mol. Cell Biol. 25, 1655 (2005)
N
C Tgl1 LD
Tgl1-GFP
Lumenal
Cytosolic
N
C Yeh1 LD
Yeh1-GFP Erg6-RFP Nomarski
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)
CAPILLARY
RF
QUADRUPOLE QUADRUPOLE COLLISION CHAMBER
MASS DETECTOR
+/-
Nano ESI-MS/MS
Scan Modes: 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-3-
Phosphatidylcholine
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
500 550 600 650 700 750 800 850 900 950 1000Da/e0
100
%
835
714
686
807
760
863
952
678742
968
The acyl chain composition of yeast lipids
Kennedy de novo
PS 34%
34:1, 64%
Plasma membrane ER
PS 10%
34:1, 37%
LP PS
5% 34:1, 22%