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%