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Cell Membranes &
Membrane Proteins
Tutorial One
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Tutorial 1 Topics
1) Determination of membrane proteinmobility
The Frye and Edidin Cell Fusion Experiment
Fluorescence Recovery After Photobleaching(FRAP)
Single particle trafficking
2) Membrane proteins Membrane isolation from erythrocytes
Membrane protein separation by SDS-PAGE
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1) Evidence of Membrane Protein
Mobility
Since phospholipid membranes are fluid
(according to the Fluid Mosaic model) then
we should see evidence of membrane
protein mobility
Transverse diffusion
(~105sec)
Leaflet to leafletMovement (Flip-flop)
Lateral diffusion
(~10-6sec)
movement
within a leaflet
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Frye and Edidin Cell Fusion
Experiment Human and mouse cells were fused together to
give a single cell with two nuclei and onecontinuous membrane
Human and mouse cells, along with two differentantibodies were used Anti-human membrane protein antibodies
Anti-mouse membrane protein antibodies
Each antibody was fluorescently labeled Human: red
Mouse: green
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Fig. 4.25
The fluorescent
micrograph cell imageshowing the human-
mouse cell fusion at
time 0 (step 3 of the
next slide) of theexperiment
Predict ion s: Aremembrane proteins
Mobile?
Immobile?
Figure 4.25
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Conclusions: After 40 minutes each speciesmembrane proteins appeared uniformly
distributed
Do you expect this to be true for all
membrane proteins?
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Fluorescence Recovery After
Photobleaching (FRAP)
Fluorescence = the light absorbed by afluorophore that is re-emitted at a higherwavelength (l) (lower energy)
Photobleaching = a fluorescent molecule that isilluminated with intense light of the appropriatewavelength that can no longer re-emit light
Photobleaching can be used to determine how fastfluorescently labelled regions of the membranecan recover
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*(N) represents the cell nucleus
A particular membraneprotein is fluorescentlylabelled
A small region of themembrane is irradiated tobleach the labelled protein
This membrane region isvisually monitored over timeto determine the extent offluorescence recovery
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FRAP shows the rate of fluorescence recoveryand provides a direct measurement of the rate ofprotein diffusion BUT:
30- 70 % of membrane proteins studied werenot able to move back into the circle
Membrane
proteins moved
much more slowly
in plasmamembranes than
in pure lipid
bilayers
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Single Particle Tracking (SPT)
Allows researchers to follow specificproteins within the plasma membrane
Proteins are tracked using antibodieslinked to gold particles (40 nm in diameter)that are visualized as dark specks
Computer-enhanced video microscopy isused to analyze patterns of protein-antibody movement
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Asome proteins moved randomly (~10-10 to10-12
cm/sec)
Bsome failed to move and were immobileCmovement in a directed fashion (non-random)
toward one part of the cell
Dlimited movementdue to crowdingeffects of otherproteins
E
fencing effectsrestricting movementin a small area
Fextracellular
material entrapment
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2) Characterization of Integral
Proteins What is the main feature that distinguishes
an integral protein from a peripheral
membrane protein? Integral proteins:-pass through both leaflets ofthe lipid bilayer
-Isolation from membrane
requires detergent solubilization
-More resistant to proteases
Peripheral proteins
-Sit at phospholipid surfaces
-Isolation from membrane with
high salt or carbonate washes
- Sensitive to proteases
peripheral
Int
egral
peripheral
Lipid
Anchored
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Characterization of Membrane
Proteins Erythrocyte plasma
membranes can be isolatedby suspending cells inhypotonic solution (distilled
water)
After cells lyse, membranes
can be pelleted bycentrifugation.
(Remember there are nointercellular membranes in
erythroctyes)
Isotonic
Hypotonic
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Polyacrylamide gel electrophoresis (PAGE)
UnknownProtein
-Add SDS
++
+
+
+
+
+
1 23
4
+
-
Electric
current
Proteins will appear as bands on the gel and
separate based on their mass/charge ratio
Lane 1 protein standard (defined sizes)
Lane 2 the unknown proteinis ~50 kDa
SDSbinds to positivelycharged and
hydrophobic residues of the protein to
give the protein a uniformly negative
charge this unfolds many regions of theprotein
SDS-
Polyacrylamide
gel
Load protein onto gel
(lane 2)
1 2
1 2
Add
loadingdye
electrophoresis
90 kDa60 kDa40 kDa20 kDa10 kDa
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Erythrocyte membrane proteins examined by SDS-
PAGE
How do you determine if a membrane protein is
integral or peripheral?
SDS-PAGE
of red blood
cell(erythrocyte)
membrane.
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Treatment of cell membranes with salts,
enzymes, and detergents can selectively isolate
the type of membrane protein using SDS-PAGE Salts help break ionic bonds between proteins
Detergents solubilize/ disrupt lipids
Enzymes such as proteases can recognize and cut
peptide bonds in proteins
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45
66
97
117
155
200
MW A B C D E
AUntreated membranes
BSalt wash solution from
cell supernatant
CSalt washed isolated
membrane pellet
DDetergent solubilization of
salt washed membrane pellet
EProtease treatment of
detergent solubilizedmembrane pellet
What types of membrane
proteins in lane A are found
within erythrocyte
membranes using SDS-PAGE?
Samples loaded onto gel:
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45
66
97
117
155
200
MW A
Integral Membrane proteins
Peripheral Membrane proteins
Possibly one Lipid-Anchored
Membrane protein?
What types of membrane proteins in lane A are
found within erythrocyte membranes using
SDS-PAGE?