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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?