The use of fluorescence resonance energy transfer (FRET) to study the
interaction between CD147 and MCT1
Potential combinations of
CFP- and YFP-tagged CD147
and MCT1
OR
The principle of FRET
(CFP)
Donor
LASER
Maximum 100Å
EXCITATION
CFP
(YFP)
Acceptor
YFP
MCT1
CFP
s
s
s
s
YFP
Emission
CD147
Membrane
N-terminus
C-terminus
YFP
Transfer
Energy
CFP
Fluorescence
FRET
CFP
YFP
CFP
OR
Fluorescence
Donor
N-terminus
CFP
YFP
OR
C-terminus
OR
Energy transfer from excitation of the donor
molecule (CFP) to the acceptor molecule
(YFP) will occur only if the molecules are less
than100Å apart. This causes quenching of
donor fluorescence and excitation of the
acceptor at its emission wavelength.
MCT1cCFP is properly targeted to the plasma membrane and
catalytically active when co-expressed with CD147cYFP
MCT1cCFP single transfected
Number of cells
14
MCT1cCFP
12
single
transfected
10
Non-transfected
8
6
MCT1cCFP /
CD147cYFP
cotransfected
4
2
0
1
3
5
7
9 11
13 15
17 19
Rate of transport
F.s-1
MCT1cCFP/CD147cYFP
co-transfected
FRET occurs between MCT1nCFP and CD147cYFP but not between
MCT1nCFP and CD147nYFP
30
intensity
25
20
Co-transfection with MCT1cCFP + CD147nYFP
MCT1
CFP laser excitation
15
10
No FRET
5
0
CFP
460
480
500
520
540
560
580
600
intensity
Emission wavelength nm
14
12
10
8
6
4
2
0
460
Single transfection
MCT1nCFP
CD147
YFP laser excitation
CD147
No FRET
480
500
520
540
560
580
600
25
CD147cYFP
intensity
CFP laser excitation
10
8
CFP
Single transfection
12
intensity
ss
ss
ss
ss
Emission wavelength nm
FRET
MCT1
6
CD147
YFP
CFP
2
Co transfection
480
500
520
540
560
580
Emission wavelength nm
600
MCT1nCFP + CD147cYFP
CFP laser excitation
20
15
10
No FRET
5
0
460
ss
ss
4
0
460
YFP
MCT1
YFP
480
500
520
540
560
580
600
Emission
wavelength
YFP laser
excitationnm
30
intensity
N
20
10
0
460
480
500
520
540
560
Emission wavelength nm
580
600
Confirmation of FRET using fluorescence bleaching
Fluorescence intensity at 480nm (CFP) and 530nm (YFP) was measured before and after
bleaching with the YFP laser. Data are given as means ± S.E.M. (error bars) of 5
separate cells co-transfected with CD147nYFP and MCT1nCFP.
Before
After
CFP
YFP
CFP
YFP
Fluorescence before bleaching/
Fluorescence after bleaching
2
CFP fluorescence
1.5
YFP fluorescence
1.0
0.5
0
FRET response of different combinations of MCT1/CD147 constructs
40
35
Intensity
30
MCT1
FRET
25
CD147
ss
ss
20
15
10
5
0
450
YFP
CFP
475
500
525
550
575
600
70
60
Intensity
MCT1
NO FRET
50
CD147
YFP
40
ss
ss
30
20
10
0
450
475
500
525
550
575
CFP
600
70
FRET
Intensity
60
MCT1
50
CD147
ss
ss
40
30
20
YFP
10
0
450
475
500
525
550
575
CFP
600
140
FRET
120
Intensity
100
MCT1
ss
ss
ss
ss
60
40
20
0
450
475
500
525
180
550
575
CFP
600
NO FRET
150
MCT1
120
Intensity
CD147
80
CD147
YFP
ss
ss
90
60
30
0
450
YFP
470
490
510
530
550
570
590
Emission wavelength nm
CFP
Excitation was with
the CFP laser
Summary of FRET data for different combinations of MCT1/CD147 constructs
The presence of FRET is indicated by a low ratio of the emission at 480nm relative to 530nm. Data are
shown as means of the number of cells indicated (error bar = S.E.M.)
YFP
MCT1
CD147
s
s
s
s
480nm/530nm fluorescence emission ratio
CFP
2
(4)
YFP
CD147
MCT1cCFP
MCT1nCFP
CD147cCFP
CD147nCFP
1.6
1.2
(5)
(11)
0.8
MCT1
s
s
s
s
CD147
YFP
No FRET
CFP
CD147
MCT1
s
s
s
s
(3)
(2)
(6)
CFP
(7)
(10)
(19)
MCT1
s
s
s
s
(5) (6)
(18)
? FRET
YFP
MCT1
0.4
CFP
CD147
s
s
s
s
s
s
s
s
MCT1
FRET
YFP
0
MCT1cYFP MCT1nYFP CD147cYFP CD147nYFP
CFP
YFP CFP
MCT1
s
s
s
s
s
s
s
s
CD147
MCT1
?
Possible topology of MCT1/CD147 complex
N
out
N
out
N1
N2
Ca
in
N
in
Cb
C1
C
C
Monomer
C2
Dimer of 2 monomers