Liet al.,Science 375 , eabi4343 (2022) 14 January 2022 4 of 10
G H(^001234)
50
100
150
pixel intensity (a.u.)
(^001234)
20
40
60
80
pixel intensity (a.u.)
IJ
distance(μm)
distance(μm)
MFN1 mitoT
MFN1 mitoT
mitoT
MFN1
uninf
Toxo
merge
merge
(^001234)
50
100
150
pixel intensity (a.u.)
(^001234)
100
200
300
pixel intensity (a.u.)
distance(μm)
distance(μm)
MFN2 mitoT
MFN2 mitoT
mitoT
MFN2
uninf
MFN2
MFN1
TgGRA7
TgMAF1
ACTB
uninf wt Toxo Δmaf1
MIRO1
MIRO2
mitoT
MFN1
mitoT
MFN2
Toxo
F
A
MFN1 MFN2MIRO1MIRO2
fold change (relative to uninf)
0.0
0.4
0.8
1.2
1.6
- uninf
w t To x o
Δmaf1
log 2 FC (mitoIP WTToxo/mitoIP uninf)
-log
(adj. p-value) 10
-2 -1 0 1 2
0
1
2
3
4
5
6
7
8
log 2 FC (mitoIPΔmaf1 Toxo/mitoIP WTToxo)
-log
(adj. p-value) 10
-2 -1 0 1 2
0
1
2
3
4
5
6
7
8
−0.11
−0.26
−0.04
−0.27
−0.28
−0.28
−0.17
−0.09
−0.03
non−MitoCarta3.0
MitoCarta3.0
mito−interacting
Matrix
OMM
IMM
IMS
unknown
Membrane
−2 −1 0 1 2
0.03
0.13
0.09
0.14
0.16
0.11
0.04
0.06
MitoCarta3.0
mito−interacting
Matrix
OMM
IMM
IMS
unknown
Membrane
−2 −1 0 1 2
B
C D
E
log 2 FC (mitoIP WTToxo/mitoIP uninf)
log 2 FC (mitoIPΔmaf1 Toxo/mitoIP WTToxo)
MitoCarta 3.0
OMM
MitoCarta 3.0
OMM
0.07
non−MitoCarta3.0
MFN2MFN 2
MFN1MFN 1
MIRO1MIRO 1
MIRO2MIRO 2
MFN2MFN 2
MFN1MFN 1
MIRO1MIRO 1
MIRO2MIRO 2
ARG2ARG 2
ACAD11ACAD 11
MARC2MARC 2
MAIP1MAIP 1
Fig. 3. TgMAF1 drives changes in the mitochondrial proteome and the
depletion of certain OMM proteins that localize to SPOTs.(Aand
C) Mitochondria were immunopurified (mitoIP) from HeLa cells that were
uninfected (uninf) (n= 4), WTToxo-infected (n= 4), orDmaf1 ToxoÐinfected
(n= 3) at 24 hours after infection and analyzed by means of mass spectromtery.
Shown are boxplots depicting the log 2 -fold change (FC) of detected MitoCarta3.0
proteins between indicated treatments according to submitochondrial localiza-
tion. Median values for each subcompartment are indicated in red. (Band
D) Volcano plot of proteins in (A) and (C). Dark gray, OMM proteins as classified
by MitoCarta3.0; white, all other MitoCarta3.0 proteins. (E) Cells were uninf,
WTToxoÐ, orDmaf1-infected for 24 hours and analyzed by means of immunoblotting
forMFN1,~80kda;MFN2,~80kDa;MIRO1,~75kDa;MIRO2,~75kDa;ACTB,
~45 kDa; TgGRA7, ~32 kDa; and TgMAF1, ~60 kDa. (F) Fold change in density of
protein bands imaged as in (E) normalized to actin and relative to mean of uninfected
samples. Data are mean ± SEM from three biological replicates; P< 0.05, P< 0.01,
P< 0.001, ****P<0.0001forWTToxoversus uninfected orDmaf1-infected
samples. Representative live-cell images of (G)Mfn1−/−:GFP-Mfn1MEFs and
(I)Mfn2−/−MEFs expressing mCh-MFN2 at 24 hours after infection withToxoplasma
(BFP). (HandJ) Corresponding pixel intensity plots for white line in (G) and (I)
insets, respectively. Scale bars, 5mm and (inset) 1mm.
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