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University of Groningen
Role of mitochondrial inner membrane organizing system in protein biogenesis of themitochondrial outer membraneBohnert, Maria; Wenz, Lena-Sophie; Zerbes, Ralf M.; Horvath, Susanne E.; Stroud, David A.;von der Malsburg, Karina; Mueller, Judith M.; Oeljeklaus, Silke; Perschil, Inge; Warscheid,BettinaPublished in:Molecular Biology of the Cell
DOI:10.1091/mbc.E12-04-0295
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Publication date:2012
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):Bohnert, M., Wenz, L-S., Zerbes, R. M., Horvath, S. E., Stroud, D. A., von der Malsburg, K., Mueller, J. M.,Oeljeklaus, S., Perschil, I., Warscheid, B., Chacinska, A., Veenhuis, M., van der Klei, I. J., Daum, G.,Wiedemann, N., Becker, T., Pfanner, N., & van der Laan, M. (2012). Role of mitochondrial inner membraneorganizing system in protein biogenesis of the mitochondrial outer membrane. Molecular Biology of theCell, 23(20), 3948-3956. https://doi.org/10.1091/mbc.E12-04-0295
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1
SUPPLEMENTARY INFORMATION
Role of MINOS in protein biogenesis of the mitochondrial outer membrane
Maria Bohnert, Lena-Sophie Wenz, Ralf M. Zerbes, Susanne E. Horvath, David A.
Stroud, Karina von der Malsburg, Judith M. Müller, Silke Oeljeklaus, Inge Perschil,
Bettina Warscheid, Agnieszka Chacinska, Marten Veenhuis, Ida J. van der Klei,
Günther Daum, Nils Wiedemann, Thomas Becker, Nikolaus Pfanner, and Martin van
der Laan
2
SUPPLEMENTAL FIGURES
FIGURE S1: Mitochondrial ultrastructure of yeast cells lacking the POTRA domain of
Sam50. Representative electron microscopy images of Fcj1ProtA cells and
Fcj1ProtA Sam50∆120 cells lacking the N-terminal POTRA domain of Sam50 are shown
(mitochondria were stained with diaminobenzidine (DAB)). Bars in the first and third
rows represent 1 µM; bars in the second and fourth rows represent 200 nm.
FIGURE S2: Steady-state levels and protein import in fcj1∆ mitochondria.
Mitochondria isolated from wild-type (WT) and fcj1∆ cells were subjected to SDS-
PAGE (A) or blue native electrophoresis (B) and mitochondrial protein content was
analyzed by immunoblotting. IMS, intermembrane space; PAM, presequence
translocase-associated motor; TIM, translocase of the inner mitochondrial membrane.
(C) [35S]Porin or (D) [35S]Mdm10 were incubated with isolated wild-type, fcj1∆ and
mio10∆ mitochondria for the indicated periods. The mitochondria were analyzed by
blue native electrophoresis and digital autoradiography.
FIGURE S3: Phospholipid composition of MINOS mutant mitochondria. Mitochondria
were isolated from wild-type (WT), fcj1∆, and mio10∆ cells and mitochondrial
phospholipids were extracted and quantified. Mean values of four measurements with
standard error of the mean are shown. LP, lysophospholipids; PI,
phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; CL, cardiolipin; DMPE,
dimethylphosphatidylethanolamine; PA, phosphatidic acid.
FIGURE S4: Mitochondrial protein content upon depletion of mitofilin/Fcj1 in yeast.
Mitochondria (µg protein) isolated from wild-type (WT) or Fcj1-depleted (Fcj1↓) cells
were subjected to SDS-PAGE (A) or blue native electrophoresis (B) and the protein
content was analyzed by Western blotting.
3
FIGURE S5: Biogenesis of outer membrane proteins in fcj1∆ mitochondria. (A) 35S-
labeled Tom22 or (B) 35S-labeled Tom5 were imported into wild-type (WT), fcj1∆ and
mio10Δ mitochondria for the indicated periods. Upon solubilization in digitonin-
containing buffer, blue native electrophoresis and digital autoradiography were
applied. (C) [35S]Tom40 was imported into wild-type, Fcj1ProtA and Oxa1ProtA (control)
mitochondria for five minutes. Mitochondria were re-isolated, lysed with digitonin-
containing buffer and subjected to IgG affinity chromatography, elution with TEV
protease, SDS-PAGE and digital autoradiography. Load, 0.5%; elution, 100%.
Fcj1ProtA
Fcj1ProtA Sam50∆120
Bohnert et al., Figure S1
SAM-Mdm10440
232
140
669
Tim9 -
Tim10 -
Tim12 -
Tim13 -
Tom22 -
Tom70 -
Tom40 -
Sam35 -
Sam50 -
Mio27 -
Fcj1 -
Tim23 -
WT
fcj1∆
WT
fcj1∆
1 2 3 4
Tim21 -
Tim44 -
MINOS
TOM
SAM
IMSchaperones
TIM23-PAM
A
440232
140
kDa669
440
232
140
kDa669
WT
fcj1∆
WT
fcj1∆
TOM
Anti-Tom40
Anti-Tom22
1 2 3 4 5 6
WT
fcj1∆
Anti-Sam50B
SAM
D
440
232
140
kDa
C1 5 20 1 5 20 1 5 20
fcj1∆ WT mio10∆[35S]Porin
min
Porin
1 2 3 4 5 6 7 8 9
kDa5 15 40 5 15 40 5 15 40
fcj1∆ WT mio10∆[35S]Mdm10
min
1 2 3 4 5 6 7 8 9
Bohnert et al., Figure S2
Bohnert et al., Figure S3
0
10
20
30
40
50
LP PI PS PC PE CL DMPE PA
WTfcj1Δmio10Δ
mol
% o
f mito
chon
dria
l pho
spho
lipid
s
SAM440
232
140
kDa669
Tim9 -
Tim10 -
Tim12 -
Tim13 -
IMSchaperones
WT
WT
TOM
Anti-Tom40
Anti-Tom22
1 2 3 4
Sam35 -
Sam50 -
Fcj1 -
Tom20 -
Bohnert et al., Figure S4
1 2 3 4
Sam37 -
MINOS
TOM
SAM
402010Mito. (μg)
Mio10 -
Mio27 -
Aim5 -
Aim13 -
Aim37 -
Tom40 -
Tom22 -
Tom70 -
402010 402010Mito. (μg) 402010
WT Fcj1 WT Fcj1
5 6 7 8 9 10 11 12
A
B
Tim44 - TIM23
440
232
140
kDa
5 6
WT
Anti-Sam50
Fcj1
Fcj1
Fcj1
TOM
1 2 3 4 5 6 7 8 9
5 15 40 5 15 40 5 15 40
fcj1∆ WT mio10∆[35S]Tom22
min
440
232
140
kDa669
Bohnert et al., Figure S5
TOM 440
232
140
kDa669
66
5 15 40 5 15 40 5 15 40
fcj1∆ WT mio10∆[35S]Tom5
min
1 2 3 4 5 6 7 8 9
A
B
Load Eluate
WT
Fcj1
Pro
tA
WT
Fcj1
Pro
tA
1 2 3 4 5 6 7 8
WT
Oxa
1 Pro
tA
WT
Oxa
1 Pro
tA
Load Eluate
[35S]Tom40 -
C