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., ... van derLaan, M. (2012). Role of mitochondrial inner membrane organizing system in protein biogenesis of themitochondrial outer membrane. Molecular Biology of the Cell, 23(20), 3948-3956.https://doi.org/10.1091/mbc.E12-04-0295

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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.

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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