University of Groningen Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane Bohnert, 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, Bettina Published in: Molecular Biology of the Cell DOI: 10.1091/mbc.E12-04-0295 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record 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 der Laan, M. (2012). Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane. Molecular Biology of the Cell, 23(20), 3948-3956. DOI: 10.1091/mbc.E12-04-0295 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 22-11-2018
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 1
SUPPLEMENTAL FIGURES FIGURE S1: Mitochondrial ultrastructure of yeast cells lacking the POTRA domain of Sam50. Representative electron microscopy images of Fcj1 ProtA cells and Fcj1 ProtA 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 mitochondria. Mitochondria isolated from wild-type () and 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) [ 35 S]Porin or (D) [ 35 S]Mdm10 were incubated with isolated wild-type, 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 (),, 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 () 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. 2
FIGURE S5: Biogenesis of outer membrane proteins in mitochondria. (A) 35 S- labeled Tom22 or (B) 35 S-labeled Tom5 were imported into wild-type (), and mio10δ mitochondria for the indicated periods. Upon solubilization in digitonincontaining buffer, blue native electrophoresis and digital autoradiography were applied. (C) [ 35 S]Tom40 was imported into wild-type, Fcj1 ProtA and Oxa1 ProtA (control) mitochondria for five minutes. Mitochondria were re-isolated, lysed with digitonincontaining buffer and subjected to IgG affinity chromatography, elution with TEV protease, SDS-PAGE and digital autoradiography. Load, 0.5%; elution, 100%. 3
Fcj1 ProtA Fcj1 ProtA Sam50 120 Bohnert et al., Figure S1
A Fcj1 - Mio27 - Sam50 - Sam35 - Tom70 - MINOS SAM Tim13 - Tim12 - Tim10 - Tim9 - Tim44 - IMS chaperones B Tom40 Tom22 SAM Sam50 Tom40 - Tim23 - TIM23-PAM Tom22 - Tim21-1 2 3 4 5 6 1 2 3 4 C [ 35 S]Porin mio10 D [ 35 S]Mdm10 mio10 min Porin 1 5 20 1 5 20 1 5 20 min SAM-Mdm10 5 15 40 5 15 40 5 15 40 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Bohnert et al., Figure S2
mol% of mitochondrial phospholipids 50 40 30 20 10 0 fcj1δ mio10δ LP PI PS PC PE CL DMPE PA Bohnert et al., Figure S3
A Mito. (μg) Fcj1-10 Fcj1 Fcj1 20 40 10 20 40 Mito. (μg) 10 20 40 10 20 40 Tom70 - Mio27 - Tom40 - Mio10 - Aim37 - MINOS Tom22 - Tom20 - Aim13 - Tim13 - Aim5 - Sam50 - Tim12 - Tim10 - IMS chaperones Sam37 - SAM Tim9 - Sam35 - Tim44 - TIM23 1 2 3 4 5 6 7 8 9 10 11 12 B Tom40 Tom22 Sam50 Fcj1 Fcj1 SAM Fcj1 1 2 3 4 5 6 Bohnert et al., Figure S4
A min [ 35 S]Tom22 mio10 5 15 40 5 15 40 5 15 40 1 2 3 4 5 6 7 8 9 B min [ 35 S]Tom5 mio10 5 15 40 5 15 40 5 15 40 1 2 3 4 5 6 7 8 9 66 C Load Eluate Load Eluate Fcj1 ProtA Fcj1 ProtA Oxa1 ProtA Oxa1 ProtA [ 35 S]Tom40-1 2 3 4 5 6 7 8 Bohnert et al., Figure S5