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. 2009 Dec 29;19(24):2133-9.
doi: 10.1016/j.cub.2009.10.074. Epub 2009 Dec 3.

Mitochondrial cardiolipin involved in outer-membrane protein biogenesis: implications for Barth syndrome

Natalia Gebert  1 , Amit S Joshi, Stephan Kutik, Thomas Becker, Matthew McKenzie, Xue Li Guan, Ved P Mooga, David A Stroud, Gnanada Kulkarni, Markus R Wenk, Peter Rehling, Chris Meisinger, Michael T Ryan, Nils Wiedemann, Miriam L Greenberg, Nikolaus Pfanner
Affiliations

Mitochondrial cardiolipin involved in outer-membrane protein biogenesis: implications for Barth syndrome

Natalia Gebert et al. Curr Biol. .

Abstract

The biogenesis of mitochondria requires the import of a large number of proteins from the cytosol [1, 2]. Although numerous studies have defined the proteinaceous machineries that mediate mitochondrial protein sorting, little is known about the role of lipids in mitochondrial protein import. Cardiolipin, the signature phospholipid of the mitochondrial inner membrane [3-5], affects the stability of many inner-membrane protein complexes [6-12]. Perturbation of cardiolipin metabolism leads to the X-linked cardioskeletal myopathy Barth syndrome [13-18]. We report that cardiolipin affects the preprotein translocases of the mitochondrial outer membrane. Cardiolipin mutants genetically interact with mutants of outer-membrane translocases. Mitochondria from cardiolipin yeast mutants, as well as Barth syndrome patients, are impaired in the biogenesis of outer-membrane proteins. Our findings reveal a new role for cardiolipin in protein sorting at the mitochondrial outer membrane and bear implications for the pathogenesis of Barth syndrome.

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Figures

Figure 1
Figure 1. Outer membrane lipids and genetic interaction of cardiolipin mutants with tom/sam mutants
(A) Purified yeast mitochondrial outer membrane vesicles were analyzed by immunoblotting (left panel) and liquid chromatography/mass spectrometry (right panels). PE, phosphatidylethanolamine; PI, phosphatidylinositol; Sss1, endoplasmic reticulum protein. (B) Cardiolipin was quantified [11] using tetramyristoyl cardiolipin as internal standard. Data from two independent outer membrane preparations are represented as mean +/− range. The purity of outer membrane vesicles was determined by a Western blot titration of outer membrane proteins (Tom40, OM45) and inner membrane proteins (Cox1, Sdh4). (C and D) Genetic interactions, synthetic growth defects. Cells were grown at 30°C in liquid YPD to the early stationary phase, serially diluted, spotted on YPD plates and incubated at the indicated temperatures. Synthetic growth defects of double deletion mutants are indicated; ‘-‘ no synthetic growth defect.
Figure 2
Figure 2. Cardiolipin mutant mitochondria are impaired in precursor accumulation at the outer membrane
(A) Steady-state protein levels. Mitochondria (Mitoch., μg protein) from wild-type (WT), crd1Δ and taz1Δ yeast grown at 21°C in liquid YPG were analyzed by SDS-PAGE and immunoblotting. (B) Isolated mitochondria were analyzed by blue native electrophoresis and immunoblotting. The band at ~230 kDa in the Tom40 Western blot is only visible after long exposure. (C) 2D-analysis. Isolated mitochondria were separated by blue native electrophoresis, followed by SDS-PAGE and immunoblotting. (D) Oxa1 was imported into isolated BY mitochondria at 24°C in the absence of a Δψ. Samples were analyzed by blue native electrophoresis and autoradiography.
Figure 3
Figure 3. Cardiolipin mutant mitochondria are impaired in the assembly of outer membrane proteins
(A) Isolated yeast mitochondria were separated by blue native electrophoresis and analyzed by immunoblotting. (B) [35S]Tom40 precursor was incubated with WT, crd1Δ and taz1Δ mitochondria (min). Mitochondria were reisolated and analyzed by blue native electrophoresis (upper panel) or SDS-PAGE (lower panel) and autoradiography. (C) Tom40 precursor was imported and analyzed by blue native electrophoresis. (D) Tom40-G354A precursor was imported in isolated mitochondria. (E) [35S]Tom20 was incubated with WT, crd1Δ and taz1Δ mitochondria at 24°C (min). Samples were analyzed by blue native electrophoresis and autoradiography. (F) Tom22 precursor was imported and analyzed as in E. (G) After import of [35S]Tom20 (lane 1–10) or Tom22 (lane 11–20), mitochondria were incubated with 0.1 M Na2CO3 for 30 min. Pellets were analyzed by SDS-PAGE and autoradiography. Samples 10 and 20 did not contain mitochondria.
Figure 4
Figure 4. Assembly of outer membrane proteins in Barth syndrome mitochondria
(A–C) Mitochondria isolated from control or Barth syndrome patient lymphoblasts were incubated at 37°C (min) in the presence of [35S]VDAC1 or [35S]Tom22. Following import, samples were analyzed by blue native electrophoresis (upper panel) and SDS-PAGE (lower panel) and autoradiography. A sample of reticulocyte lysate (representing 50% of added precursor/import) is also shown on the SDS-PAGE gel. For B, assembled VDAC1 was quantified from four independent experiments (three for 2.5 min). Data are represented as mean +/− SEM. (D) Control and Barth syndrome mitochondria were analyzed by SDS-PAGE and immunoblotting. SDHA, 70 kDa subunit of succinate dehydrogenase.
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