The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

Abstract

Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein.

Figure 1.

LCA stimulates Ugo1 biogenesis in vivo and in organello. (A) Immunoblot analysis of mitochondria isolated from wild-type yeast cultures that were incubated in the presence or absence of LCA. (B) Quantification of protein levels of mitochondria from LCA-treated or control yeast cultures (−LCA values were set to 100%). Quantifications represent mean ± SEM (error bars; n ≥ 3). (C) Assembly reaction of radiolabeled Ugo1, Por1, and Aac2 precursors in isolated mitochondria from yeast cultures that were incubated in the presence or absence of LCA. Samples were solubilized in digitonin buffer and separated via blue native electrophoresis. Assembly of precursor in mature complexes was visualized by autoradiography. Δψ, membrane potential. (D) Quantification of assembly reactions from C. Values represent mean ± SEM (error bars; n = 3). (E) Two-dimensional blue native/SDS-PAGE of digitonin-lysed mitochondria from a wild-type yeast strain that was cultured in the absence or presence of LCA. TOM, translocase of the outer membrane (MW ∼400 kD); Ugo1, Ugo1 complex (MW ∼140 kD).

Figure 2.

Analysis of Ugo1 in ups1Δ mitochondria. (A) Immunoblot analysis of protein levels in isolated mitochondria from wild-type (WT) and ups1Δ strains. (B) Quantification of Ugo1 and control protein levels of WT and ups1Δ mitochondria from A (WT values were set to 100%). Quantifications represent mean ± SEM (error bars; n ≥ 3). (C) Two-dimensional blue native/SDS-PAGE of digitonin-lysed WT and ups1Δ mitochondria. TOM, translocase of the outer membrane (MW ∼400 kD); Ugo1, Ugo1 complex (MW ∼140 kD).

Figure 3.

Assay to test protein import in liposomes mimicking the lipid composition of the mitochondrial outer membrane (OMLs). (A) Experimental setup for testing of import and assembly of radiolabeled precursor proteins into liposomes mimicking the mitochondrial outer membrane lipid composition (OMLs). (B) Import of ³⁵S-labeled precursor proteins into mitochondria and OMLs as described in A. Precursors and accessible protein parts were digested by addition of Proteinase K (Prot. K) where indicated. Samples were analyzed by SDS-PAGE and autoradiography.

Figure 4.

The fusion machinery component Ugo1 assembles into its mature complex in OMLs. (A) Membrane topology comparison of ³⁵S-labeled Ugo1 in the mitochondrial outer membrane (Mito.) and OMLs assayed by Trypsin accessibility. f1 and f2, protease-protected fragments of imported Ugo1. Prec., Ugo1 precursor. (B) Sodium carbonate extraction (pH 11.5) was performed after import of radiolabeled Ugo1 into wild-type mitochondria (Mito.) or OMLs. Mock control, radiolabeled Ugo1 precursor only. Lanes 3 and 4 show control proteins after carbonate extraction of mitochondria (Tom70, integral outer membrane protein; Mge1, soluble matrix protein). P, pellet; SN, supernatant. (C) ³⁵S-labeled Ugo1 was imported into isolated mitochondria (Mito.) or OMLs at 25°C for the indicated time periods. Mitochondria and OMLs were lysed with digitonin and analyzed by blue native electrophoresis and digital autoradiography. (D) Import of radiolabeled Ugo1 in the presence of increasing amounts of OMLs. Sample analysis was performed as in C.

Figure 5.

Assembly of Ugo1 requires PA. (A) ³⁵S-labeled Ugo1 was imported into isolated mitochondria (Mito.) and OMLs lacking or containing PA (0.4%). Samples were lysed in digitonin buffer and separated by blue native electrophoresis and analyzed by digital autoradiography. (B) Quantification of the 140 kD assembly of Ugo1 in OMLs analyzed as described in A. Values were adjusted to the lipid amount of the samples analyzed and set to 100% for OMLs containing PA. Quantifications represent mean ± SEM (error bars; n = 3). (C) Radiolabeled Ugo1 was imported into mitochondria (Mito.) or OMLs with increasing concentrations of PA. Samples were solubilized in digitonin buffer and analyzed by blue native electrophoresis and digital autoradiography.

Figure 6.

In vivo stimulation of Ugo1 biogenesis in mim1Δ cells by LCA treatment or UPS1 deletion. (A) Immunoblot analysis of mim1Δ mitochondria from cells that were cultured in the absence or presence of LCA. (B) Quantification of Ugo1 and control protein levels from A. Quantifications represent mean ± SEM (error bars; n = 3). (C) Two-dimensional blue native/SDS-PAGE of digitonin-lysed mitochondria from mim1Δ yeast that was cultured in the absence or presence of LCA. TOM, translocase of the outer membrane (MW ∼400 kD); Ugo1, Ugo1 complex (MW ∼140 kD). (D) Immunoblot analysis of mitochondria from mim1Δ and mim1Δups1Δ strains. (E) Quantification of Ugo1 and control proteins from D as described in B (n ≥ 3). (F) Two-dimensional blue native/SDS-PAGE of digitonin-lysed mitochondria from mim1Δ and mim1Δups1Δ strains. SDH, succinate dehydrogenase complex (MW ∼230 kD).