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. 2013 Nov;90(4):824-40.
doi: 10.1111/mmi.12402. Epub 2013 Oct 10.

Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae

V A Samuli Kursu  1 , Laura P Pietikäinen, Flavia Fontanesi, Mari J Aaltonen, Fumi Suomi, Remya Raghavan Nair, Melissa S Schonauer, Carol L Dieckmann, Antoni Barrientos, J Kalervo Hiltunen, Alexander J Kastaniotis
Affiliations

Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae

V A Samuli Kursu et al. Mol Microbiol. 2013 Nov.

Abstract

Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell.

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Figures

Fig. 1
Fig. 1
Mitochondrial fatty acid synthesis (mtFAS)/ lipoic acid (LA) synthesis and attachment pathways and synthetic mutants identified in the screen. (A) Schematic depiction of the mtFAS and LA synthesis and attachment pathways. (B) Graphic presentation of the results of the synthetic petite screen. Mutated factors are color-coded to indicate functional groups. Blue: mtFAS enzymes, magenta: post-transcriptional gene expression related proteins, purple: mutations related to LA attachment (Lip3), LA-dependent complexes (Kgd1) or the pyruvate dehydrogenase complex (PDH) bypass (Ald4).
Fig. 2
Fig. 2
Characterization of yeast strains carrying deletion mutations in genes required for lipoic acid synthesis and attachment and mitochondrial fatty acid synthesis deletion. (A) Endogenous cellular respiration, (B) cytochrome spectra, (C) cytochrome c oxidase complex (COX) activity, (D) NADH cytochrome c reductase activity (complex I + III) (NCCR) activity, (E) succinate cytochrome c reductase (complex II + III) (SCCR) activity, and (F) ATPase activity. Data are shown as the mean of three independent repetitions ± SD. (G) Mitochondrial membrane potential in etr1Δ and FabI strains inhibited by triclosan, assessed by measuring JC-1 fluorescence with FACS (fluorescence-activated cell sorting). Orange fluorescence (FL2) indicates high membrane potential, while green fluorescence (FL-1) is observed when the mitochondrial membrane potential is strongly decreased (see Experimental Procedures). FCCP (carbonyl cyanide 3- trifluoromethoxy phenylhydrazone) is an uncoupler that causes dissipation of membrane potential.
Fig. 3
Fig. 3
Mitochondrial DNA gene expression and OXPHOS enzyme subunit levels in intron-containing mitochondrial fatty acid synthesis mutant strains. (A) Steady state levels of indicated proteins by western blot analysis of isolated mitochondria. Loading control: Porin. (B) De novo translation of mtDNA-encoded proteins. (C) Processing of mitochondrial transcripts. A cpb2Δ strain was used as a non-lipoylation/non-mtFAS-defective respiratory deficient control. Total RNA extracted from the strains was hybridized with a probe complementary to COX1, COX2 and CYTB. Loading control: SCR1, the RNA subunit of the Signal Recognition Particle (SRP), a housekeeping gene.
Fig. 4
Fig. 4
Mitochondrial gene expression, OXPHOS enzyme subunit levels, COX activity, respiration and growth of intronless mtFAS mutant strains. (A) Processing of mitochondrial transcripts in mitochondrial fatty acid synthesis deletion strains with or without mitochondrial introns. Total RNA extracted from the strains was hybridized with a probe complementary to COX1, COX2 or CYTB. Loading control: SCR1. (B) Analysis of the steady state levels of indicated proteins from mitochondrial extracts by western blot. Loading control: Porin. (C) De novo protein synthesis in intronless and control strains. (D) Cytochrome oxidase activity measurements and cell respiration in intron-containing and intronless W1536 5B etr1Δ strains and the ietr1Δ strain expressing one extra copy of either mss51F199I or wild-type MSS51. Endogenous cell respiration was measured polarographically. Data are shown as the mean of three independent repetitions ± SD. (E) Dilution series testing for growth of strains analyzed in panels C and D on non-fermentable and fermentable carbon sources.
Fig. 5
Fig. 5
Effect of δ-aminolevulenic acid (δ-ALA) supplementation and FAM1-1 overexpression on the OXPHOS phenotypes of mtFAS and LA attachment mutant strains. (A) β-Galactosidase assay. Medium was supplemented with 100 µg ml−1 δ-ALA. Asterisk indicates statistical significance determined by Student's t test (***p < 0.0001, *p < 0.05). Data are represented as the mean of at least 17 measurements ± SD. (B) Supplementation with δ-ALA does not improve accumulation of respiratory complex subunits. Western blot analysis of Cox1 and Sdh2 steady state levels in mitochondria from wild-type, mtFAS and LA attachment-deficient cells grown in the presence or absence of 400 µg ml−1 δ-ALA. Loading control: Porin. (C) Western blot analyses of the steady state levels of the indicated proteins in control and mtFAS or LA attachment deficient strains overexpressing FAM1-1. Lat1 and Kgd2 are the lipoylated E2 subunits of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex, respectively. Loading control: Porin. (D) Processing of the RPM1 RNA subunit of RNase P is improved in mtFAS deficient strains overexpressing FAM1-1. Total RNA extracted from the indicated strains was hybridized with a probe complementary to RPM1. The arrow indicates mature RPM1.
Fig. 6
Fig. 6
Effect of δ-aminolevulenic acid (δ-ALA) supplementation and FAM1-1 overexpression on heme content in mitochondrial fatty acid synthesis and lipoic acid attachment mutant strains. Hemes were extracted from isolated mitochondria (left panel) or spheroplasts (right panel) and analyzed by HPLC on a reverse phase C18 column. Hemoglobin was used to calibrate the column. The peaks corresponding to heme B were quantified by calculating the areas under the peaks and expressed in µV*sec. The peaks corresponding to heme O and heme A were basically undetectable at the represented scale and have been omitted from the figure.
Fig. 7
Fig. 7
Simplified model of mtFAS-dependent regulatory circuits controlling mitochondrial gene expression. MtFAS is needed for octanoyl-ACP production, the sole octanoate source for lipoic acid (LA) synthesis. The pyruvate dehydrogenase complex (PDH) activity is part of a positive feedback loop (red and yellow circular arrow loop) with LA production and acetyl-CoA substrate production. The Krebs cycle and α-ketoglutarate dehydrogenase complex (α-KGDH) provide reducing power, succinate and precursors for heme biosynthesis. The arrows represent the flow of material and the requirement of products of the mtFAS pathway in LA synthesis, RPM1 RNA processing and respiratory complex assembly. RPM1 must be processed to assemble fully active RNase P for processing of the tRNA-containing RPM1 precursor RNA (second proposed positive feedback loop). Translation is needed for mRNA splicing, possibly resulting in mtFAS signal amplification (third proposed feedback loop). Some subunits of the cytochrome c reductase complex (III), cytochrome c oxidase complex (IV) and F1Fo ATP synthase (V) are translated in the mitochondria. The succinate dehydrogenase complex (II), as well as complexes III, IV and cytochrome c (c), require heme for their assembly and function.
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