Biogenesis of mitochondria: The effects of altered steady-state membrane lipid composition on mitochondrial-energy metabolism in Saccharomyces cerevisiae

A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression.The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated.The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%.In cells which contained lipids with 22–28% unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the levels of cytochromes aa₃ and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.     

The effect of altered membrane sterol composition on the temperature dependence of yeast mitochondrial ATPase

The sterol content of cells of $\text{Saccharomyces cerevisiae}$ was manipulated by growing the organism anaerobically in a medium containing excess supplements of unsaturated fatty acids and a range of supplements of ergosterol. Anaerobic mitochondrial precursor structures were isolated whose membrane lipids contain the same fatty acid composition but whose sterol content varies from 7 to 105 mg/g mitochondrial protein. Arrhenius plots of the mitochondrial ATPase activity of the different preparations show a discontinuity with Arrhenius activation energies of about +40 and +80 KJ/mole, respectively, above and below the transition temperature. However, the temperature of the transition is markedly dependent on sterol composition, and increases by up to 17° as the sterol content of the mitochondria is progressively decreased. These results support the concepts that membrane lipid composition influences the activity of membrane-bound enzymes, and that sterols promote the gel to liquid phase transition in biological membranes.     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

Lipid content of Saccharomyces cerevisiae strains with different degrees of ethanol tolerance

Summary We analysed the fatty acid and sterol compositions of various Saccharomyces cerevisiae strains with ethanol tolerance varying from 4% to 12% (v/v) ethanol and at different concentrations of ethanol. The results we obtained agree with the existence of a relationship between membrane fluidity and ethanol tolerance but they do not support a direct role of unsaturated fatty acids in this tolerance. On the other hand, they support the importance of ergosterol in this phenomenon.     

Induction of respiratory incompetent mutants by unsaturated fatty acid depletion in Saccharomyces cerevisiae

Constant levels of cellular unsaturated fatty acids were obtained by growing a fatty acid desaturase mutant of $\text{Saccharomyces cerevisiae}$ in glucose limited chemostat cultures supplemented with various concentrations of Tween 80. An increase in the frequency of cytoplasmic respiratory incompetent mutants was observed in cultures growing at low cellular levels of unsaturated fatty acids. This effect has been shown to result from an increase in the rate of mutation as the cellular unsaturated fatty acid level is decreased. The majority of induced petite mutants are ?° (contain no mitochondrial DNA).     

Proton magnetic resonance spectroscopy of promitochondrial membranes from yeast grown under different regimes of lipid supplementation

Summary Promitochondrial membranes, prepared fromSaccharomyces cerevisiae grown anaerobically under different conditions of lipid supplementation, have been examined by PMR spectroscopy. Promitochondria from cells cultured anaerobically in media containing both unsaturated fatty acid and sterol supplements, or containing unsaturated fatty acid alone, yield high resolution spectra similar to those which are characteristic of aerobic mitochondria. By contrast, promitochondrial membranes from cells grown only with sterol supplementation in order to deplete unsaturated fatty acid and total phospholipid content of the organelles, yielded PMR spectra which were very substantially broadened. These spectra are similar to those obtained with rat liver mitochondria.PMR spectra of promitochondria from each cell type dispersed in trifluoroacetic acid, or of extracted lipids or residual proteins similarly dispersed, were different only in detail. It appears, therefore, that in the native state membranes of unsaturated fatty acid-depleted promitochondria are structurally different from promitochondria of the other two cell types. The difference may be a consequence of altered lipid-to-protein ratios, and thus of changes in the extent of lipid domain formation in the membranes of these organelles.     

Isolation of Saccharomyces cerevisiae Mutants Defective in Acyl Coenzyme A Synthetase

Mutants of Saccharomyces cerevisiae defective in acyl-CoA synthetase (EC 6.2.1.3) were isolated. The mutants were concentrated by the radiation-suicide technique with the use of tritiated palmitic acid. Selection of the mutants was based on the premise that acyl-CoA synthetase activity would become indispensable when yeast cells in which fatty acid synthesis de novo is blocked are grown in a medium supplemented with fatty acid. The mutant strains isolated exhibited low acyl-CoA synthetase activity in vitro. Furthermore, they accumulated markedly more of the incorporated palmitic acid in the nonesterified form than did the wild- type strain. Some of the mutants showed thermosensitive acyl-CoA synthetase activity, indicating a mutation of the structural gene of the enzyme. Genetic studies on these mutants indicated that their phenotype resulted from a single, recessive mutation of a nuclear gene, designated faa 1 (fatty acid activation).     

Thermostability of Pseudomonas hydrogenothermophila Cytochrome c-552

The alcohol-fermenting yeast Torulaspora delbrueckii No. 3110 was less tolerant to high temperature than Saccharomyces cerevisiae IFO 0224 as measured by alcohol fermentation during mild agitation: at 40°C, ethanol production of the two yeasts was 0.8 and 5.2 wt% respectively. The No. 3110 cells had much unsaturated fatty acid (C18:2) and little ergosterol, which suggests that the low tolerance might be caused by high membrane fluidity. Two types of miconazole-resistant mutants were isolated and characterized. Strain M47 had less unsaturated fatty acid and was found to be more temperature tolerant than No. 3110. Strain M59 was defective in ergosterol synthesis and was less temperature tolerant than No. 3110. These results indicate the importance of membrane rigidity in temperature tolerance.

M59 aaccumulated much less trehalose than No. 3110 did. Addition of trehalose to the permeabilized cell system of M59 restored the temperature sensitivity, but not when the trehalase inhibitor deoxynojirimycin was also added, which suggests that the accumulation and metabolism of trehalose is important for the expression of temperature tolerance.     

Mitogenic Effect of the Mannans from Saccharomyces cerevisiae on Mouse Spleen Lymphocytes

The DNA synthetic activities of mannans isolated from two Saccharomyces cerevisiae strains were examined in vitro using spleen cells obtained from normal or nude BALB/c strain mice. A highly branched mannan isolated from the S. cerevisiae wild type strain induced a greater increase in mitogenic activity than those displayed by the mannan of the S. cerevisiae X2180–1A-5 mutant strain which possessed fewer branching moieties. Acid-hydrolyzed wild type strain mannan with two-thirds of the molecular weight of the parent intact mannan showed weak mitogenicity. Increases in the DNA synthetic activities of nude and normal spleen cells were almost the same as that of wild type strain mannan, while nylon wool column-passed spleen cells obtained from both normal and nude mice did not show mitogenicity with this mannan. The results indicated that the mitogenic activity was responsible for the highly branched structure of the wild type strain mannan, and that this mannan is a B-cell mitogen.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both pro-mitochondria and mitochondria obtained from O₂-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N₂) to elaidic (O₂)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O₂-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

The effects of selenium,vitamin E and their combination on the composition of fatty acids and proteins in Saccharomyces cerevisiae

The aim of our studies was to test the effect and role of vitamin E and selenium supplements on yeast cell. In this study, the effects of selenium (Se), vitamin E (Vit. E), and their combination (Se plus Vit. E) on the composition of fatty acids and proteins were examined in Saccharomyces cerevisiae strains WET136 and 522. S. cerevisiae cells were grown up in YEPD medium supplemented with Se, Vit. E or their combination. It was found that the level of stearic acid was increased in all supplemented groups (p<0·05; p<0·001). The content of saturated and unsaturated fatty acids was decreased (p<0·05; p<0·01; p<0·001) in Vit. E and Vit. E plus Se supplemented S. cerevisiae. On the other hand, Se alone caused an increase (p<0·001) in the saturated fatty acids but a decrease (p<0·05; p<0·001) in the unsaturated fatty acids. Total proteins in S. cerevisiae were significantly increased (p<0·001) by Vit. E supplement. There was no significant change observed in S. cerevisiae supplemented with Se. These findings indicate that membrane composition of S. cerevisiae is affected by both Vit. E and Se supplements. © 1997 John Wiley & Sons, Ltd.     

The key role of the energized state of Saccharomyces cerevisiae mitochondria in modulations of the outer membrane channels by the intermembrane space proteins

Mitochondria of the yeast Saccharomyces cerevisiae constitute a perfect model to study the outer membrane channel modulation as besides the TOM complex channel they contain only a single isoform of the VDAC channel and it is possible to obtain viable mutants devoid of the channel. Here, we report that the fraction of the intermembrane space isolated from wild type and the VDAC channel-depleted yeast mitochondria, except of the well-known VDAC channel modulator activity, displays also the TOM complex channel modulating activity as measured in the reconstituted system and with intact mitochondria. The important factor influencing the action of both modulating activities is the energized state of mitochondria. Moreover, the presence of the VDAC channel itself seems to be crucial to properties of the intermembrane space protein (s) able to modulate the outer membrane channels because in the case of intact mitochondria quantitative differences are observed between modulating capabilities of the fractions isolated from wild type and mutant mitochondria.     

Unsaturated fatty acid composition of wild type and respiratory deficient yeasts after aerobic and anaerobic growth

An analysis is given of the fatty acid composition of 18 yeast species, predominantly of the genus Saccharomyces; respiratory deficient mutant strains are included. The results are discussed from chemotaxonomical and physiological viewpoints, with special attention to unsaturated fatty acids and their relation to the petite mutation. The fatty acid composition of anaerobically grown Saccharomyces cerevisiae remains restricted, as far as unsaturated fatty acids are concerned, to those added to the medium and it may thus differ considerably from the composition after aerobic growth. Depending on the acids added, the cells may contain either palmitoleic or linoleic acids as the sole unsaturated fatty acid after anaerobic growth and as the predominant unsaturated fatty acid after aerobic growth. In contrast to all other known eukaryotes, Schizosaccharomyces japonicus seems to possess an anaerobic pathway for synthesis of unsaturated fatty acids.     

Ionophores and intact cells II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg²⁺ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.

     

Products of Mitochondrial Protein Synthesis in Yeast

Analysis of membrane proteins of Saccharomyces cerevisiae mitochondria in cycloheximide-inhibited cells shows that they are encoded on mitochondrial DNA.     

Developing a genetic approach to investigate the mechanism of mitochondrial competence for DNA import

Mitochondrial gene products are essential for the viability of eukaryote obligate aerobes. Consequently, mutations of the mitochondrial genome cause severe diseases in man and generate traits widely used in plant breeding. Pathogenic mutations can often be identified but direct genetic rescue remains impossible because mitochondrial transformation is still to be achieved in higher eukaryotes. Along this line, it has been shown that isolated plant and mammalian mitochondria are naturally competent for importing linear DNA. However, it has proven difficult to understand how such large polyanions cross the mitochondrial membranes. The genetic tractability of Saccharomyces cerevisae could be a powerful tool to unravel this molecular mechanism. Here we show that isolated S. cerevisiae mitochondria can import linear DNA in a process sharing similar characteristics to plant and mammalian mitochondria. Based on biochemical data, translocation through the outer membrane is believed to be mediated by voltage-dependent anion channel (VDAC) isoforms in higher eukaryotes. Both confirming this hypothesis and validating the yeast model, we illustrate that mitochondria from S. cerevisiae strains deleted for the VDAC-1 or VDAC-2 gene are severely compromised in DNA import. The prospect is now open to screen further mutant yeast strains to identify the elusive inner membrane DNA transporter.     

Overexpression of the OLE1 gene enhances ethanol fermentation by Saccharomyces cerevisiae

 The fermentation characteristics of Saccharomyces cerevisiae strains which overexpress a constitutive OLE1 gene were studied to clarify the relationship between the fatty acid composition of this yeast and its ethanol productivity. The growth yield and ethanol productivity of these strains in the medium containing 15% dextrose at 10 °C were greater than those of the control strains under both aerobic and anaerobic conditions but this difference was not observed under other culture conditions. During repeated-batch fermentation, moreover, the growth yield and ethanol productivity of the wild-type S. cerevisiae increased gradually and then were similar to those of the OLE1-overexpressing transformant in the last batch fermentation. However, the unsaturated fatty acid content (77.6%) of the wild-type cells was lower than that (86.2%) of the OLE1-recombinant cells. These results suggested that other phenomena caused by the overexpression of the OLE1 gene, rather than high unsaturated fatty acid content, are essential to ethanol fermentation by this yeast. Received: 11 June 1999 / Received last revision: 12 November 1999 / Accepted: 28 November 1999