Regulation of mitochondrial biogenesis: enzymatic changes in cytochrome-deficient yeast mutants requiring delta-aminolevulinic acid.

Yeast cells almost completely deficient in all cytochromes were obtained by introducing two defective nuclear genes, cyd1 and cyc4, into the same haploid strain. The action of the two mutant genes is synergistic, since either gene acting singly results in only partial cytochrome deficiency. Normal synthesis of all cytochromes can be restored in the double mutant by adding delta-aminolevulinic acid to the growth medium. The optimum concentration of delta-aminolevulinate for restoration of cytochrome synthesis is about 40 muM; when higher concentrations are used, synthesis of cytochromes is partially suppressed, particularly that of cytochrome a.a3. Growth yield of the double mutant is stimulated by ergosterol and Tween 80, a source of unsaturated fatty acid. Methionine stimulates further. None of these nutrients is required for growth when sufficient delta-aminolevulinic acid is present in the growth medium. With respect to nutritional responses, the single-gene, cytochrome-deficient mutant, ole3, behaves like the double mutant. The frequency of the p-mutation in the double mutant grown in the absence of ergosterol, Tween 80, and delta-aminolevulinic acid is at least 15%. The frequency can be reduced to less than 1% by either delta-aminolevulinic acid or Tween 80. Ergosterol alone does not decrease the p- frequency. The ole3 mutant does not exhibit increased p-frequency under similar conditions of unsaturated fatty acid deficiency.     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.     

Separation of a protein factor necessary for the oxidative desaturation of fatty acids in the rat.

Microsomes from rat liver were extracted by low ionic strength solutions. Extracted microsomes lost most of the linoleic acid desaturation activity. The addition of the extract back into the extracted microsomes was necessary to restore full desaturation activity. The soluble fraction had no desaturation activity. The existence of a soluble factor loosely bound to the microsomes, stable to sonication, and unstable to heat and trypsin digestion was recognized. This protein could not be replaced by albumin. The factor was also essential for the oxidative desaturation of palmitic, stearic, linoleic, and gamma-linolenic acid. The present experiment suggests that the protein factor is not NADH-cytochrome b5 reductase, cytochrome b5, or the cyanide-sensitive factor.     

Trypanosoma brucei oleate desaturase may use a cytochrome b5-like domain in another desaturase as an electron donor.

An open reading frame with fatty acid desaturase similarity was identified in the genome of Trypanosoma brucei. The 1224 bp sequence specifies a protein of 408 amino acids with 59% and 58% similarity to Mortierella alpina and Arabidopsis thaliana Delta12 desaturase, respectively, and 51% with A. thaliana omega3 desaturases. The histidine tracks that compose the iron-binding active centers of the enzyme were more similar to those of the omega3 desaturases. Expression of the trypanosome gene in Saccharomyces cerevisiae resulted in the production of fatty acids that are normally not synthesized in yeast, namely linoleic acid (18:2Delta9,12) and hexadecadienoic acid (16:2Delta9,12), the levels of which were dependent on the culture temperature. At low temperature, the production of bi-unsaturated fatty acids and the 16:2/18:2 ratio were higher. Transformed yeast cultures supplemented with 19:1Delta10 fatty acid yielded 19:2Delta10,13, indicating that the enzyme is able to introduce a double bond at three carbon atoms from a pre-existent olefinic bond. The expression of the gene in a S. cerevisiae mutant defective in cytochrome b5 showed a significant reduction in bi-unsaturated fatty acid production, although it was not totally abolished. Based on the regioselectivity and substrate preferences, we characterized the trypanosome enzyme as a cytochrome b5-dependent oleate desaturase. Expression of the ORF in a double mutant (ole1Delta,cytb5Delta) abolished all oleate desaturase activity completely. OLE1 codes for the endogenous stearoyl-CoA desaturase. Thus, Ole1p has, like Cytb5p, an additional cytochrome b5 function (actually an electron donor function), which is responsible for the activity detected when using the cytb5Delta single mutant.     

The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.)

The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.     

Evidence for the different responses of delta9-, delta6- and delta5-fatty acyl-CoA desaturases to cytoplasmic proteins.

Microsomes prepared from the livers of 4-week-old rats were, after extraction with 0.1 M potassium phosphate buffer, pH 7.4, unable to catalyse either the delta6 desaturation of alpha-linolenic acid (9c.12c.15c., 18 : 3) into 6c.9c.12c.15c., 18 : 4 or the delta5 desaturation of eicosatrienoic acid (8c.11c.14c., 20 : 3) into arachidonic acid (5c.8c.11c.14c., 20 : 4). Both these enzymes only showed full activity after incubation of the microsomes with either the 100 000 X g supernatant fraction or with purified bovine catalase. Bovine serum albumin, while capable of restoring 50% of the delta5 desaturase activity has no effect on the delta6 desaturase. In contrast the delta9 desaturase activity of microsomes was never completely lost after extraction with buffer but could be stimulated by optimum concentrations of both bovine serum albumin and catalase. The significance of the different responses of the three desaturases to the cytoplasmic components is discussed.     

Increased activity of stearoyl-CoA desaturation in liver from rat fed clofibric acid

Male rats were fed a diet containing 0.5% (w/w) p-chlorophenoxyisobutyric acid (clofibric acid), a hypolipidemic drug. Activities of stearoyl-CoA desaturation in hepatic microsomes were increased approx. 4 times following the administration of clofibric acid for 7 days. An increase in the activity of desaturation of stearic acid was also observed in the liver of clofibric acid-fed rats in vivo. The increase in the activity of microsomal stearoyl-CoA desaturation by clofibric acid-feeding was due to the increase in the activity of terminal desaturase as measured by the rate constant for cytochrome b5 reoxidation, but not due to the changes in cytochrome b5 content and NADH-cytochrome b5 reductase activity. Increases in the activity of stearoyl-CoA desaturation by clofibric acid-feeding were also observed in rats of hormonally altered state, such as diabetic rats, hyperthyroid rats and hypothyroid rats. Percentages of octadecenoic acid in total fatty acid of hepatic lipid were increased with the increase in the activity of stearoyl-CoA desaturation.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

Stimulation of hepatic cytochrome b5-mediated lipid desaturation by renal microsomes

Although microsomes prepared from rat kidney cortex contained significant concentrations of both NADH cytochrome b₅ reductase and cytochrome b₅, they did not catalyze cytochrome b₅-dependent Δ⁹ oxidative lipid desaturation. However, incubation of kidney microsomes in the presence of control liver microsomes resulted in a two-fold increase in fatty acid desaturase activity over that seen with liver microsomes alone. Addition of kidney microsomes to liver microsomes prepared from animals maintained on a fat free diet resulted in an increased desaturase activity which was twice that seen with the control liver preparation. Kidney microsomes alone did not catalyze the cytochrome P-450-dependent N-demethylation of aminopyrine, and in contrast to the desaturate, no increase in demethylase activity was observed when kidney microsomes were added to liver microsomes.     

Regulation of rat hepatic stearoyl coenzyme A desaturase. The roles of insulin and carbohydrate.

The rat hepatic stearoyl-CoA desaturation decreased by 3.7-fold in streptozotocin-induced diabetes. Insulin treatment of diabetic rats increased the enzyme activity by 7-fold. In marked contrast to glucose administration, fructose feeding in diabetic rats resulted in 20-fold stimulation of stearoyl-CoA desaturation, although both carbohydrates stimulated stearoyl-CoA desaturation in normal rats. Measurement of the microsomal electron transfer components showed no significant changes in the NADH-cytochrome b5 reductase activity or in the concentration of cytochrome b5. However, the activity of the terminal desaturase changed in a parallel fashion as the amount of terminal desaturase reflect changes in the overall desaturation. Supplementation of various microsomes with the saturating amount of purified terminal desaturase resulted in the formation of similar amounts of catalytically active complex and increased the stearoyl-CoA desaturation to the same level suggesting that the changes in the amount of terminal desaturase reflect changes in the overall desaturation. The results support the suggestion that both insulin and the intermediates of carbohydrate metabolism are involved in the regulation of terminal desaturase.     

The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p

The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta-king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings.     

Site of participation of cytochrome b5 in hepatic microsomal fatty acid chain elongation. Electron input in the first reduction step

The present study provides strong evidence for the involvement of rat liver microsomal cytochrome b5 in the first reduction step of fatty acid chain elongation. The rate of reoxidation of NADH-reduced microsomal cytochrome b5 was markedly stimulated (up to 3-fold) by the addition of increasing concentrations of beta-ketohexadecanoyl-CoA (1-8 microM). A quantitative analysis of product formation, the effect of cyanide, and anaerobiosis completely exclude the possibility that desaturase activity accounted for the beta-ketohexadecanoyl-CoA-induced stimulation of the cytochrome b5 reoxidation rate. Using liver microsomes from untreated rats, the beta-keto substrate was found to stimulate the rate of reoxidation of cytochrome b5 by 30%. However, when liver microsomes from fat-free diet rats were employed the stimulation was more than 3-fold, suggesting that the beta-ketoacyl-CoA reductase is inducible by a high carbohydrate, fat-free diet. This study also provides evidence for the noninvolvement of cytochrome b5 in the terminal reaction step (second reduction step of chain elongation), which is catalyzed by the trans-2-enoyl-CoA reductase. Although trans-2-hexadecenoyl-CoA significantly stimulated the NADH-reduced cytochrome b5 reoxidation rate under aerobic conditions, it did not have any stimulatory effect under anaerobic conditions. One interpretation of these results is that the trans-2-hexadecenoyl-CoA is substrate for the microsomal delta 9 desaturase system. Consistent with this conclusion was the fact that the trans-2-hexadecenoyl-CoA inhibited the liver microsomal delta 9 desaturation of stearoyl-CoA to oleoyl-CoA.     

Inhibition of the microsomal stearoyl coenyme A desaturation by divalent copper and its chelates

Divalent copper and copper complexes of tyrosine, histidine and lysine inhibited at low concentrations the stearoyl-CoA desaturation reaction in both chicken liver microsomes and in a purified system consisting of chicken liver delta 9 terminal desaturase, cytochrome b5, ascorbate and liposome. Although the copper chelates lowered the steady-state level of ferrocytochrome b5 by 20%, and partially inhibited the NADH-ferricyanide reductase activity, the availability of the ferrocytochrome b5 during the time course of desaturation was not affected, indicating that the site of inhibition of desaturation was at the terminal step, i.e., on the delta 9 terminal desaturase. The presence of chalates during catalysis was essential for the observed inhibition. Based on the observation that O2 is involved in the desaturation and that there is an initial electron reduction of desaturase iron, it is plausible that the copper chelates are inhibiting by acting as superoxide scavengers.     

Studies on the delta 5-desaturation in ergosterol biosynthesis in yeast.

Studies were carried out on the delta 5-desaturation reaction in ergosterol biosynthesis with a particulate fraction of cell-free extract of yeast. A reduced pyridine nucleotide coenzyme and molecular oxygen were required for the reaction. It was shown that the enzyme activity is located in a fraction corresponding to microsomes. The reaction was inhibited by KCN, but not by CO. Menadione and potassium ferricyanide inhibited the NADPH- and NADH-dependent reactions, respectively, and cytochrome c inhibited both of them. These results suggested an involvement in delta 5-desaturation of a mixed function oxidase system resembling that for the fatty acyl-CoA desaturation reaction.     

The sensitivity of a commercial lager yeast to novel inhibitors of unsaturated fatty acid and ergosterol biosynthesis

Summary Cyclopropenoid fatty acids inhibited fatty acid desaturation in a lager strain ofSaccharomyces uvarum. N,N-dimethylazasqualene prevented ergosterol biosynthesis in the same yeast. Inhibitions took place under conditions favourable for ergosterol and unsaturated fatty acid formation, each independently of the other.     

Partial purification and characterization of lathosterol 5-desaturase from rat liver microsomes

The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-aminohexyl-Sepharose column chromatography. The final enzyme preparation was free from other electron transfer components and phospholipids in microsomes, and the desaturation reaction was reconstituted with the following components: NADH, molecular oxygen, phospholipids and three proteins, i.e., NADH-cytochrome b5 reductase, cytochrome b5 and the terminal oxidase. Omission of one of these components led to an almost complete loss of the desaturase activity. Under the reconstitution conditions, the desaturase activity was significantly inhibited by potassium cyanide but was not affected by -SH reagents such as N-ethylmaleimide and dithiothreitol.     

Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes

According to Ohba et al. (Ohba, M., Sato, R., Yoshida, Y., Nishino, T. and Katsuki, H. (1978) Biochem. Biophys. Res. Commun. 85, 21-27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of [1,7,15,22,26,30-14C]lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) in the presence of NADPH, NAD+ and molecular oxygen, concomitant with the liberation of 14CO2 derived from C-30 (one of the two methyl groups at the C-4 position). In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system. In this study it was found that the 14CO2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b5 and by palmitoyl-CoA, a substrate of the cytochrome b5-containing fatty acyl-CoA desaturase system of yeast microsomes. However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol). It is concluded that cytochrome b5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14 alpha-demethylation of lanosterol, by yeast microsomes. It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation. The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

Characterization of a membrane-bound phospholipid desaturase system of candida lipolytica.

Several characteristics of the microsomal phospholipid desaturase of Candida lipolytica are described. The phospholipid desaturase reaction required molecular oxygen and reduced pyridine nucleotides as essential cofactors and was inhibited by cyanide but not by carbonmonoxide, indicating that it required cytochrome b5. Desaturation of both 1-acyl-2-[14-C]oleoyl-sn-glycero-3-phosphorylcholine and 1,2-di-[14C] oleoyl-sn-glycero-3-phosphorylcholine appeared to follow Michaelis-Menten kinetics, with apparent Km values of 2.5 10-minus 4 M and 9.5 10-minus 4 M, respectively. Desaturation of the di-[14C] oleoylphosphatidylcholine took place at both position-1 and position-2; the distearoyl or dielaidoyl phosphatidylcholines were not desaturated. Rate of desaturation of the 1=acyl-2-[14-C] oleoyl-glycerophosphorylcholine by microsomes from cold-grown cells was equal to or slightly less than that by microsomes from cells grown at the normal growth temperature of 25 degreesC, measured in the temperature range 10-30 degrees C. However, the rate of desaturation of [14-C]-oleoyl-CoA desaturase was greater with the microsomal preparation from cold-grown cells than with that from 25 degreesC grown cells. These data suggest that the observed increase of diunsaturated fatty acids in cold-grown cells may perhaps be explained by the increased activity of the oleoyl-CoA desaturase acting at the low temperature.