Lipid Composition of Saccharomyces Cerevi- Siae Defective in Mitochondria Due to Pantothenic Acid Deficiency

J. Gen. App!. Microbial., 20, 47-58 (1974) LIPID COMPOSITION OF SACCHAROMYCES CEREVI- SIAE DEFECTIVE IN MITOCHONDRIA DUE TO PANTOTHENIC ACID DEFICIENCY KUNIAKI HOSONO AND KO AIDA The Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo (Received October 15, 1973) Lipid composition of Saccharomyces cerevisiae defective in mitochondria and respiratory activity due to pantothenic acid deficiency and its normal- ization were studied. Quantitatively, the deficient cells contained about one-half of total lipid, one-fifth of fatty acids, one-fourth of ergosterol, and one-half of phospholipids compared with normal cells. Unsaturat- ed fatty acids, such as palmitoleic acid and oleic acid, were decreased in the deficient cells. The composition of fatty acids and phospholipids in mitochondria in both types of cells was the same as in whole cells. Phospholipids, such as phosphatidylethanolamine and glycerol phospha- tides, decreased in deficient cells, but the percentage of phosphatidylcholine increased. Intracellular coenzyme A content was one-fifth of the normal. By the addition of pantothenic acid to the deficient cells, unsaturated fatty acids, especially palmitoleic acid, were synthesized, but other fatty acids were synthesized to only a small extent. The recovery of phospholipids preceded that of fatty acids. Along with the recovery of these lipids, the respiration rate of the deficient cells also reached the normal level. When yeast, Saccharomyces cerevisiae, is grown on glucose under anaerobic conditions, synthesis of several respiratory enzymes in the cells is strongly repressed, resulting in the inability to respire (1), and the structures corresponding to mitochondria of aerobic cells seem to be considerably reduced or become primitive (2- 5). During the aeration of anaerobically grown yeast cells, the respiratory activity gradually emerges and the primitive mitochondrial structures develop into fully functional mitochondria (6, 7). In the yeast, synthesis of unsaturated fatty acid and ergosterol is an oxygen-dependent process (8, 9). Such anaerobically grown cells have a high content of short-chain saturated fatty acids and an abnormal lipid composition, as compared with aerobically grown cells (10). These changes in lipid, to which an important role in the structure of membrane is assigned, might 47 48 HosoNo and AIDA VOL. 20 cause an inadequate synthesis of the membrane, especially mitochondrial inner membrane. Actually, mitochondrial profiles of anaerobically grown cells supplemented with unsaturated fatty acids and ergosterol approach those of aerobically grown cells in structure and lipid composition, while the mitochondrial profiles of non-supplemented cells are very primitive, with poor inner membrane organi- zation, and have an abnormal lipid composition (10, 11). Our previous paper (12) reported that the respiratory activity of pantothenic acid-deficient Saccharomyces cerevisiae was significantly lower and at the same time the cytochrome contents were lower in these cells than normal cells; cytochrome a--a3 and b were not detected. Cytochrome oxidase is well known to be localized at the inner membrane of mitochondria (13) and bound to phospholipid tightly (14-16). As pantothenic acid is closely related to lipid metabolism through coenzyme A (17, I8), the correlation between pantothenic acid deficiency and lipid, especially fatty acid and phospholipid, has been examined in the present study. MATERIALSAND METHODS Organism and growth conditions. A diploid strain of Saccharomyces cerevisiae, BA-l, which essentially requires pantothenic acid, was used throughout. The cells were aerobically grown under the same conditions and on the same medium as described previously (12). Recovery from pantothenic acid deficiency. The cells grown on a pantothenic acid-deficient medium (10 pg/liter) up to the stationary phase were transferred to a fresh complete medium containing 200 ,ug of calcium pantothenate. After incubation, recovery of the cells from pantothenic acid-deficiency was examined by changes in lipid and CoA content, and respiration rate. Extraction of lipids. The cells were completely disrupted by means of a Braun homogenizer for 2 min and extracted with 20 volumes of chloroform: methanol (2: 1, v/v). The residue was further extracted with 10 volumes of a mixture of chloroform : methanol : 10 N HCl (66: 33: 2, v/v) by the method of JOLLOWet al. (10). The extracts were separately washed as described by FOLCH et al. (19) and evaporated in vacuo. Analysis of fatty acids. Total fatty acid was extracted from lyophilized cells. The cells were suspended in 15 volumes of 15 % KOH in 50% aqueous methanol and refluxed for 4 hr. The resultant alkaline suspension of the digested cells was treated twice with 3 volumes each of hexane to remove non-saponifiable material, and the hexane layer was discarded. The aqueous layer was acidified to pH 2.0 with 6 N H2SO4 and extracted 4 times with 3 volumes each of ether. The ether extracts were combined and dehydrated over anhydrous Na2SO4 overnight. The fraction containing fatty acids was evaporated in vacuo and the residue was dis- solved in a small volume of ether. 1974 Lipid Composition of Pantothenic Acid-Deficient Yeast 49 Fatty acids were methylated with diazomethane (20) and the products were separated by gas chromatography. The column was a glass tube (4 mm x 150 cm) packed with Shimalite W (60-80 mesh) coated with 10 % diethylene glycol suc- cinate. A gas chromatograph, Shimadzu GC-4BF, provided with a flame ioniz- ation detector and nitrogen as the carrier gas was used. The fatty acids were identified from their relative retention time, comparing with those of authentic standards. For the determination of the fatty acid content and composition of the cells, a known weight of pentadecanoic acid was added as the internal standard to the fatty acids extracted from the cells. Analysis of phospholipids. Total phospholipid was determined by assaying the phosphorus content of lipid extracts using the method of ALLEN(21). Indi- vidual phospholipids were separated by thin-layer chromatography with the solvent system of chloroform : methanol: acetic acid: water (25: 15 : 4 : 2, v/v) as described by SKIPSKIet al. (22), and identified their Rf values relative to appropriate phospholipid standards and their specific staining behavior. For quantitation, separated phospholipids were eluted from the silica gel plate as described by SKIPSKIet al. (22), and their phosphorus content was determined by the method of CHENet al. (23). Analysis of ergosterol. Sterol was extracted with ether after alkaline hydro- lysis (24) and estimated as ergosterol by the Liebermann-Burchard reaction (25). Estimation of Coenzyme A. To extract the intracellular CoA, the centrifuged cells (100 mg dry weight) were suspended in 1 ml of water in a stoppered tube, and boiled for 10 min. After centrifugation, CoA in the aqueous fractions was analyzed by the use of phosphotransacetylase according to the method of ABIK0(26). Chemicals. Phosphatidylcholine, lyso-phosphatidylcholine, phosphatidyl- inositol, phosphatidylserine, phosphatidylethanolamine, and cardiolipin were obtained from Nutritional Biochemicals Co., U.S.A., capric acid, lauric acid, myristic acid, pentadecanoic acid, palmitic acid, stearic acid, and ergosterol from Tokyo Kasei Co., Tokyo, acetyl phosphate, arsenate, CoA, palmitoleic acid, and oleic acid from Sigma Chemical Co., U.S.A., and phosphotransacetylase from Boehringer-Mannheim, Tokyo. RESULTS AND DISCUSSION Total lipid content The lipid contents of both normal and pantothenic acid deficient cells are given in Table 1 in terms of total lipid, total fatty acid, ergosterol, and phospholipid. It is clear that there is a significant difference in lipid composition between normal cells and pantothenic acid-deficient cells. Normal cells contained total lipid and phospholipid about twice and total fatty acid about 5 times as much as 50 HosoNo and AmA VOL. 20 Table 1. Lipid composition of pantothenic acid-deficient and -sufficient cells. Table 2. Fatty acid composition of yeast cells. the deficient cells. It was interesting that at stationary phase the normal cells contained about 4 times as much ergosterol as the deficient cells, and the normal cells at log phase contained even more ergosterol. The effect of pantothenic acid appears to be related to the biosynthesis of ergosterol. Fatty acid content and composition The effect of pantothenic acid on the fatty acid composition of yeast cells is shown in Table 2. Generally, normal yeast cells had a large amount of unsaturated fatty acid, mainly palmitoleic acid and oleic acid, and their content oc- cupied more than 80% of the total fatty acids. However, the total amount of fatty acids was lower in the deficient cells, though the percentage of palmitoleic acid and oleic acid was still high. The amount of palmitoleic acid was more than that of oleic acid in normal cells. Conversely, the deficient cells contained much oleic acid, accounting for about 60% of total fatty acids. These results showed that pantothenic acid drastically influenced the fatty acid composition of yeast. If pantothenic acid is closely related to the biosynthesis of fatty acids, the recovery of fatty acid synthesis will be observed when pantothenic acid is added to the deficient cells. The deficient cells were then transferred to a fresh complete medium and biosynthesis of fatty acids was examined with time. The change in the fatty acid composition

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