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 phosphatides, 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, phosphatidylinositol, 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 is shown in Fig. 1, in which each component is expressed as the percentage of total fatty acids of cells. Abnormal composition of fatty acids 1974 Lipid Composition of Pantothenic Acid-Deficient Yeast 51

Fig. 1. Changes of the fatty acid composition in whole cells after addition of pantothenic acid. The amounts of individual components are expressed as percentages of total fatty acid. contained in the deficient cells recovered to the normal level in a short time, in about 5 hr. Furthermore, the quantity of individual fatty acids was calculated and is shown in Fig. 2. It is interesting that the quantity of fatty acids reached the normal level after 15 hr, although the composition of fatty acids contained in the deficient cells was regained about 5 hr after incubation. These results revealed that pantothenic acid influenced the biosynthesis of fatty acids. The fatty acid composition of mitochondria was further investigated. The mitochondrial fraction was prepared as described previously (12), and the fatty acids were analyzed in the same manner. These results are shown in Figs. 3 and 4. The percentage of fatty acids and the recovery of fatty acid composition in mitochondrial fraction were similar to those of yeast cells. However, we noted that the amount of oleic acid in mitochondrial fraction prepared from the deficient cells was at almost the same level, compared with the amount in mitochondria from normal cells. The biosynthesis of fatty acid in mitochondria prepared from the deficient cells regained in a short time, e.g. about 5 hr. This was one-third as long as the recovery time of fatty acids in yeast cells. In other word, the effect of pantothenic acid was more sensitive to the fatty acids contained in mitochondria than that of the whole yeast cells. Anaerobically grown cells contained a large amount of saturated fatty acids with chain length of 10, 12, and 16 carbon atoms and a small amount of unsaturated fatty acids because they could not synthesize unsaturated fatty acids in the 52 HosoNo and AIDA VOL. 20

Fig. 2. Changes of the fatty acid content in whole cells after addition of pantothenic acid. See Fig. 1 for the fatty acid symbols. absence of oxygen. In this point, the composition of fatty acids in the pantothenic acid-deficient cells differed from the composition of fatty acids in anaerobic cells. The amount 'of oleic acid was not so influenced as other fatty acids by the pantothenic acid-deficiency in regard to both cells and mitochondria. It is probable that the synthetic pathways of these fatty acids are different and the effect of pantothenic acid varies. It is well known that the biosynthesis of oleic acid is different from that of palmitoleic acid (27).

Phospholipid content and composition When yeast was grown on a pantothenic acid-deficient medium, the fatty acid composition in both cells and mitochondria was abnormal, compared with that in normal cells. The membrane, especially the mitochondrial inner membrane, is known to be composed of protein-phospholipid matrix. Therefore, we assumed that the composition of phospholipids present in the deficient cells would be different from that in normal cells. The composition of phospholipid in the 1974 Lipid Composition of Pantothenic Acid-Deficient Yeast 53

Fig. 3. Changes of the fatty acid composition in mitochondria after addition of pantothenic acid. The amounts of individual components are expressed as percentages of total fatty acid. See Fig. 1 for the fatty acid symbols.

Fig. 4. Changes of the fatty acid content in mitochondria after addition of pantothenic acid. See Fig. 1 for the fatty acid symbols. 54 HOsoNo and AIDA VOL. 20

Table 3. Phospholipid composition of yeast cells. Abbreviations: lyso PC=1yso phosphatidylcholine, PC= phosphatidylcholine, PI=phosphatidylinositol, PS ==phosphatidylserine, PE=phosphatidylethanolamine, GP=glycerol phosphatides (includes cardiolipin and phosphatidylglycerol).

Fig. 5. Changes of the phospholipid composition in whole cells after addition of pantothenic acid. The amounts of individual components are expressed as percentages of total phospholipid. cells is shown in Table 3. Incomplete resolution was found between phosphatidylserine and phosphatidylinositol and between phosphatidylglycerol and cardiolipin, and combined figures are given for these two pairs. In the deficient cells, the percentage of phosphatidylcholine was high and conversely that of phosphatidylethanolamine and glycerol phosphatides was low, compared with those of normal cells. In normal cells, the content of phosphatidylcholine decreased and the content of glycerol phosphatides increased as the growth proceeded. Further, we examined the biosynthesis of phospholipids after addition of pantothenic acid to the medium, and these results are shown in 1974 Lipid Composition of Pantothenic Acid-Deficient Yeast 55

Fig. 6. Changes of the phospholipid content in whole cells after addition of pantothenic acid. See Fig. 5 for the phospholipid symbols.

Fig. 7. Changes of the phospholipid composition in mitochondria after addition of pantothenic acid. The amounts of individual components are expressed as percentages of total phospholipid. See Fig. 5 for the phospholipid symbols. 56 HosoNo and AIDA VOL. 20

Fig. 8. Changes of the ergosterol content after addition of pantothenic acid.

Fig. 9. Changes of the intracellular CoA content after addition of pantothenic acid. 1974 Lipid Composition of Pantothenic Acid-Deficient Yeast 57

Figs. 5 and 6. The recovery of phospholipid composition reached the normal level after about 5 hr. Their biosynthesis arose more rapidly than that of fatty acids. Phospholipids in mitochondrial fractions were examined in the same manner and the results were approximately similar (Fig. 7). Ergosterol content The amount of ergosterol in the deficient cells was about one-fourth of the level in normal cells (see Table 1). Therefore, the deficient cells were transferred to a complete medium and the change in ergosterol content was examined. As shown in Fig. 8, the amount of ergosterol in yeast cells increased gradually for 10 hr and then decreased. The amount contained in the log phase cells was more than the amount in the stationary phase cells, as shown in Table 1. In the same manner, the amount of ergosterol in the transferred cells had a tendency to decrease over a long period. For anaerobic growth, yeasts require external source of unsaturated fatty acids and sterol, such as ergosterol. From our examination, the deficient cells contained ergosterol about one-fourth of that in normal cells. In order to reveal the relation between ergosterol and the respiratory activity, we checked the respiration rate of the cells grown in a pantothenic acid-deficient medium supplemented with much ergosterol. However, ergosterol did not affect the respiration rate of yeast cells directly.

CoA content LIPMANNet al. proved that pantothenic acid was a component of coenzyme A, which took an important part in the fatty acid metabolism (17, 18). From their reports and results, it is necessary to examine the content of coenzyme A, and its change by adding pantothenic acid. The content of coenzyme A in normal cells was about 5 times greater than that in the deficient cells (Fig. 9). After addition of pantothenic acid, the cellular CoA increased as the time proceeded, but its amount did not reach the normal level. The reason why coenzyme A did not reach the normal level is unknown.

Table 4. Recovery of the respiration rate after addition of pantothenic acid.

From our present studies, we found that the cells grown on a pantothenic acid-deficient medium contained less coenzyme A, and the lipid metabolism was abnormal, compared with normal cells. Furthermore, these abnormal pheno- mena were recovered on addition of pantothenic acid. Corresponding to the recovery of these components, the respiration rate of the cells recovered as the time proceeded (Table 4). However, the respiration rate increased gradually for 10 hr and then decreased as shown in the recovery of other components. 58 HosoNo and AIDA VOL. 20

REFERENCES

1) B. EPHRUSSIand P. P. SLONIMSKY,Biochim. Biophys. Acta, 6, 256 (1950). 2) A. W. LINNANE,E. VITOLs, and P. G. NOWLAND,J. Cell. Biol., 13, 342 (1962). 3) E. S. POLAKIS,W. BARTLEY,and G. A. MECK, Biochem. J., 90, 364 (1964). 4) G. MORPURGO,G. SERLUPI-CRESCENZI,G. TECCE, F. VALENTE,and D. VENETACCI,Nature, 201, 897 (1964). 5) G. SCHATZ,Biochim. Biophys. Acta, 96, 342 (1965). 6) Y. YOTSUYANAGI,J. Ultrastruct. Res., 7, 141 (1962). 7) P. G. WALLACEand A. W. LINNANE,Nature, 201, 1191 (1964). 8) D. K. BLOOMFIELDand K. BLOCH,J. Biol. Chem., 235, 337 (1960). 9) A. A. ANDREASENand T. J. B. STIER,J. Cell. Comp. Physiol., 41, 23 (1953). 10) D. JOLLOW,G. M. KELLERMAN,and A. W. LINNANE,J. Cell. Biol., 37, 221 (1968). 11) K. WATSON,J. M. HASLAM,and A. W. LINNANE,J. Cell. Biol., 46, 88 (1970). 12) K. HosoNo, K. AIDA, and T. UEMURA,J. Gen. Appl. Microbiol.,18, 189 (1972). 13) H. A. LARDYand S. M. FERGUSON,Annu. Rev. Biochem., 38, 1005 (1969). 14) S. FLEISCHER,H. KLOURVEN,and G. BRIERLEY,J. Biol. Chem., 236, 2936 (1961). 15) D. G. MCCONNEL,A. TZAGOLOFF,D. H. MACLENNANE,and D. E. GREEN, J. Biol. Chem., 237, 3264 (1962). 16) Y. C. AWASTHI,T. F. CHUANG,T. W. KEENAN, and F. L. CRANE, Biochim. Biophys. Acta, 226, 42 (1971). 17) G. D. NoVELLI and F. LIPMANN,Arch. Biochem., 14, 23 (1947). 18) H. P. KLEIN and F. LIPMANN,J. Biol. Chem., 203, 95 (1953). 19) J. FoLCH, M. LEES,and G. H. S. STANLEY,J. Biol. Chem., 226, 497 (1957). 20) H. SCHLENKand J. L. GELLERMAN,Anal. Chem., 22, 1412 (1960). 21) R. J. A. ALLEN,Biochem. J., 34, 858 (1940). 22) V. P. SKIPSKI,R. F. PETERSON,and M. BARCLAY,Biochem. J., 90, 374 (1964). 23) P. S. CHEN, T. Y. ToRIBARA,and H. WARNER,Anal. Chem., 28, 1756 (1956). 24) W. H. C. SHAWand J. P. JEFFERIES,Analyst, 78, 514 (1953). 25) T. S. STADTMAN,Methods Enzymol., 3, 392 (1957). 26) Y. ABIKO,Methods Enzymol.,18A, 314 (1970). 27) K. BLOCH, In Lipid Metabolism, ed. by K. BLOCH,Academic Press Inc., New York (1964) p.1.