A structure-function correlation for fatty acids inSaccharomyces cerevisiae

EXPERIMENTAL MYCOLOGY 8, 55-62 (1984)

A Structure-Function Correlation for Fatty Acids in Saccharomyces cerevisiae W. DAVID *Plant

NES,*T~

JOHN H. ADLER,

AND WILLIAM

R. NES

Physiology and Chemistry Research Unit, WRRC-ARSIUS. Department Berkeley, California 94710 and Department of Biological Sciences, Drexel Philadelphia, Pennsylvania 19104

of Agriculture, University,

Accepted for publication October 20, I983 NES, W. D., ADLER, J. H., AND NES, W. R. A Structure-Function Correlation for Fatty Acids in Saccharomyces cerevisiae. Experimental Mycology 8, 55-62. A relationship which distinguishes those fatty acids that support the growth of oxygen-deprived Saccharomyces cerevisiae from those that do not was found. To function properly, a long chain of saturated carbon atoms appears to require interruption by an appropriate chemical group such that only a maximum number of contiguous saturated carbon atoms is present anywhere in the chain. A double bond was found to serve as an interrupting group, and for 19 cis-unsaturates studied, ranging from CM:,A9 to C22:6-A4,7,10,13,16,1g, the number of saturated C atoms was 9. The chain length and the position and number of double bonds had no influence in determining whether the acids were active or inactive except as these structural features related to the interruption rule. Although less extensively examined, a hydroxyl group or trans-double bond also appeared to act as an interrupting group with allowed numbers of 9 and 7, respectively, for saturated C atoms. Oxygen deprivation did not result in a shift to shorter chain length of the saturates formed, and the presence of unsaturates did not prevent biosynthesis of saturates. INDEX DESCRIPTORS:oleic acid; polyunsaturated fatty acids; membranes; yeast.

Studies of the ability of various fatty acids to support the growth of both oxygendeprived Saccharomyces cerevisiae (Alterthum and Rose, 1973; Andreasen and Stier, 1954; Light et al., 1962; Myer and Bloch, 1963) and yeast mutants with a lesion in fatty acid biosynthesis (Barber and Lands, 1973; Eletr and Keith, 1972; Holub and Lands, 1975; Keith et al., 1973; Vandenhoff et a/., 1975; Williams et al., 1973; Wisnieski and Kiyomoto, 1972) have shown unequivocally that unsaturation plays a vital role in this organism. Presumably the crucial function is to permit an appropriate degree of fluidity in the yeast’s membranes which effect biochemical properties such as transport (Keenan et al., 1982) but neither the

growth studies nor the concept of have permitted the development of prehensive structural correlation w which one could predict whether or not a particular fatty acid would be effective. As an approach to the solution of this problem we reexamined and extended the earlier work on the relationship of structure to growth by making a systematic study of the effects of chain length and of the positive, number, and configuration of double We used a technique for the growth ygen-deprived yeast which was recently employed successfully in a study of the structure-function problem with sterols (Nes et al., 1976, 1978b; Pinto and Nes, 1983). From our work with fatty acids an empirical rule which depends on the ability of a double bond to segregate groups of saturated carbon atoms from each other by interrupting the sequence of CH, groups emerged. The rule correctly predicts

l Reference to a company and/or product named by the Department is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. 55

0147-5975184 $3.00 Copyright 0 1984 by Academic &ess, Inc. Ali rights of reproduction in any form resewed.

56

NES,

ADLER,

observed activity or inactivity of each of 33 fatty acids which were studied. MATERIALS

AND

METHODS

A wild-type diploid S. cerevisiae (ATCC 18790) was grown in continuous culture on a synthetic medium (Nes et al., 1978b; Sekula, 1979) at 27°C as described previously (Nes et al., 1978). For examination of the effect of the different fatty acids, an aliquot of the continuous culture in log phase of growth was used to inoculate 1.0 liter of medium containing ergosterol and fatty acid. The ergosterol (10.0 mg) in 2.0 ml of ethanol was added to 100 mg of the fatty acid in 3.0 ml of Tiiton X-100 and this mixture was then added to the medium prior to incubation. The vessels were flushed with nitrogen scrubbed by chromous chloride (Nes, et al., 1978). The number of cells used as inoculum gave 100,000 cells/ml in the experimental vessels, which were sealed with wax at all joints and maintained at 27°C in a water bath as described earlier (Nes et al., 1978). After 72 h the cells were counted and sized on a Coulter counter, Model TArr equipped with a population accessory unit, and a fatty acid analysis was made. After centrifugal separation of the cells from the medium they were lyophilized and extracted with methanol-chloroform (2:l v/v). The extracted material was chromatographed on a thin layer of silica gel G in benezene-ether-ethanol-acetic acid (6:4:2:2 v/v). The material at the origin was esterified (CH,OH-H,SO,), and the esters were obtained from the hexane layer after the reaction mixture was partitioned between hexane and water. The esters were then chromatographed on a Varian instrument (Aerograph, Series 2100) in the vapor state at 175°C on ethylene-glycol succinate deposited on Chromosorb W of mesh size lOO- 120. Authentic fatty acids used both as chromatographic standards and as substrates were obtained commercially or through the generosity of Dr. A. D. Keith of Pennsylvania State University. They

AND

NES

showed no evidence of significant ties by chromatographic analysis.

impuri-

RESULTS

The growth induced by the various fatty acids is shown in Table 1. Previous studies (Alterthum and Rose, 1973; Andreasen and Stier, 1954; Light et al., 1962 Myer and Bloch, 1963) with anaerobic yeast have dealt with several saturated and cis-unsaturated fatty acids, one trans-unsaturate, and three hydroxy acids. Most of these were encompassed in the 33 acids we used. The presence of less oxygen in the present experiments may account for our failure to observe any growth at all with the 12-hydroxy-C,,-acid which was found to be weakly active in earlier work (Light et al., 1962), but in other cases of overlap our work agreed with the literature. A correlation was also possible with the results obtained with mutants having a defect in the desaturation system (Barber and Lands, 1973; Eletr and Keith, 1972; Holub’and Lands, 1975; Keith et al., 1973; Vandenhoff et al., 1975; Williams et al., 1973; Wisnieski and Kiyomoto, 1972). In all but two cases (cis-(&-A6 and trans-trans-C,8,2-A9~12) about which questions have already been raised (Keith et al., 1973), there again was agreement with the literature. The data reveal (a) that saturates with more than 12 carbon atoms are ineffective; (b) that some unsaturates but not others support growth; (c) that the configuration of the double bond is not itself a determinant of activity; and (d) that the presence of an OH-group may but does not necessarily confer activity. This apparently confused pattern of biological behavior becomes rational through what weshall call the “interruption rule” as presented under Discussion. The fatty acid compositions of the polar lipids of representative cultures which grew sufficiently to permit analysis are given in Table 2. Several interesting points are revealed:

FATTY TABLE

ACIDS

1

Particles per ml of Anaerobic Yeast after 72 h in the Presence Ergosterol and Various Fatty Acids Fatty acid Saturates Even numbered C,, through CXO HO Saturates C,,-Z-OH C,,-12-OH cis-Monounsaturates

Particles per mid (in millions)

0 0 0 98

‘&-A9 &-A9 C,,-A6 C La-A9 C,,-AiL C,,-A5

112 0 112 90 0

Cz,,-A”

24

Cz2-A9 Cz2-A13 C,,-Ai5 C,,-A922-OH

0 0 0

22

all cis-Polyunsaturates C,,-A9J2 Cl,-A6~%“2 C;,-A9,‘2,‘5 f&,-A11,14 +#“.‘4,‘7 &eA5.8.11,14

(&,&~8>‘%‘7 ~22~~47A13>‘6.19 truns-Unsaturates Cr4-A9 c,,-A9 &-A6 C1*-A9 C18-A9,12 C,,-A” C,,-Ai

107 100 112

54 109 103

68 70 48 27 0 0 0 0 0

a Data were obtained with a Coulter counter and represent averages of two or more experiments. The number zero implies no growth but with varying effects in detail (Iysis of the inoculum, perturbation of the morphology of the inoculum, no effect on the inoculum, and on occasion slight growth (ca. 1 x lo6 particles/ml) which was thought to have been due to traces of oxygen.

(a) The addition of free unsaturated fatty acid did not prevent the biosynthesis of saturated fatty acids. (b) The addition of fatty acids in the form of Tween 80 led to a distribution in the polar

IN YEAST

57

lipids of the cells which was similar to that in Tween 80 itself. In particular, there was a high (81%) value for oleate and a low (12%) value for saturates. Similar resuhs have been found for the acyl lipid profile of promitochondria (Paltauf and Schatz, 1969). Tween 80 is reported (Jollow et al., 1968) to be 71 mol% oleate, 13% palmitoleate, 3% myristoleate, and 13% saturates. The polar lipid composition of the yeast suggests that either the Tween the saturates present in it inhibit thesis, since otherwise an increase in the ratio of saturates to unsaturates wou been expected. It is, of course, p that our ‘“polar lipid” was primarily Tween 80 which was absorbed by the cell but which had not been metabolized. ~~wev~~~ in other studies (Hosack and R Jollow et al., 1968) the various lipid classes were actually isolat high oleate level was also fo Tween 80 was the source of fatty acid. (c) Anaerobiosis did not induce a shift to shorter chain length as suggested earher (Jollow et ad., 1968; Myer and Blo 963; Paltauf and Schatz, 1969). Such a s also has not been observed by others et al., 1972). As seen from Table 2, t inant saturate was always C16:0 r of the efficacy of the added fatty acid. It seems unlikely, therefore, that a strong mechanism exists in yeast for Ghana-shortening as a compensation for the i~ab~~ty to form unsaturates, although this matter might profit from further investigation. (d) Endogenous biosynthesis of saturate acids occurred under anaerobiosis did not lead to consequential bios of unsaturated fatty acids. There was either no desaturation or the acid formed an this manner amounted to g% or less ture. The principal unsaturated c of the mixture was always to the medium which in t cases apparently underwent s elongation as observed with (fa (Orme et al., 1972).

73 4 1 4 2 6 6 3

18-A6,9,‘2, erg, 18-A9,*2,15, erg, T-X T-X 16-Ag, erg, T-X 14-A9, erg, T-X 16-A9-t, erg, T-X 14-A9-t, erg, T-X 20-A”, erg, T-X 20-A1*,14, erg, T-X 20-A*1,‘4,17, erg, T-X 20-A5*8,11J4,erg, T-X ‘JyJ&A”JW, erg, T-X

8 27

-13 19 9 9 11 1 3

2 13 5 10 11

9 26 9 31 55 36

387 21 56 24 35 42 28 32 59 21

18:O

16:0

2

-22 41 -

-

-

2 3

14:l

-

1

-

5

12 -

24

53 10 5

23 -

28 81 ,565

33

18:l

-5 46 7 55

51 26 6 2 -

16:l

Yeast

-_ _ -

_ _ 50

18:2

37 82 _ -

_ -

18:3

% Fatty acid in polar lipids

TABLE 2 of the Polar Lipids of Anaerobic

-_ _ 38 -

_ _ -

2o:l

-_ _ _ 68 -

_ _ _ _ -

20:2

_ 59 -

_ -

20~3

27 -

-

20:4

-43

_ -

20:.5

a FA is fatty acid; erg is ergosterol; T-80 is Tween 80; and T-X is Triton-X-100. Unless noted by T for t~ult~, all double bonds are cis. Oxygen was eliminated to the best of our ability except as noted by “02.” The % is that of the FA in the mixture. A dash means less than 1%.

1 3 3 4 7 3

14:o

No FA, no erg, 0, No FA, erg, limited 0, No FA, erg, T-80 18-A9, erg, T-X 18-A*‘, erg, T-X 18-A9,i2, erg, T-X

Contents of medium0

Fatty Acid Composition

“! b 3 Z !z

z ”E! * F

FATTY

ACIDS

IN YEAST

5

rule.” The special case in which only nine saturated C atoms are allowed will be referred to as “the rule of 9,” and when only In addition to the necessity for small amounts of saturated fatty acids (Adler et seven are allowed the term “rule of 7” will al., 1981; Keith et al., 1973; and Otoguro be used. Each of the acids we studie et al., 1981) it is well known that unsatu(Table 1) obeyed the “interruption rule” in rated fatty acids are necessary for memthe form of either the “rule of 9” or the “rule of 7.” Furthermore, based on earlier brane assembly (Eletr and Keith, 1972; Buttke et al., 1980, 1982). Since some un- work (Myer and Bloch, 1963) with anaersaturates with chain lengths as small as C,, obic yeast, the 9- and IO-hydroxy C,,:,and some as large as C,, were both active, acids are active. These facts, which the number of C atoms per se cannot be a the “rule of 9,” indicate that the hy group may also act as an interruptive agent determining factor for growth support. Similarly, consideration of the position of the (as apparently can halogens or the cyclodouble bond in terms of count either from propyl group at least in other systems (Silthe carboxyl or methyl ends of the chain bert et al., 1968, 1973). Conversely, one would not expect the 2- or 12hydroxyfailed to lead to a satisfactory correlation. For instance, all A9-acids were not active, C,szO-acids to be active, since in both cases, and, as shown by the inactivities of Czzzl- the “rule of 9” is violated. Our own results A9, C22:1-A13, and C,,:,-A15 compared to the agree with this prediction, but in an earlier activities of C1s,2-A9~12, C22:6-A4’7,9713716,19, report (Light et al., 1962) some small acand C1s:3-A9~12~15,double bonds which did tivity (half of that with the 9- or 1 not confer activity did not necessarily pre- analog) for the 12-hydroxy-C,,, vent it. Furthermore, neither a considerfound in supposedly anaerobic ation of the number of CH, groups between may stem from somewhat less stringent oxthe carbonyl group and the double bond ygen deprivation. closest to it in polyunsaturates nor a conIt is not clear why the ““rule of 7” should sideration of the number of C atoms be- operate in the tralzs-case in contrast to the tween the methyl group and the double “rule of 9” with the c&-acids. ~owev~r~ bond closest to it gave any coherence to the the difference between the cis- and trarasspectrum of activities. However, as shown acids seems to manifest itself at a level in Fig. 1, when the largest number of sat- which is below that which governs t terruption rule” in general. In ver urated C atoms anywhere in the molecule was plotted against the extent of growth, a the configuration from cis to trans was also ctear relationship emerged for all of the sat- attended by other subtle changes, Lower urates, &-unsaturates, and hydroxy-satucell counts were consistently observed with rates A break in the curve occurred at nine the active tvans-acids compared to their cisC atoms; above this number none of the analogs, e.g., the cis- and trQ~s-C~~:~~~~acids was active, while at or below nine all acids, and Lands and his associates (Graff were active. Similarly, although the data and Lands, 1976; Tsao and Lands, 1 have found that certain tvans-fatty aci are less extensive, only the trans-unsaturales with less than seven contiguous satu- while providing sufficient fluidity to ce rated atoms were active (Fig. 2). membranes (Lauds et DE., 1978), alter other We believe the data show that a double parameters which depend on ate of is inbond functions to interrupt the sequence of cellular metabolism in which saturated C atoms and that there is a limit volved. Related results have been ~bta~~~d to the number of such atoms which is per- in mammalian systems (Karney an homitted. We shall call this the “interruption peshwarkar, 1979). DISCUSSION

60

NES, ADLER,

AND NES

18:lA”

i

18:lA’

12-OH+.?

2 NO.

CH2-

AND

CHj

GROUPS

FIG 1. Relationship between the growth-supporting activity of a fatty acid and the largest number of contiguous saturated C atoms anywhere in the molecule. AU unsaturates are cis. The identity of points not labeled can be determined by reference to Table 1. The number of particles was determined at 72 h with a Coulter counter. There is a question mark by the point for the 18:1-A9-l-12-hydroxyacid, because we had difficulty in solubihzing the material.

In conclusion, the “interruption rule” leads to a rational qualitative relationship between the effects and structures of the fatty acids as measured by the growth of yeast. Regulation of membrane fluidity (either directly in the bilayer or indirectly

7sat.Gatoms

27

sat.C-atoms

;

:

through the specificity of the acyltransferases which presumably reflect the bilayer’s requirements) seems to be the most plausible reason why the rule operates. To further elucidate the physiological basis for this empirical correlation it would be of interest to determine the subcellular distribution of the dietary acids and relate this information to physicochemical properties of the fatty acids themselves and of the membranes containing these lipids. Additional studies such as substrate specificity determinations of the acyltransferase and K, values for the uptake of these molecules would also be helpful. ACKNOWLEDGMENTS

I

;

;

14

;

:

;

;

:

16 18 20 CARBON NUMBER

:

!

22

FIG. 2. Relationship between the growth-supporting activity of trans-unsaturates and the total chain length of the acid. The number of particles was determined at 72 h with a Coulter counter.

We are grateful for support through NIL-I Grant AM12172 and should like to thank Dr. B. C. Sekula for stimulating discussions. We also should like to thank Dr. A. D. Keith for the gift of various fatty acids. A preliminary report on a portion of this study was presented (cf. Nes et al., 1978a).

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