On the relationship between respiratory activity and lipid composition of the yeast cell

BIOCHIMICA

94

ET BIOPHYSICA

ACTA

BB* 55336

ON

THE

LIPID

L.

RELATIONSHIP

COMPOSITION

KovAt,

J.

Department

SuBiIi,

G.

of Biochemistry,

OF

mm

BETWEEN THE

AND

Komensky

RESPIRATORY

YEAST

K.

AND

CELL

KoLLh

University,

(Received November rgth, 1966) (Revised manuscript received February

ACTIVITY

Bvatislava

(Czechoslovakia)

z4th, 1967)

SUMMARY

I.

Phospholipids,

total fatty

acids, sterols

and hydrocarbons

were determined

in respiratory-competent and respiratory-deficient cells of Saccharomyces cerevisiae grown aerobically and anaerobically. 2. In a respiratory-competent strain, the previously-reported reduction in the content of phospholipids, fatty acids, and sterols of cells grown anaerobically on glucose in medium supplemented cally-grown cells, was confirmed.

with ergosterol and Tween 80, relative to aerobiAnaerobically-grown cells from medium lacking

ergosterol contained more phospholipids and fatty acids, and from medium lacking both ergosterol and Tween 80 contained more phospholipids, than cells from the supplemented medium. 3, Changes in the lipid fractions during respiratory adaptation of anaerobicallygrown respiratory-competent yeast were followed. The level of phospholipids rose during the first 2 h of adaptation, the level of other fractions rising during several hours approximately parallel with the development of respiratory activity. 4. When anaerobically-grown respiratory-competent yeast was aerated in the presence of acriflavin or chloramphenicol, no respiratory adaptation took place. However, the content of phospholipids and of total fatty acids increased in a manner similar to that in cells during respiratory adaptation, whereas the content of sterols increased less. The cells aerated in the presence of cycloheximide had no respiratory ability, the content of phospholipids was similar to that in the cells aerated with acriflavin or chloramphenicol, the content of fatty acids was intermediate between aerobically- and anaerobically-grown cells, and the content of sterols was markedly reduced and approximated that of non-aerated anaerobically-grown cells. 5. The respiratory-deficient mutant, derived from the competent strain, showed the same pattern of lipid composition as the original strain. The amount of lipids was reduced in anaerobically-grown cells. The lipid composition of aerobically-grown mutant cells was the same as that of aerobically-grown wild yeast, except for the phospholipid content which was lower in the mutant.

Biochim.

Biophys.

Acta,

144 (1967)

94-101

RESPIRATION AND LIPIDS IN YEASI

95

INTRODUCTION When baker’s yeast was grown on glucose under anaerobic conditions, synthesis of several respiratory enzymes in the cells is strongly repressed, resulting in their inability to respireI, and structures corresponding to mitochondria of aerobic yeast seem to be considerably reduced or totally absent2-5. During aeration of anaerobicallygrown yeast, ability to respire gradually emergesI, and typical mitochondria are formed6$‘. By a cytoplasmic

mutation,

apparently

affecting

mitochondrial

DNA8, the

respiratory ability is permanently reducedg, and mitochondria of such a respiratorydeficient mutant have an aberrant StructurelO. Although the reduction or absence of some respiratory enzymes is an observable manifestation both of anaerobic repression of respiration and of the cytoplasmic mutation to respiratory inability, several investigators have suggested that synthesis of some structural components of the cell may be primarily affected, changes in respiratory enzymes being a secondary consequencell-l”. A change in lipids to which an important role in structures of aerobic cells, electron transport in the respiratory chain and in oxidative phosphorylation is assigned15, might be such a primary event in the repression or mutation manifestation. Even if not, a correlation might be expected between the state of the respiratory apparatus and mitochondria in yeast and its lipid content and composition. Such a correlation has been suggested by many previous reports showing that lipid composition in yeast is strongly influenced

by aerobiosis1+18.

KLEIN’* proposed

that the synthesis

of lipids and of en-

zymes concerned with respiration in yeast are related events. However, such factors as energy dependence of lipid synthesis and requirements of 0, for desaturation of fatty acids and for sterol formation1a-21 preclude a simple conclusion from these observations that there is a functional interdependence between respiration activity and lipid content in yeast. An attempt was made in the present work to elucidate such a possible interdependence. MATERIALS AND METHODS Chemicals Chemicals originated from Lachema, Brno, except for acriflavin Houses, London), chloramphenicol (Spofa, Prague), and cycloheximide Kalamazoo). Solvents were freshly distilled in the laboratory.

(British Drug (Upjohn Co.,

Micro-organisms and their cultures The yeasts were a laboratory strain Saccharomyces cerevisae DT XII originating from a baker’s yeast factory in TrenEin, and a respiratory-deficient mutant prepared from it by trypaflavin treatment22. They were grown in synthetic medium containing per 1: I g KH,PO,, 1.2 g (NH,),SO,, 0.7 g MgSO,*7H,O, 0.5 g NaCl, 0.4 g CaCl,, 5 mg FeCl,, 50 g glucose, 2 ,ug biotin, 400 ,ug calcium pantothenate, 2 mg inositol, 400 ,ug niacin, 400 ,ug pyridoxal, 400 ,ug thiamine, 200 ,ug riboflavin and 200 l_cgp-aminobenzoic acid. For anaerobic growth, the medium was supplemented with 12 mg ergosterol, a.6gTween 80 and 3.5 g ethanol. Tween 80 served as a source of unsaturated fatty acids. In some experiments, either ergosterol or Tween 80 or both were omitted. When Tween 80 was omitted, ergosterol was kept in dispersion by gelatin (final Biochim.

Biophys.

Ada,

144 (1967)

g4-IOI

L. KOV&

96 concn. 0.5%). The vitamins, sterilized by filtration, added separately to the bulk of the medium. Inocula were prepared by static cultivation

and the lipid supplements of cells transferred

et al.

were

from stock

nutrient agar slopes in 3 ml medium at 30’ for 24 h. For aerobic culture, the inoculum was poured into 300 ml of the medium in a 3-l erlenmeyer flask which was then incubated on a shaker at 30’. Anaerobic culture was made in a 2-l flask, equipped with a Hg trap allowing bubbling and escape of CO,, which was filled with medium almost to the neck and bubbled for I h with purified Ar, then inoculated, left at 3o”, and shaken occasionally by hand.

bubbled

again for 30 min, closed,

For both aerobic and anaerobic growth, the amounts of inocula (IO~--IO~cells/ ml) were selected to reach the stationary phase after about 35 h. The number of dead cells (stained by methylene blue) at the end of growth was negligible in aerobic culture and in anaerobic culture in the presence of Tween 80 and ergosterol; if, in anaerobic culture, one or both lipid supplements were omitted, the number of cells stained with the dye was 25 and 33%, respectively. The yield of cells from such deficient media was much lower (IO times or more) than from the supplemented medium. After growth ceased, the contents of cultivation flasks were cooled to o0 and centrifuged

in the cold. The cells were washed 4 times with ice-cold

with Ar, and, for lipid analyses,

water bubbled

lyophilized.

Respiratory adaptation of anaerobically-grown yeast The cells from the stationary phase of growth (2.4 g dry wt.) were suspended in 300 ml of 0.11 M citrate-phosphate buffer (pH 4.3) and 3% glucose, and aerated by shaking in a 3-l erlenmeyer flask at 30’. Samples were taken for measurement of respiration,

dry weight determination

and lipid analysis.

Respiration Respiration was measured at 30’ in 40 mM citrate-phosphate with 50 mM glucose, manometrically or polarographically with electrode. Lipid

buffer (pH 4.3) a vibrating Au

analysis Various methods of lipid extraction from lyophilized yeast were tried (according to refs. 18, 23-25, and with chloroform-methanol in a Soxhlet apparatus). Although two extraction procedures gave identical results for phospholipids23p25, only the saponification method18 was found satisfactory for other lipids. The method does not allow the differentiation of free and esterified fatty acids; it has been found, however, that even in extracts from a Soxhlet apparatus more than a half the total fatty acids were represented by free fatty acids (see also ref. 16). The following procedure was used. Two aliquots of yeast were analysed separately, one for phospholipids, and one for other lipids. For phospholipids, a modified extraction procedure25 was employed: 100 mg lyophilized yeast were mixed in a centrifuge tube with 5 ml of 10% trichloroacetic acid. After 30 min, the suspension was centrifuged, the sediment resuspended in 7 ml of methanol and warmed at 55” for 15 min in a tightly-stoppered tube. After the tubes were cooled, 14 ml of chloroform were added, the mixture was shaken vigorously and left to stand overnight. It was then centrifuged, the sediment washed Biorhim.

Bio$hys.

Acta,

144

(1967) 9q-101

RESPIRATIONAND LIPIDS IN YEAST

97

with 3 ml of chloroform-methanol (z : I, v/v) and the pooled supernatants purified by the FOLCH procedureZ4. After mineralization a3, Pi was determinedaB. It was assumed that I mg of phosphate corresponds to 25 mg of phospholipidz’. The second aliquot of IOO mg lyophilized yeast was saponified and divided into fractions of total fatty acids and non-saponifiable lipids according to KLEIN’~. Sterols were determined by the Lieberman-Burchard reaction2s, and total non-saponifiable lipids photometrically with dichromate 27. By subtracting sterols from the total nonsaponifiable lipids the content of hydrocarbons was obtained’s, Total fatty acids were analysed volumetrically by titration with 0.01 M KOH (ref. 29) and also photometrically with dichromatez7; for calculation, it was assumed that I mg of total fatty acids reduces 17.6 mg of dichromate 27. Iodine titrimetric procedure30.

number

was determined

by a

RESULTS Content and composition

of lipids in yeast grown under aerobic and anaerobic conditions The results of lipid analysis in yeast Saccharomyces cerevisiae DT XII and its respiratory-deficient variant are summarized in Table I. The reduction in the content of the lipid fractions in anaerobically-grown respiratory competent yeast as compared with aerobically-grown cells confirmed previously reported data+18. As expected, cells grown anaerobically in media unsupplemented with ergosterol or Tween 80 were highly deficient in that essential component which was omitted from the medium; the minute amounts present can be accounted for as being derived from the original inoculum. But other differences between cells grown anaerobically in media supplemented or non-supplemented with lipids were also observed. When compared with cells from medium supplemented with both ergosterol and Tween 80, the following differences were noted. Cells from medium with ergosterol but lacking Tween 80 had less sterols; cells from medium with Tween 80 but without ergosterol containedmorefattyacids; and cells from media lacking one or both lipid components had a significantly higher content of phospholipids. TABI,E I CONTENT

AND

COMPOSITION

OF

YEAST

LIPIDS

Several lyophilized batches of yeast grown under the same conditions were pooled and analysed. 8-14 independent analyses were made, except for the determinations of the iodine number and of equivalents of fatty acids which were performed on 4 samples. The standard error of the mean of the values presented here has not exceeded 0.5% for phospholipids and sterols, 2.5% for hydrocarbons and total fatty acids and 4% for the iodine number. If not stated otherwise the values are expressed as per cent of yeast, dry weight.

strain

S. cerevisiae

DT XII (respiratory competent)

Respiratorydeficient mutant

Growth Supplenzents cow.ditions to medium

Phospholipids (%o)

Aerobic

3.80

-

Anaerobic Anaerobic Tween 80 Anaerobic Ergosterol Anaerobic

Tween 80+ ergosterol

Aerobic Anaerobic

Tween 80+ ergosterol

Sterols (%)

Hydrocarbons (%)

Total fatty acids

pquiv

Iodine

num(Ojoj(iuequwIg yeast) KOH Img o_ffatty acids ber ~~~.~.

0.10

1.21

2.07

0.15

1.19

8.65 1.78 4.15 I.69

I .69

0.25

0.89

2.56

I22

4.79

9.0

2.66 1.62

3.48

1.16 1.03

8.49 2.99

368 140

4.33 4.67

65.5 IO.4

2.00 2.18

3.14

I.29

0.12

I.25

0.25

378 88 197 85

4.37 4.93 4.75 5.02

~

__ Biochim. Biophys.

_~____

Acta, 144 (1967) g+-‘o’

62.5 3.5 15.0 3.7

Differences in iodine number indicate lower unsaturation in lipids of anaerobically-grown yeast. A higher proportion of fatty acids with shorter chains in fat from anaerobically-grown yeasP fatty acids; it was highest

is indicated by an increased ratio pequiv in cells from the medium lacking Tween

KOH/mg of 80 and thus

deficient in unsaturated fatty acids. Important conclusions can be drawn from Table I concerning the respiratorydeficient mutant. Anaerobically-grown cells of the mutant had the same lipid content as anaerobic cells of the original respiratory-competent strain. Although, after aerobic growth, the state of the respiratory apparatus of the mutant is obviously similar to that of anaerobically-grown cells, the lipid content was entirely different and was practically the same as in aerobically-grown cells of the respiratory-competent Only the phospholipid level was significantly lower in the mutant even considerably Changes

higher

in lipids

than

in anaerobically-grown

i~t the cowse

of respiratory

strain. though

cultures.

adaptation

It has been reported that the sterol content of yeast increased during aeration of washed cells in the presence of gluco_e c 32--35.The kinetics of changes in the content of sterols and other lipids in the course of respiratory adaptation and its correlation with the kinetics of respiratory system synthesis was followed, and the results are shown in Fig. I. In the course of respiratory adaptation, the lipid content typical for anaerobically-grown cells changed gradually to the content characteristic of aerobically-grown yeast. The content of phospholipids rose during the first 2 h of adaptation while other fractions increased during several hours together with the respiration capacity. In an attempt to clarify whether the increase in lipid content amd the development of respiratory ability are functionally incubated under the same conditions as during respiratory presence of inhibitors preventing the synthesis of lipid analysis from such cells are presented

of respiratory in Table II.

linked, the ceils wtre adaptation but in the enzymes. The results After aeration in the

presence of the inhibitors the cells had no respiratory activity, which indicates that the inhibitors made the synthesis of a complete respiratory apparatus impossible. Despite this, the lipid content of the cells increased. Regarding their fatty acid and phospholipid contents, cells aerated in the presence of acriflavin or chloramphenicol approached control cells aerated in the absence of the inhibitors; however, the sterol content was considerably reduced, even though it was higher than in non-aerated cells. When cycloheximide was present during aeration, the content of phospholipids was similar to that in the aerated control, the content of fatty acids was intermediate between the similar to that in produced/mg cells mained unchanged

aerated and non-aerated controls while the content of sterols was anaerobically-grown cells. It should be added that the Q$ (,ul CO, per h) of the cells aerated with acriflavin or chloramphenicol rewhile it decreased for the cells aerated with cycloheximide.

DISCUSSION

The following conclusions can be drawn from the results presented. (I) The lower content of lipids in anaerobically-grown yeast, as compared with aerobically-grown cells, is not due to a limited supply of energy for lipogenesis under anaerobic conditions. This follows from the finding that the content of lipids in the Biochim.

Biophys.

Acta,

144 (1967)

94-101

RESPIRATION

AND LIPIDS

IN YEAST

99

Fig. I. Changes in lipid content in the course of respiratory adaptation. Respiratory competent yeast was grown anaerobically in medium supplemented with ergosterol and Tween 80. The values are means from two experiments. A. l , -Qo2 (~1 0,/h per mg dry wt.; 0, phospholipids. B. 0, total fatty acids; o, sterols. C. l , iodine number; 0, ,uequiv KOH/mg of fatty acids.

respiratory-deficient mutant was subjected to the same variation depending on aerobiosis as in wild yeast, despite the fact that energy production in the mutant is the same aerobically and anaerobically, being limited to glucose fermentation. (2) There is no simple relation between the content of respiratory enzymes and the content of lipids in the yeast cell. The aerobically-grown respiratory-deficient mutant, with a reduced level of cytochromes and other respiratory enzymes, had a similar content of lipids to that of aerobic wild yeast containing a complete assembly of respiratory enzymes. Inhibitors of respiratory enzyme synthesis, chloramphenicol and acriflavin3sp37, although preventing respiratory adaptation, did not substantially affect the synthesis of fatty acids and phospholipids in the course of aeration of anaerobically-grown yeast. The latter observation differs from a previous reporP that Mn2+ and acriflavin inhibited both respiratory adaptation and fatty acid synthesis in anaerobic yeast. (3) Cytoplasmic mutation to respiratory deficiency in yeast does not lead to Biochim.

Biophys.

Acta.

144 (1967)

94-101

L. KOVk et al.

100

profound changes in lipid composition. Tnis conclusion extends some previous observations18,3BP40; it does not exclude, however, the possibility that a temporary disturbance in lipogenesis

might be involved in mutant formation, as suggested by SARAcontent of phospholipids observed in aerobically-grown mutant cells relative to the original wild yeast may be connected with structural defects in the CHEK~~. The decreased

mitochondria

of the mutantlo.

TABLE II LIPID CONTENTOF

ANAEROBICALLY-GROWN

YEAST

(RESPIRATORY-COMPETENT

STRAIN)

AFTER

AERATION

Yeast grown anaerobically in medium supplemented with ergosterol and Tween 80 has been used. The values are means from two analyses, and are expressed in per cent of yeast dry weight. Aeration

Inhibitor

(h)

(pg Wl

Phqbholipids (%)

: 8 8 8

0

Acriflavin (5) Chloramphenicol (4000) Cycloheximide (25) _____ -~ The increase

in lipid content

1.87 2.87 2.60 2.64 2.58

stero1s

Hydrocarbons

Total fatty acids

(Yb)

f”/,)

I.44 0.68 0.56

0.98 I.10 0.90 0.78

2.83 6.96 6.11

0.32

I.01

(“W 0.25

of aerobically-grown

relative

5.77 4.55

to anaerobically-

grown cells may be related to a more complicated structural organization of aerobic cells, especially to the presence of mitochondria. At least one of the inhibitors which prevented respiratory adaptation but not synthesis of fatty acids and phospholipids (Table II), namely chloramphenicol, allows the development of mitochondrial profiles, even though they be different from normal mitochondria42. The formation of aerobic structures might control lipogenesis in yeast. An inverse possibility, that limitation in lipid synthesis under anaerobic conditions may be one of the factors determining the depression of mitochondrial structures, remains an attractive alternative. Additional points introduce complexity into the problem. First, the functional relationship between cell structures and lipids may be obscured because part of the lipids in baker’s yeast can function not as structural components but as nutritional reservesIB. Second, a link between protein synthesis and lipid formation during aeration of anaerobically-grown yeast has been suggested by the inhibition of both syntheses with cycloheximide (Table II), an inhibitor of general protein synthesis in yeast43-46. Third, a complex and apparently paradoxical relation between sterol and the respiratory system seems to be indicated by two observations. First, inhibitors preventing respiratory enzyme formation at the same time considerably depressed the synthesis of sterols (Table II). Second, cells grown anaerobically in a medium supplemented with Tween 80 but lacking ergosterol and thus deficient in sterols, contained more fatty acids and phospholipids than control cells, as if they were intermediate between aerobic and anaerobic cells (Table I) ; and it has been confirmedd, although not reported in the RESULTS, that, immediately after being harvested from an anaerobic culture, they showed a marked respiratory activity (Qo, = 13-20). These results are in line with other observations indicating a possible involvement of sterol in the organization of respiratory activity in yeast351*7. Biochim.

Biophys.

Acta,

144 (1967)

94-101

RESPIRATION

101

AND LIPIDS IN YEAST

Changes in lipid composition of anaerobically-grown yeast from lipid-unsupplemented media (Table I) are difficult to evaluate, and require further studies. A more detailed analysis of isingle lipid fractions with regard to their intracellular distribution also seems to be necessary. REFERENCES

9 10 II

12 I3 I4 15 16 17 18 I9 20 *I 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

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Biochim. Biophys.

Acta, 144 (1967) 94-101