Lipids of the yeast Hansenula anomala

BIOCH1M1E, 1977, 59, 97-104.

Lipids of the yeast Hansenula anomala. KIM HENG NG ( ' ) a n d MARIE-ANTOINETTE LAN~ELLE(>. Centre de Recherche de Bioehimie et Gdndtique Cellulaires, 118, route de Narbonne, 31077 Toulouse Cedex, France.

(4-10-1976). Summary. - - An analysis of the free lipids of Hansenula anomala was performed. The main fatty acids obtained by saponification of whole cell crude lipids ~.ere palmitic, Cxs:a, C~se and C,s:8 acids. In mitoehondrial lipids the tri-unsaturated acid was present as traces. Fatty acid composition of each class of lipids was also determined. Phosphatidylcholine and phosphatidylethanolamine were the main phospholipids ; phosphatidylserine, phosphatidylinositol and cardiolipin 'were also characterized. The most abundant sterols were ergosterol and lanoslerol. An acetate of a 2l-ethyl cholesterol was also isolated. Two glycolipids, a galactosyl diglyceride and a glucosyl eeramide nvere identified ; concerning the galactosyl diglyceride, the content of C~s:3 acid 'was higher than in other lipid classes. In the glucosyl eeramide, the main fatty acid was a-hydroxy C~s~ acid ; C~6:0, C,8:~, C~s~_,and C~:8 acids were present too. The long chain base was sho~'n to be C~8-phytosphingosine (4-hydroxy C~s sphinganine). Some similarities and differences avith Saccharomyces cereoisiae are discussed.

INTRODUCTION. F e w w o r k s exist on t h e g e n e r a l l i p i d c o m p o s i t i o n of y e a s t s ; e x c e p t s t u d i e s c a r r i e d out w i t h t h e g l u c o s e r e p r e s s i b l e y e a s t Saccharomyces cerevisiae, o n l y f r a g m e n t a r y a n a l y t i c a l d a t a a r e a v a i l a ble on s o m e o t h e r g e n e r a [11. Yeasts p r e s e n t t a x o n o m i c p r o b l e m s y e t u n r e s o l v e d d u e to t h e l a c k of c o m p l e t e i n f o r m a t i o n on e a c h genus. C h e m i c a l a n a l y s i s h a v e p r o v i d e d s u p p l e m e n t a r y d a t a in t h e c h a r a c t e r i z a t i o n of d i f f e r e n t f a m i l i e s : f o r e x a m ple, c e l l w a l l a n a l y s e s w e r e u s e d [2J, a n d J o h n s o n a n d B r o w n [31 h a v e s h o w n in y e a s t s a p a r a l l e l i s m between polyunsaturated fatty acid composition of t h e cells a n d the r e p r e s s i v e effect of g l u c o s e ( C r a b t r e e effect) u n d e r a e r o b i c c o n d i t i o n s . W e a n a l y s e d l i p i d s of w h o l e cells a n d of m i t o c h o n d r i a of t h e g l u c o s e n o n - r e p r e s s i b l e y e a s t Hansenula anomala.

M A T E R I A L AND 5 I E T H O D S . ORGANISM.

T h e y e a s t s t u d i e d w a s Honsenula anomala f r o m t h e W a i t e r J. N i c k e r s o n C o l l e c t i o n [4]. (*) This work is a part of thesis presented at the Universit6 Paul Sabatier of Toulouse, France, for the ¢ Doetorat de Sp6cialit~ >> on J u l y l l t h 1975. To 'whom a l l correspondence should be addressed.

MEDIA AND CULTURE CONDITIONS.

T h e y e a s t s w e r e c u l t i v a t e d in a f e r m e n t o r (50 l i t r e s m e d i u m p e r b a t c h ) f o r l i p i d e x t r a c t i o n , a n d in s h a k e n flasks (300 m l p e r flask) for m i t o chondrial preparations. T h e m e d i u m c o n s i s t e d of : y e a s t e x t r a c t 1 g, KHuPO 4 10 g, NH~C1 2.5 g, MgSO 4 0.2 g, NaC1 I g, 60 p e r c e n t s o d i u m l a c t a t e 25 m l , d i s t i l l e d w a t e r w a s a d d e d u p to one litre. T h e p H of t h e m e d i u m w a s a d j u s t e d to 5.2-5.4 b y t h e a d d i t i o n of d i l u t e d s u l f u r i c a c i d a n d g r o w t h w a s c a r r i e d out at 27°C. (Jells w e r e c o l l e c t e d at t h e b e g i n n i n g of t h e s t a tionary growth phase. LIPID EXTRACTION AND SOLVENT FRACTIONATION.

All s o l v e n t s u s e d f o r e x t r a c t i o n s a n d c h r o m a t o g r a p h y w e r e r e d i s t i l l e d p r i o r to use. Cells w e r e s u s p e n d e d in CHCI 3 : CH,~OH 2/1 ( v o l / v o l ) c o n t a i n i n g 0.2 p e r c e n t a c e t i c a c i d . T h e o r g a n i c p h a s e w a s w a s h e d w i t h w a t e r to e l i m i n a t e w a t e r - s o l u b l e c o m p o u n d s , d r i e d o v e r Na2SO ~ a n d evaporated under vacuum. Lipid extracts were s t o r e d u n d e r n i t r o g e n at - - 20°C. T o t a l l i p i d s diss o l v e d in a m i n i m u m of d i e t h y l e t h e r w e r e c r u dely separated by precipitation with cold acetone y i e l d i n g an a c e t o n e - s o l u b l e f r a c t i o n of <>. COLUMN CHROMATOGRAPHY.

Neutral lipids were eluted from a column of F l o r i s i l 60/100 m e s h ( T o u z a r t a n d M a t i g n o n ,

98

K i m H e n g Ng and M.-A. LandelIe.

Paris, F r a n c e ) b y s t e p w i s e i n c r e a s i n g c o n c e n t r a tions of d i e t h y l e t h e r in p e t r o l e u m ether b.p. 35 °50 ° (1 p e r cent to 100 p e r cent). G l y c o l i p i d s w e r e eluted on the s a m e c o l u m n w i t h CHCIs w i t h inc r e a s i n g c o n c e n t r a t i o n s of CH3OH (5 p e r cent to 50 p e r cent). P h o s p h o l i p i d s w e r e s e p a r a t e d on s i l i c i c a c i d (Mallindkrodt, G e r m a n y ) a n d DEAE-cellulose (Cellex D, Bio-Rad l a b o r a t o r i e s , California) p r e p a r e d a c c o r d i n g to R o u s e r et al [5].

( S e r d a r y R e s e a r c h L a b o r a t o r i e s , L o n d o n , Ontario, Canada), sterols (Mann R e s e a r c h L a b o r a t o r i e s , New York) and p h y t o s p h i n g o s i n e (Calbiochem, San Diego, California). Relative p e r c e n t a g e s of l i p i d s w e r e e s t i m a t e d b y p h o t o d e n s i t o u i e l r y of c h a r r e d t h i n - l a y e r c h r o m a t o g r a m s d e v e l o p p e d w i t h solvent system (c), (h) o r (i). GAS LIQUID CHROMATOGRAPHY,

T h i n l a y e r plates w e r e p r e p a r e d w i t h s i l i c a gel G (Merck) for a n a l y t i c a l studies ( t h i c k n e s s 0.2.5 ram) w h i l e p r e p a r a t i v e t h i n - l a y e r plates (thickness 1 rain) w e r e p r e p a r e d w i t h s i l i c a gel P F 254 (Merck) [61.

Analysis of fatty a c i d 1nethyl esters was c a r r i e d out w i t h a V a r i a n A e r o g r a p h 1400 a p p a r a t u s e q u i p p e d w i t h columns p a c k e d w i t h s i l i c o n e SE 30 6 p e r cent (2.5 m) or DEGS 20 p e r cent (3 m) on c h r o m o s o r b W. Retention times w e r e c o m p a r e d to those of i n t e r n a l s t a n d a r d s and p e r c e n t a g e s w e r e c a l c u l a t e d from p e a k areas.

F o r s e p a r a t i o n of free a n d a c e t y l a t e d sterols ; t h i n - l a y e r plates w e r e i m p r e g n a t e d w i t h 5 p e r cent AgNO:~ a c c o r d i n g to C o p i u s - P e e r e b o o m a n d Beekes [7].

Sterols, a c e t y l a l e d sterols, t r i m e t h y l s i l y l sterols [10] a n d t r i m e t h y l s i l y l p h y t o s p h i n g o s i n e !11] were identified by chromatography with standards on a silicone SE 30 10 p e r cent (1 nl) column.

THIN LAYER CHROMATOGRAPHY.

The f o l l o w i n g solvent systems w e r e u s e d for the s e p a r a t i o n of the a p p r o p r i a t e groups of ]ipids : (a) p e t r o l e u m e t h e r b. p. 350-50 ° - d i e t h y l e t h e r 9 : 1 (vol/vol) ( m e t h y l esters) ; (b) p e t r o l e u n i ether b. p. 350-5.0 ° - d i e t h y t ether 7 : 3 (vol/vot) ( h y d r o x y l a t e d c o m p o u n d s ) ; (c) p e t r o l e u m e t h e r b. p. 3.50-5.0° - d i e t h y l e t h e r - a c e t i c a c i d 9,0 : 10 : 1 ( v o l / v o l / v o l ) ( n e u t r a l l i p i d s ) ; (d) CHCl:~- d i e t h y l ether-acetic a c i d 97:2.5:0.5 ( v o l / v o l / v o l ) (sterols) ; (e) CHCl:s-petroleum ether b. p. 35°-50°-acetic a c i d 25:75:0.5 ( v o I / v o l / v o l ) ( a c e t y l a t e d s t e r o l s ) ; (f) CHCla-CH3OH 9:1 (vol/vol) (glycolipids) ; (g) CHCla-CH3OH 85:15 (vol/vol) (glycolipids) ; (h) CHCls-GHaOH-HsO 65:2'5:4 ( v o l / v o l / v o l ) ( p h o s p h o l i p i d s ) ; (SHC13-CHaOH-N~H~OH 7N 60':35:5 ( v o l / v o l / vol) ( p h o s p h o l i p i d s ) ; b u t a n o l - a c e t i c a c i d - w a t e r 60:20:20 ( v o l / v o l / v o l ) (plasnialogens). I n the t w o d i m e n s i o n a l t h i n - l a y e r c h r o m a t o g r a p h y a n a l y s i s of p h o s p h o l i p i d s , solvent system (h) w a s used in the first d i r e c t i o n a n d solvent s y s t e m (i) in the s e c o n d d i r e c t i o n . L i p i d s w e r e v i s u a l i s e d b y r h o d a m i n e B, sterols w e r e d e t e c t e d w i t h 20 p e r cent sulfuric a c i d f o l l o w e d b y heating, g l y c o l i p i d s w e r e d e t e c t e d w i t h t h e p e r i o d a t e - S c h i f f r e a g e n t [8], p h o s p h o l i p i d s w i t h the D i t t m e r - L e s t e r r e a g e n t [9], ninh y d r i n for the a m i n o groups a n d the D r a g e n d o r f f r e a g e n t [61 for c h o l i n e groups. S p e c i f i c v i s u a l i s a t i o n a n d Rf values w e r e comp a r e d to those of c o m m e r c i a l s a m p l e s : p h o s p h a t i d y l i n o s i t o l (PI) a n d p h o s p h a t i d y l s e r i n e (PS)

BIOCHIMIE, 1977, 59, n ° 1.

PAPER CHROMATOGRAPHY.

W h a t m a n p a p e r n ° 1 was used for c h r o m a t o g r a p h y of w a t e r - s o l u b l e p r o d u c t s w i t h t w o solvent s y s t e m s ; (k) b u t a n o l : p y r i d i n e : w a t e r 6 / 4 / 3 ( v o l / v o l / v o l ) ; (1) b u t a n o l : a c e t i c a c i d : w a t e r 4 / 1 / 1 ( v o l / v o l / v o l ) . F r e e a m i n o groups w e r e d e t e c t e d b y n i n h y d r i n (0.2 p e r cent in acetone), p o l y h y d r o x y l a t e d c o m p o u n d s b y A g N O J N a O H and aldoses b y p - a n i s i d i n e , HC1 (3 p e r cent in acetone) f o l l o w e d b y heating. ACIDIC HYDROLYSIS.

Phospholipids and glycolipids were hydrolysed in 1 N H,C] at 100°C o v e r n i g h t in sealed tubes. S p h i n g o l i p i d s w e r e t r e a t e d w i t h aqueous m e t h a nolic HC1 [12]. ALKALINE HYDROLYSIS.

F a t t y a c i d s of l i p i d s of w h o l e cells w e r e obtained b y s a p o n i f i c a t i o n in b e n z e n e : m e t h a n o l 2/8 (vol/vol) c o n t a i n i n g 5 p e r cent KOH u n d e r r e f l u x d u r i n g 2 hours. Mild a l k a l i n e h y d r o l y s i s w a s c a r r i e d out in m e t h a n o l i c 1 N NaOH at r o o m temp e r a t u r e for 2 hours. MITOCHONDRIAL PREPARATION.

M i t o c h o n d r i a w e r e p r e p a r e d a c c o r d i n g to K o v a c et al. [13] a n d p u r i f i e d b y c e n t r i f u g a t i o n in a disc o n t i n u o u s s a c c h a r o s e g r a d i e n t (20 to 60 p e r cent).

99

Lipids of the yeast H a n s e n u l a a n o m a l a . Tile r e s p i r a t i o n w a s m e a s u r e d e l e c t r o d e (Gilson o x y g r a p h ) .

with

a b u n d a n t in galactosyl d i g l y c e r i d e s . F r e e fatty acids of the a c e t o n e soluble f r a c t i o n h a d a c o m p o sition sinlilar to that of total lipids : t h e y could have been released by h y d r o l y s i s d u r i n g the extractions.

a Clark

I~ESULTS.

Hansenula anomala cells c o n t a i n e d about 9 p e r cent of lipids of t h e i r d r y w e i g h t at s t a t i o n a r y p h a s e w h e n g r o w n in the c o n d i t i o n s m e n t i o n e d above. This is c o m p a r a b l e to o t h e r yeasts (7 p e r cent to 15 p e r cent d r y weight) [131. Neutral lipids r e p r e s e n t e d 40 p e r cent of the total lipids ; t h e i r c o m p o s i t i o n was estimated by p h o t o d e n s i t o m e t r y of t h i n - l a y e r c h r o l n a t o g r a m s (solvent c) : h y d r o c a r b o n s 1 p e r cent, t r i g l y c e r i d e s 75 p e r cent, dig l y c e r i d e s 10 p e r cent, ergosterol 10 p e r cent, l a n o s t e r o l 3 p e r cent, free fatty acids and glycolipids 1 p e r cent. T h e acetone-insoluble f r a c t i o n c o n t a i n e d p h o s p h o l i p i d s and small quantities of glycolipids.

STEROLS.

After s e p a r a t i o n of n e u t r a l lipids by c o l u m n c h r o m a t o g r a p h y on Florisil, ergosterol o b t a i n e d by c r y s t a l l i s a t i o n in cold diethyl ether f r o m the sterol fraction, was c h a r a c t e r i z e d by its b e h a v i o u r on t h i n - l a y e r c h r o m a t o g r a m s and its ultra-violet s p e c t r u m El4!. In t h i n - l a y e r c h r o m a t o g r a p h y on silica gel G in solvent (b), sterols h a d the same Rf, but t h e i r c o l o u r r e a c t i o n s w h e n h e a t e d after s p r a y i n g w i t h sulfuric acid w e r e different. By using AgNO:~ imp r e g n a t e d t h i n - l a y e r plates, free or acetylated sterols w e r e s e p a r a t e d m o r e efficiently (table II).

TABLE [.

Relative percentages of fatty acids in lipid classes and whole cells. 16:0

16:1

17:1

13.2

3.7

1.0

18:0

18:1

18:2

18:3

a-OH t8:0

i I

[

Whole cells

2.0

27.0

13.0

37.0

1--

Total lipids

12.9

4.6

1.6

1.3

17.6

42.8

Neutral lipids

46.8

8.0

3.2

2.7

36.5

2.8

2.6

4.7

15.6

36.5

18.5

--

18.2

30.4

2.5

1.5

0.8

30.0

40.0

9.3

2.6 i 1.4

3.3

29.3

19.5

3.9

3.9 L 1.9

3.4

ii 37.2

15.8

Free fatty acids

14.6

6.5

Phosphatidylcholine

38.3

10.5

Phosphatidylethanolamine

16 0

2.4

Phosphatidylinositol

--!

]-

Phosp ha tidylserine Monogalaetosyl diglyeeride Glucosyl ceramide

37.8

. . . . . . .

i

9.2

3.4

16.0

1.8

2.2 --

19.2

I

19.8 i 40.0

23.6

i 10.3

11.1

8.6

54.0

Fatty acids are calculated as percentages of total fatty acid content of each class of lipid. Fatty acid chain length is indicated bv the first figure ; the second one indicates the number of double bonds, u-'OH 18:0 stands for ct-hydroxy stearic acid.

FATTY ACIDS. Table I shows the d i s t r i b u t i o n of fatty acids in the different classes of lipids. N e u t r a l lipids w h i c h c o n t a i n e d 75 p e r cent of t r i g l y c e r i d e s h a d a l o w c o n t e n t of p o l y u n s a t u r a t e d fatty acids. Di-unsatur a t e d fatty acids w e r e m a i n l y f o u n d in the phosp h o l i p i d s w h e r e a s t r i - u n s a t u r a t e d fatty acid w a s

BIOCHIMIE, 1977, 59, n ° 1.

Gas l i q u i d c h r o m a t o g r a p h y analysis of t r i m e thyl sterol d e r i v a t i v e s c o n f i r m e d the p r e s e n c e of l a n o s t e r o l and ergosterol, i d e n t i f i e d by r e t e n t i o n times and c o c h r o m a t o g r a p h y w i t h c o m m e r c i a l samples. Column c h r o m a t o g r a p h y f o l l o w e d by p r e p a r a tive t h i n - l a y e r c h r o m a t o g r a p h y gave a c o m p o u n d 8

K i m H e n g Ng and M.-A. Landelle.

100

TABLE II.

Sterols analyses by AgNOs impregnated thin lager chromatography R(cholest~rol) Of [ r e e ster01s

R(acetyl cholesterol)

of "~cetylated sterols

Solvent (d) color after visualisation (a) Cholesterol Ergosterol Lanosterol

standards

H. anomala slandards

red

1.0

greenish grey

0.4

0.4

1.38

1.35

I

I orange

24 - ethyl - choles- [ terol I red

Solvent (e)

not observed,

H. anomala [i not observed

1.0

0.3 .

0.6

0.7

0.72

i - -

0.76

not observcd (b)

Sterols 'were analysed under their free and chemically acetylated forms (except for 24-ethyl cholesterol naturally aeetylaled in H. anomala. Plates were coated w i t h silica gel G impregnated 'with 5 per cent AgNO:, (a) visualisation by heating after sulfuric acid spray. (b) observed only after saponification.

i d e n t i f i e d to an a c e t y l a t e d 2 4 - e t h y l a c c o r d i n g to t h e f o l l o w i n g r e s u l t s :

cholesterol

i) a f t e r s a p o n i f i c a t i o n , a f r e e s t e r o l w a s obtained, t h e c h r o m a t o g r a p h i c b e h a v i o u r a n d t h e color a t i o n of w h i c h w e r e s i m i l a r to ~-sitosterol.

PAO

GL G CL ® 0 °.,. 6/

it) a b s o r p t i o n b a n d s at 8.1 ~ ( a c e t y l b a n d ) a n d at 5.8 ~ (ester b a n d ) , a n d t h e a b s e n c e of v i n y l g r o u p a b s o r p t i o n b a n d s in tlle i n f r a - r e d s p e c t r u m of t h e c o m p o u n d . iii) gas l i q u i d c o c h r o m a t o g r a p h y w i t h an a c e t y l ated commercial sample. iv) t h e m a s s s p e c t r u m of this c o m p o u n d e x h i b i t e d a p e a k at m / e 396 (M-60) ; p e a k s at m / e 2.1.3 a n d 2,55 a r e in a g r e e m e n t w i t h t h e m o n o - u n s a t u r a t e d t e t r a c y c l i c s t e r o l s k e l e t o n [15]. T h i s s p e c t r u m w a s i d e n t i c a l to t h a t of a s a m p l e of 8-sitosterol acetate. T h e p r e s e n c e of 24 e t h y l c h o l e s t e r o l in y e a s t s h a s n o t b e e n o b s e r v e d so f a r a l t h o u g h t h i s s t e r o l is f r e q u e n t l y e n c o u n t e r e d in h i g h e r plants. T h e a c e t y l a t e d f o r m c o u l d be an a r t e f a c t d u e to t h e p r e s e n c e of 0.2 p e r c e n t a c e t i c a c i d i n t h e e x t r a c t i o n s o l v e n t s y s t e m ; ho~vever, n o o t h e r a c e t y l a t e d sterol was detected.

Q%..,. 1 LPC ,2

FIe. 1. - - Phospholipids of Hansenula anomala. Total phospholipids 'were separated by two dimensional thin-layer chromatography on Silica Gel G ; first direction with solvent system (h), second direction with solvent system (i). O visualised 'with Dittmer-Lester reagent. ~-' visualised only after charring. A b b r e v i a t i o n s : glyeolipid (GI~) ; phosphatidic acid (PA) ; eardiolipid (CL) ; phosphatidylethanolamine (PE) ; phosphatidylglyeerol (PG) ; phosphatidyleholine (PC) ; lyso phosphatidyleholine (LPC) ; phosphatidylserine (PS) ; phosphatidylinositol (PI) ; non identified (n.J.).

PHOSPHOLIPIDS. The two dimensional thin-layer chromatograp h y (figure 1) s h o w e d t h a t Hansenula anomala

BIOCHIMIE, 1977, 59, n ° 1.

c o n t a i n e d t h e s a m e t y p e s of p h o s p h o l i p i d s as t h o s e f o u n d in Saccharomgces cerevisiae [16].

Lipids of the yeast H a n s e n u l a a n o m a l a . C a r d i o l i p i n (CL), p h o s p h a t i d y l c h o l i n e (PC), p h o s p h a t i d y l e t h a n o l a m i n e (PE), p h o s p h a t i d y l i n o sitol (PI) a n d p h o s p h a t i d y l s e r i n e (PS) w e r e the m a j o r c o m p o n e n t s (See <> p a r a g r a p h ) . No e v i d e n c e of the existence of alkyl or a l k e n y l analogues (plasmalogens) w a s found. Major p h o s p h o l i p i d s w e r e i s o l a t e d b y p r e p a r a tive t h i n - l a y e r c h r o m a t o g r a p h y a n d h y d r o l y s e d for t h e i r i d e n t i f i c a t i o n . The p r e s e n c e of m e t h y l ated e t h a n o l a m i n e c o m p o u n d s in the w a t e r soluble f r a c t i o n s w a s not detected.

101

p h y (as t r i m e t h y l s i l y l derivatives). The i d e n t i f i c a tion w a s c o n f i r m e d b y mass s p e c t r o m e t r y of the p e r a c e t y l a t e d c o m p o u n d s . The s p e c t r a of the acet y l a t e d c o m m e r c i a l s a m p l e a n d the u n k n o w n base w e r e i d e n t i c a l (figure 2) : the m o l e c u l a r ion (M = 485) w a s p r e s e n t in the t w o s p e c t r a ; intense p e a k s w e r e f o u n d c o r r e s p o n d i n g to 1-2 cleavage ( m / e 412) and 2-3 cleavage ( m / e 144) [19]. P e a k s r e s u l t i n g of the loss of one o r m o r e acetyl units w e r e f o u n d at m / e 425, 365, 305 a n d at m / e 352 (412-60), 292 (412-120).

GLYCOLIPIDS.

F a t t y a c i d c o m p o s i t i o n is s h o w n in table I. The a - h y d r o x y s t e a r i c a c i d w a s i d e n t i f i e d as p r e v i o u s l y d e s c r i b e d [20].

After c o l u m n c h r o m a t o g r a p h y of acetone-soluble l i p i d s , f r a c t i o n s c o n t a i n i n g c a r b o h y d r a t e s were pooled and purified by preparative thinl a y e r c h r o m a t o g r a p h y . F u r t h e r studies w e r e carr i e d out on two c o m p o u n d s .

The s t r u c t u r e of this g l y c o l i p i d is p r o b a b l y analogous to the b r a i n c e r e b r o s i d e s . This is s u p p o r t e d b y the f o l l o w i n g a r g u m e n t s :

1. Monogalactosyl diglyceride (MGDG). This m a j o r c o n s t i t u e n t of the g l y c o l i p i d f r a c t i o n gave a blue colour w i t h the p e r i o d a t e - S c h i f f reagent on t h i n - l a y e r c h r o m a t o g r a p h y (Rf 0.67 in solvent g). A c i d h y d r o l y s i s y i e l d e d g l y c e r o l a n d galactose d e t e c t e d on p a p e r c h r o m a t o g r a p h y (solvent sysreins k a n d 1). Mild a l k a l i n e h y d r o l y s i s gave a w a t e r - s o l u b l e c o m p o u n d w i t h a Rglucose : 0.8 in p a p e r c h r o m a t o g r a p h y (solvent system k) ; this Rglucose is i n t e r m e d i a t e b e t w e e n the Rgl,eo~e of m o n o - a n d disacc h a r i d e s . A c i d i c h y d r o l y s i s of this w a t e r - s o l u b l e c o m p o u n d gave g l y c e r o l a n d galactose. F a t t y a c i d d i s t r i b u t i o n is s h o w n in table I : C18:2 a n d Cls:8 a c i d s are m o r e a b u n d a n t t h a n in o t h e r l i p i d fractions, suggesting t h a t this glycol i p i d c o u l d be i m p l i c a t e d in the b i o s y n t h e t i c p a t h w a y of p o l y u n s a t u r a t e d fatty a c i d s as has been suggested in Chlorella vulgaris [17]. G l y c o s i d e s of 1,2-diacyl g l y c e r o l are c o m m o n l y f o u n d in plants, g r a m - p o s i t i v e b a c t e r i a a n d in a n i m a l s /18].

2. Glacosyl ceramide (cerebroside). The o t h e r g l y c o l i p i d s t u d i e d (Rf 0,31 in solvent system g) y i e l d e d b y a c i d i c m e t h a n o l y s i s m e t h y l esters, a long c h a i n base a n d glucose. The l o n g c h a i n base w a s i d e n t i f i e d as C18-phytosphingosine (4-hydroxy-sphinganine) by compar i s o n w i t h a c o m m e r c i a l s a m p l e in t h i n - l a y e r c h r o m a t o g r a p h y a n d in gas l i q u i d c h r o m a t o g r a -

BIOCHIMIE, 1977, 59, n ° 1.

i) fatty a c i d s w e r e l i n k e d to the long c h a i n base b y an a m i d e b o n d as d e t e r m i n e d b y the i n f r a - r e d s p e c t r u m a n d b y the r e s i s t a n c e to alkaline h y d r o lysis. it) the two s e c o n d a r y h y d r o x y l groups of 4-hyd r o x y s p h i n g a n i n e w e r e free in the i n t a c t glycol i p i d as s h o w n b y the f o r m a t i o n of a l o n g c h a i n aldehyde by periodic oxydation. MITOCHONDRIAL LIPIDS.

C o n s i d e r i n g that Hansenula anomala was a glucose n o n - r e p r e s s i b l e yeast, it s e e m e d i n t e r e s t i n g to c o m p a r e the l i p i d c o m p o s i t i o n of the cells w i t h that of m i t o c h o n d r i a . The p r e s e n t s t u d y w a s carr i e d out w i t h m i t o c h o n d r i a p r e p a r e d f r o m cells g r o w n on lactate. R e s p i r a t i o n of the p a r t i c l e s was m e a s u r e d for each p r e p a r a t i o n : succinate, p y r u vate-malate, ethanol, 9 a n d L-lactate ~Tere good s u b s t r a t e s ; NADH a n d ct-ketoglutarate w e r e also oxidized. P o l a r l i p i d s w e r e e s s e n t i a l l y p h o s p h o l i p i d s : the m a i n l i p i d s w e r e the same as those in w h o l e cells. The e s t i m a t i o n of each class of p h o s p h o l i p i d s b y d e n s i t o m e t r y of t h i n - l a y e r c h r o m a t o g r a m s gave the f o l l o w i n g values (values w i t h i n b r a c k e t s a r e r e l a t e d to w h o l e c e l l s ) . p h o s p h a t i d y l c h o l i n e 3:3 p e r cent (37 p e r cent) - - p h o s p h a t i d y l e t h a n o l a m i n e 29 p e r cent (31 p e r cent) phosphatidylserine and phosphatidylinositol 14 p e r cent (17 p e r cent) - - l y s o p h o s p h a t i d y l c h o l i n e 10 p e r cent (7 p e r cent) - - c a r d i o l i p i n 14 p e r cent (6 p e r cent).

K i m H e n g Ng and M.-A. Lan6elle.

102

T h e s e v a l u e s w e r e c o m p a r a b l e w i t h w h o l e cells phospholipids, the only significant difference b e i n g t h e h i g h e r a m o u n t of c a r d i o l i p i n in m i t o chondria.

c u l t i v a t e d in t h e s a m e c o n d i t i o n s . c r e a s e in t h e u n s a t u r a t e d f a t t y g r o w n w i t h s h a k i n g c o m p a r e d to p o s i t i o n of w h o l e cells g r o w n

W e n o t e d a dei n w h o l e cells fatty acid comin a f e r m e n t o r

100

60. Z

Z4o.

,i2

,...I 305

m~, 2o_

2~2

,,IhlPl ,,,]rill

100

15o

Ih

I

/i

200

Ir

I[

250

h

[I

~ 3OO

i

352

iI

fl

382

II

35o

IIEr

]1

4oo

rl

II

45o

~

,.

A

41~

8O.

.:,5

I C,~C,21,~,+C,~-LC~, 3

Iolo:;: I

~o~

Ill

~3 ~c~

Z40

1144

292

II

,,,;

mo

,I

goo

Iii

g~o

II

~,

,I

352

',1

~oo

I

550

382

II

"i

412

. . . . . . hi

4'oo

15'o

I

~00

Mass spectra of the peracel!tlaled derivatives of commercial ~-hydroxy-sphinganine (A) and $-hgdroxy-sphinganine isolated from cerebroside of Hansenula anomala (B).

FIG. 2.

-

-

Mass spectrometry was performed with a Varian MAT 311A Mass spectrometer using the direct inlet system. Ion source temperature : 110°C. Electron energy : 90 eV. Peaks below 100 were not represented.

T h e o t h e r l i p i d s w e r e c o m p o s e d of e r g o s t e r o l , g l y c e r i d e s a n d m a i n l y f r e e f a t t y acids. T h e f r e e f a t t y a c i d c o m p o s i t i o n of m i t o c h o n d r i a w a s i d e n -

(table I) ; t h i s m a y be due to a b e t t e r c o n t r o l l e d a e r a t i o n in t h e f e r m e n t o r . T h e p e r c e n t a g e of uns a t u r a t e d fatty a c i d s w a s h i g h e r in m i t o c h o n d r i a

TABLE III.

Relative percentages of fatty acids in whole cells and in mitochondria (growth in shaken flasJcs). 16:0 Whole cells Mitochondria

t i c a l to t h a t o b t a i n e d d i r e c t l y b y s a p o n i f i c a t i o n of t h e s e o r g a n i t e s . I n t a b l e III, f a t t y a c i d c o m p o s i t i o n of m i t o c h o n d r i a w a s c o m p a r e d to t h a t of c e l l s

BIOCHIMIE, 1977, 59, n ° 1.

16:1 9

17:1

18:0 8

t8:1 t t8:2 35

18:3

9

10 _

(85 p e r cent) t h a n in w h o l e cells (63 p e r cent), n e v e r t h e l e s s , w h o l e cells h a v e a h i g h e r c o n t e n t in C18:3 f a t t y acid.

Lipids of the yeast H a n s e n u l a DISCUSSION.

Hansenula anomala d i d not a p p e a r s t r i k i n g l y d i f f e r e n t f r o m Saccharomyces cerevisiae w i t h reg a r d s to its l i p i d c o m p o s i t i o n , a p a r t f r o m t h e p r e -

anomala.

103

It w a s s h o w n t h a t Hansenula anomala m i t o c h o n d r i a h a s a h i g h e r a m o u n t of u n s a t u r a t e d f a t t y a c i d s t h a n in w h o l e cells, as in Saccharomyces cerevisiae [1~. It is w o r t h w h i l e to note t h a t t h e t r i - u n s a t u r a t e d C~s:a f a t t y a c i d is a b s e n t f r o m Han-

TABLE IV.

Constituents of glycosyl ceramides of yeasts and [unyi. Fungi

Yeasts

H. anomala Long chain base

Iphytospbingosinei

Fatty acids

16:0 18:1 18:2 18:3

~-hydroxy fatty acids Sugar

s e n c e of p o l y u n s a t u r a t e d

S. cerecisiae

12'2[

1231

sphingosine !

1"2~1

!phytosphingosine ~hytosphingosine

16:0 16:1 18:0 18:1 18:2 i8:3

26 : 0

15:0 16:1 18:1 18:2

18:0

18:0

26:0

16:0

glucose

glucose

Cls f a t t y a c i d s in t h e

former. Concerning the phospholipids, phosphatidylcholine and phosphatidylethanolamine were the main components followed by phosphatidylserine and phosphatidylinositol. The main sterol was e r g o s t e r o l as in Saecharomyces eerevisiae [21~. T w o k i n d s of g l y c o l i p i d s w e r e i d e n t i f i e d : i) a galactosyl-diglyceride it) a glucosylceramide. T a b l e IV i n d i c a t e s t h e s i m p l e c o n s t i t u e n t s of c e r e b r o s i d e s of y e a s t s a n d fungi. F a t t y a c i d c o m p o s i t i o n is t h e s a m e i n Hansenula anomala a n d HansenuIa ci[errii [22], w h i l e Saccharomyces c o n t a i n s hexacosanoic and a-hydroxy hexacosanoic acids [23]. H o w e v e r , Hansenula anomala d i f f e r s f r o m Hansenula ciferrii b y the n a t u r e of t h e l o n g c h a i n base. I n Hansenula anomala t h e l o n g c h a i n b a s e is p h y t o s p h i n g o s i n e ( 4 - h y d r o x y s p h i n g a n i n e ) as in f u n g i [24] w h e r e a s in Hansenula ciferrii it is s p h i n g o s i n e ( 4 - s p h i n g e n i n e ) . It is i n t e r e s t i n g to n o t e t h a t Hansenula ciferrii w h i c h p r o d u c e s e x t r a cellular acetylated phytosphingosine contains only s p h i n g o s i n e i n its c e r e b r o s i d e s [25]. W i t h Hansenula anomala, no d e t e c t a b l e e x t r a - c e l l u l a r l i p i d s w e r e o b s e r v e d e x c e p t f a t t y a c i d s p r o b a b l y due to c e l l a u t o l y s i s . M o r e o v e r , w e d i d n o t o b s e r v e in Hansenula anomala t h e i n o s i t o l p h o s p h o r y l c e r a m i d e s as h a s b e e n d e s c r i b e d f o r Saccharomt.lces cerevisiae [26].

BIOCHIMIE, 1977, 59, n ° 1.

H. ciferrii

glucose

senula anomala m i t o c h o n d r i a , w h i l e a n i m a l c e l l s mitochondria f a t t y acids.

are

enriched

in

polyunsaturated

Acknawledgments. We are indebted to Dr. J. C. Prome for mass spectrometry analyses. R~SUML Les lipides libres de Hansenula anomala ont 6t6 analysds. Par saponification des lipides cellulaires totaux, nous avons identifi~ eomme aeides gras pr~pond6rants l'acide C~8~ et les acides en Cas:a, C~s.-_, et Cls:3. Dans les lipides des mitoehondries l'acide C~8:3 est pr6sent h l'6tat de traces. La composition en aeides gras de ehaque classe de lipides a 6t6 d6termin6e. La phosphatidylcholine et la phosphatidyl6thanolamine sont les phospholipides majeurs ; la phosphatidyls6rine et le phosphatidylinositol ont ~galement ~t~ identifi~s, ainsi que le cardiolipide. Les st6rols Ies plus abondants sont l'ergost5roi et le lanost~rol. Un st6rol ae6tyl6, de type 5thyl 24 cholest6rol, a ~t6 isol6. Deux glyeolipides, un galactosyldigtyc6ride et un glucosyle6ramide, ont 6t6 identifi6s ; d a n s le galaetosyldiglyc6ride, la proportion d'acide C~s:3 est plus 51ev6e qne dans les autres lipides. Dans le glucosyle~ramide, l'acide principal est t'aeide a-hydroxy-C18:o ; l'acide C1~ et les acides en Cash, Cas~ et C~s:.~sont 6galement presents. La base h tongue ehalne a ~t6 identifi~e la C~s phytosphingosine (4-hydroxy Cns sphinganine). Des diffSrences et ressemblances avec Saccharomyces

cerevisiae sont diseut6es.

K i m Heng Ng and M.-A. Landelle.

104 REFERENCES.

1. Weete, J. D. (1974) Fungal lipid biochemistry, dislribntion and metabolism, in Monograph in lipid research, Vol. 1, edited by D. Kritchevski, Plenum Press, New York. 2. Bartnicki-Garcia, S. (1968) Ann. Rev. Microbiol., 22, 87-108. 3. J o h n s o n , B. ~a Bro'wn, M. C. (1972~ Anlonie van Leeuiwenhoek J. Microbiol., 38, 137-144. 4. Silver, W. S. (1957) J. Bacteriol., 73, 241-246. 5. Rouser, C., Kritchevski, G. a Yamamoto, A. (1967) in Lipid chromato.qraphic analysis, vol. 1, edited by G. V. Marinetti, Marcel I)ekker Inc. New York, p. 99-16.2. 6. Stahl, E. (1969) Thin layer chromatography, Springer Verlag, Berlin. 7. Copius-Peereboom, J. W. ~a Beekes, H. W. (1964) J. Chromalo.q., 17, 99-113. 8. Sha~w, N. (1968) Biochim. Biophys. Acta, 164, 435436. 9. Dittmer, J. C. a Lester, R. L. (1964) J. Lipid Res., 5, 126-127. 10. Lisboa, B. P. (1969) in Lipid Chromatoyraphie analysis, vol. 2, edited by G. V. Marinetti, Marcel Dekker Inc. New Ym~k, p. 57-147. 11. Gaver, R. C. ,¢ Sweeley, C. C. (1965) J. Amer. Oil Chemists Soc., 42, 294-298. 12. Kates, M. (1972) in Techniques of Lipidolo.q.u, edited bv T. S. Work and E. Work,. North Holland Publi. Co. Amsterdam.

BIOCHIMIE, 1977, 59, n ° 1.

13. Kovac, L., Groot, G. S. P. & Racker, E. (1972) Biochim. Biophys. Acta, 256, 55-62. 14. Hunter, K. a Rose, A. H. (1971) in the Yeasts, Vol. 2, edited by A. H. Rose ~ J. S. Harrison, Academic Press, New York, p. 211-271. 15. Hugel, M. F., Vetter, W., Audier, H., Barbier, M. & Lederer, E. (1964) Phytochemistry, 3, 7-16. 16. Letters, R. (1968) Bull. Soe. Chim. Biol., 50, 13851393. 17. Nichols, B. W., James, A. T. & Breuer, J. (1967) Biochem. J., 104, 486-496. 18. Sastry, P. S. (1974) in Advances in lipid research, Vol. 12, Academic Press New York, p. 251-310. 19. Karlsson, K. A. (1966) Acta Chem. Scand., 20, 28842885. 20. Lan6elle, M. A., Asselineau, J. ,¢ Castelnuovo, G. (1968) Ann. Inst. Pasteur, 114, 305-312. 21. Barton, D. H. R., Kempe, U. M. ~ ~Viddowson, D. A. (1972) J. Chem. Soc. Perkin Trans., 1, 513-522. 22. Kaufman, B., Basu, S. a Roseman, S. (1971) J. Biol. Chem., 246, 4266-4~71. 23. Weinert, M., Kljaic, K. & Prostenik, M. (1973) Chem. Phys. of Lipids, 11, 83-88. 24. Weiss, B..& Stiller, R. L. (1972) Biochemistry, 11, 4552-4557. 25. Stodola, F. H., Deinema, M. H. ~ Spencer, J. F. T. (1967) Bacteriol. Rev., 31, 194-213. 26. Smith, S. W. ~ Lester, R. L. (1974) J. Biol. Chem., 249, 3395-3405.