Ecology and Metabolism of Plant Lipids - American Chemical Society

Chapter 16

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Structure-Function Relationships for Sterols in Saccharomyces cerevisiae William R. Nes Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104

Ergosterol has two kinds of function which can be distin guished from each other by differing sensitivities to the 24β-methyl moiety of the sterol's structure. The "bulk membrane" function which requires most of the sterol, is only slightly influenced either by the presence, absence, or size of the substituent at C-24 or by the chirality of C-24, while the "regulatory" function has an absolute requirement for the 24β-methyl group. In agreement with the assignment of most of the ergosterol to an architec tural role in membranes, the sterol's overall length is close to the distance from the polar to the nonpolar side of the lipid monolayer when the sterol's side chain is in the staggered conformation with C-22 positioned to the right. However, when the side chain is altered so that it has significantly different spatial character istics, S. cerevisiae is unable to utilize the resulting sterol. Deleterious changes are brought about for instance by removal of the carbon atoms on the right by direct chain shortening or by shifting them to the left rigidly with a 17(20)-double bond or conformationally by inversion of C-20. V a r i a b i l i t y of S t e r o l Structure Cholesterol, s i t o s t e r o l , and ergosterol are among the names of s t r u c t u r a l l y d i f f e r e n t s t e r o l s which are not only widely recognized but are also commonly associated with animals, plants, and fungi, respectively. This i l l u s t r a t e s an important point: The structure of s t e r o l s i s v a r i a b l e , and there i s some sort of association between structure and biology. A c t u a l l y , the s t r u c t u r a l v a r i a b i l i t y turns out to be quite large when one examines the problem i n d e t a i l (1-3). More than a hundred s t e r o l s have been found i n l i v i n g systems. They f a l l i n t o two major categories i n terms of amount. There are those which accumulate and presumably play a functional r o l e (1) and secondly those which do not accumulate and remain i n trace amounts because they are only transient precursors to the functional

0097-6156/87/0325-0252$06.00/0 © 1987 American Chemical Society

Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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compounds. On occasion there i s some overlap between the two cate gories, e s p e c i a l l y with respect to saturated-unsaturated pairs when one of them i s a precursor to the other. This l a t t e r s i t u a t i o n i s probably represented by accumulation of the pairs i s o f u c o s t e r o l / s i t o s t e r o l and s i t o s t e r o l / s t i g m a s t e r o l , but f o r the most part accumu lated or dominant s t e r o l s tend to be end-products rather than i n t e r mediates . Good examples of dominant s t e r o l s are cholesterol and i t s homologs, 24a-methylcholesterol (campesterol) and 24a-ethylcholesterol ( s i t o s t e r o l ) . These along with 243-methylcholesterol and the 22Edehydro d e r i v a t i v e of s i t o s t e r o l (stigmasterol) comprise the main s t e r o l component of most tracheophytes. However, there are many more s t e r o l s to consider than j u s t these three even among higher p l a n t s . I f we concentrate only on the s t e r o l s which accumulate, there are as many as three dozen of them which are known to be present i n t e r r e s t r i a l (i_.e_., non-marine) l i v i n g systems (Table I ) . Most of them vary Table I. Variable Features of the P r i n c i p a l Sterols of T e r r e s t r i a l L i v i n g Systems 3

5

7

Nuclear double bond: Δ or Δ Carbon a d d i t i o n to side chain: 24-CH or 24-C2H5 Configuration of substituent at C-24: α or 3 Ε-Double bond at C-22,C-23 Double bond at C-24,C-28 Double bond at C-25,C-27 (only 243-Series) 3

b

a) The features l i s t e d are added cumulatively (except where i n d i cated by "or" or "only") to 5a-cholestan-33-ol. Thus, a d d i t i o n of Δ gives c h o l e s t e r o l , or cumulative a d d i t i o n of Δ , 24a-ethyl, and Δ gives s p i n a s t e r o l . Thirty-two s t e r o l s (including cholesterol and l a t h o s t e r o l ) can be constructed from t h i s l i s t . Stanols, e s p e c i a l l y 5a-cholestan-33-ol and 24a-ethyl-5a-cholestan-33-ol ( s i t o s t a n o l ) , also occur. In addition, A > - s t e r o l s accumulate e s p e c i a l l y i n many fungi and i n some algae. The most common one i s i n the 243-methyl-22E-dehydro series (ergosterol). Others include e r g o s t e r o l s 243-ethyl analog, 7-dehydroporiferasterol. 5

7

2 2 Ε

5

7

1

b) In the 24-ethylidene series both the E- and the Z-configurations are known. by changes i n unsaturation i n r i n g Β and the side chain and by sub s t i t u t i o n at C-24. For instance, we have i s o l a t e d f i f t e e n of the possible Δ - and A - s t e r o l s from the seeds of a single plant (the squash, Cucurbita maxima) (Table II) (4,5). Although i n most plants a smaller number of s t e r o l s accumulate, i t i s i n t e r e s t i n g that as many as f i v e s t e r o l s of the squash comprising 81% of the mixture are each present at l e v e l s > 10.0% of the t o t a l . Six more s t e r o l s are present at l e v e l s from 1.0 to 6.4%. This suggests a function f o r each of the s t e r o l s . The l i s t of f u n c t i o n a l s t e r o l s i s made s t i l l longer by the a d d i t i o n of s t e r o l s from the marine environment where v a r i a t i o n can also be based on lengthening or shortening of the side chain as w e l l as on additions of methyl and cyclopropyl groups to several p o s i t i o n s i n the side chain (6-8). Changes also occur i n the skeleton of r i n g A. 5

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Table I I . The 4-Desmethylsterols of Cucurbita maxima (squash) Seeds (Garg and Nes, r e f s . k_ and 5) Sterol 24a-Ethyl-5a-cholesta-7,22E-dien-33-ol

Amount (mg/100 g) 10.55

% of Sterol 26.2

243-Ethyl-5a-cholesta-7,25(27)-dien-33-ol

6.96

17.3

243-Ethyl-5a-cholesta-7,22E,25(27)-trien-33-ol

6.59

16.4

24a-Ethyl-5a-cholest-7-en-33-ol

4.29

10.7

24Z-Ethylidene-5a-cholest-7-en-33-ol

4.19

10.4

243-Methylcholesta-5,25(27)-dien-33-ol

2.56

6.4

243-Ethylcholesta-5,25(27)-dien-33-ol

1.43

3.6

24a-Ethylcholesta-5-en-33-ol

1.20

3.0

24a-Ethylcholesta-5,22E-dien-33-ol

0.79

2.0

24Z-Ethylidenecholes t-5-en-3 3-ol

0.54

1.3

24a-Methylcholest-5-en-33-ol

0.41

1.0

24ξ-Μβ^1-5α-^οΐ63^7-θη-33-ο1

0.33

0.8

243-Ethylcholesta-5,22E,25(27)-trien-33-ol

0.31

0.7

0.07

0.1

trace

-

243-Methyl-5a-cholesta-7,25(27)-dien-33-ol 243-Methylcholest-5-en-33-ol


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255

Implications of Sterol V a r i a b i l i t y Now, the question which has motivated much of the recent work i n my laboratory, and which I should l i k e to discuss here, i s t h i s : What r e a l l y i s the reason why c e r t a i n s t e r o l s accumulate? Is one s t e r o l as good as another? Are we j u s t observing a more or less random d i s t r i b u t i o n based on chance mutations? Or i s a s e l e c t i o n operative, a mating of structure to function, suggesting, f o r instance, i n squash the existence of multiple functions f o r sterol? One way to get at t h i s problem would be to delineate what s t e r o l s a c t u a l l y do, to f i n d a way to quantitate t h e i r value, and then to determine the extent to which deviations i n structure a f f e c t function. I t was t h i s course that was s e t t l e d on, but the question which arose immediately was: What l i v i n g system should be used experimentally? Early Work With Yeast I t so happens that at the time I was f i r s t thinking seriously about t h i s problem (the early to mid 1970s) the l i t e r a t u r e already revealed an apparent s o l u t i o n . The primary s t e r o l (ergosterol) of the fungus, Saccharomyces cerevisiae (the common yeast of bakers and brewers), has a 243-methyl group, as we have confirmed using ^-NMR (9), and the way t h i s group i s introduced b i o s y n t h e t i c a l l y i s f a i r l y complicated (l.,2_,lû). I t requires preservation (rather than reduction) of the terminal double bond of squalene (Δ of s t e r o l s ) so that trans fer of a Ci-group can occur to i t from S-adenosylmethionine with elimination of a proton from the incoming Ci-group (C-28) and 1,2hydride migration (C-24 to C-25). This gives a 24-methylene group which then undergoes a stereospecific reduction to y i e l d the 243methyl group. Other a l t e r n a t i v e s could have occurred, e.g., elimina t i o n from C-27 instead of C-28, reduction to the a- rather than 3methyl, or a second Cχ-transfer to the 24-methylene group might have occurred. A l l of the biosynthetic steps which a c t u a l l y produce the 243methyl group are w e l l documented (1,2,10), and much more than a single point mutation i n fungal evolution would be necessary to account f o r t h i s involved sequence of reactions. Despite t h i s , w i l d type S_. cerevisiae seemed, according to the l i t e r a t u r e (11-13) » to have l i t t l e use f o r the 243-methyl group. Ergosterol could be r e placed, f o r instance, by cholesterol which has no substituent at C-24 or by s i t o s t e r o l which has an ethyl group at C-24 and no serious loss of function was observable. In p a r t i c u l a r , growth s t i l l occurred with cholesterol or s i t o s t e r o l and almost at the same rate as with ergosterol. This strongly suggested either that the manner i n which the experiments were carried out l e f t the functional question more or less moot or that biosynthesis and function (and by implication e v o l u t i o n a r i l y surviving mutants and function) are not c l o s e l y correlated. Yeast thus presented an i n t r i g u i n g challenge. I t was chosen f o r study both f o r t h i s reason and because i t has some c h a r a c t e r i s t i c s which make i t very useful as an experimental t o o l . 24



256

U s e f u l c h a r a c t e r i s t i c s of y e a s t . Y e a s t , jL.e_., S_. c e r e v i s i a e , i s , f i r s t o f a l l , e u k a r y o t i c and a t l e a s t t o some degree s h o u l d be r e p r e s e n t a t i v e of h i g h e r forms o f l i f e i n g e n e r a l . S e c o n d l y , i t i s n a t u r a l l y s i n g l e c e l l e d and can be grown i n a l i q u i d c u l t u r e . I f needed, l a r g e q u a n t i t i e s (100 g) can be o b t a i n e d . A g r e a t d e a l i s known about t h i s m i c r o o r g a n i s m , which i s not p a t h o g e n i c , and i t can be e a s i l y o b t a i n e d i n an a x e n i c c o n d i t i o n , f o r i n s t a n c e from the American Type C u l t u r e C o l l e c t i o n . Perhaps o f most importance i s y e a s t ' s u n u s u a l a b i l i t y among e u k a r y o t e s t o be a b l e to l i v e a n a e r o b i c a l l y and to d e r i v e i t s ATP by nonrespiratory glycolysis. Under these c o n d i t i o n s , as seen under the e l e c t r o n m i c r o s c o p e , the m i t o c h o n d r i a change m o r p h o l o g i c a l l y t o what a r e c a l l e d p r o m i t o c h o n d r i a ( 1 4 ) , and the c e l l s become a u x o t r o p h i c f o r s t e r o l (11) and u n s a t u r a t e d f a t t y a c i d such as o l e a t e ( 1 5 ) . Aerob i c a l l y y e a s t d e r i v e s i t s s t e r o l and u n s a t u r a t e s by b i o s y n t h e s i s , and f o r b o t h types o f compound t h e r e a r e b i o s y n t h e t i c s t e p s i n v o l v i n g mixed f u n c t i o n o x i d a s e s , , f o r the f o r m a t i o n o f s q u a l e n e o x i d e (1,2). T h i s means t h a t m o l e c u l a r oxygen i s r e q u i r e d , and, when a v a i l a b i l i t y o f oxygen i s d e n i e d , i t i s o b v i o u s t h a t n e i t h e r s t e r o l n o r u n s a t u r a t e s can be formed. By o p e r a t i n g a n a e r o b i c a l l y we can t h e r e f o r e d i c t a t e what s t e r o l i s i n the c e l l s s i m p l y by a d d i n g the s t e r o l o f our c h o i c e to the medium. Andreasen and S t i e r (11) were the f i r s t to demonstrate t h a t t h i s c o u l d be done and they were the f i r s t a l s o to show t h a t w i t h o u t s t e r o l (11) and u n s a t u r a t e d f a t t y a c i d (15) y e a s t w i l l not grow. T h i s was an e l e g a n t d e m o n s t r a t i o n that these l i p i d s p l a y a v i t a l r o l e . The

F u n c t i o n of

Sterols

There i s e x t e n s i v e e v i d e n c e , which I have reviewed e a r l i e r (1,16)» to i n d i c a t e t h a t the p r i n c i p a l r o l e o f s t e r o l s throughout n a t u r e i s t o a c t as a r c h i t e c t u r a l components o f some though n o t n e c e s s a r i l y a l l membranes. The plasma membrane i s f r e q u e n t l y the p r i n c i p a l r e s i d e n c e of the s t e r o l ( 12,17., 1 8 ) . E u k a r y o t i c as w e l l as most p r o k a r y o t i c membranes a r e b i l a y e r s o f p h o s p h o l i p i d and p r o t e i n . Each monolayer, which i s about 2.1 nm t h i c k (19,20), i s b e l i e v e d to c o n t a i n the s t e r o l i n a nonhomogeneous d i s t r i b u t i o n , and a t l e a s t i n some c a s e s s t e r o l can move between the monolayers. This process i s c a l l e d " f l i p - f l o p " . S t e r o l has been found b o t h i n t h e m i t o c h o n d r i a (21) and the plasma membrane o f . c e r e v i s i a e (13,18), and the a b i l i t y t o s u p p o r t the growth o f a n a e r o b i c y e a s t presumably i s a s s o c i a t e d w i t h i t s membranous function. I t i s n o t f u l l y c l e a r how s t e r o l i s i m p o r t a n t a r c h i t e c t u r a l l y , but much e v i d e n c e p o i n t s t o a m o d u l a t i o n o f the p h y s i c a l p r o p e r t i e s of the membrane, e s p e c i a l l y o f the t r a n s i t i o n from the g e l t o the l i q u i d c r y s t a l s t a t e of the p h o s p h o l i p i d t h e r e b y c o n t r o l l i n g the r i g i d i t y , u s u a l l y e x p r e s s e d i n the r e v e r s e as f l u i d i t y , o f the membrane. T h i s has been c a l l e d the " b u l k membrane f u n c t i o n " (22) o f s t e r o l to d i s t i n g u i s h i t from a n o t h e r f u n c t i o n o r f u n c t i o n s which require(s) less sterol. The n a t u r e o f the l a t t e r f u n c t i o n ( s ) i s more e l u s i v e , a l t h o u g h we (.23,24) and o t h e r s (2*18,25.) have p r e s e n t e d good e v i d e n c e f o r i t s e x i s t e n c e i n y e a s t , and a s i m i l a r phenomenon e x i s t s a l s o i n p r o k a r y o t i c mycoplasmas (22,26). We have c a l l e d the f u n c t i o n


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requiring less s t e r o l "regulatory" (23), while Parks and h i s group use the word "sparking" (25), and Bloch and h i s associates apply the term " s y n e r g i s t i c " (18). The Experimental System We discovered very early that the growing of yeast anaerobically, i.e_., r e a l l y anaerobically, i s quite d i f f i c u l t . Or, to put i t another way, i t i s f a r from easy to remove enough oxygen to prevent at l e a s t some growth from occurring on a synthetic medium containing no s t e r o l . We achieved success by c a r e f u l l y sealing i n l e t s into our growth chamber with p a r a f f i n wax and p r i o r to i n o c u l a t i o n by using a prolonged f l u s h of e l e c t r o n i c grade nitrogen (< 0.2 ppm of oxygen) which had been passed through a s o l u t i o n of chromous c h l o r i d e to reduce r e s i d u a l oxygen to the water stage. Under these conditions i t became possible to take a log phase inoculum of yeast from a continu ous c u l t u r e and f i n d i t would not grow (< 10 from 1 0 c e l l s ) i n the presence of Tween-80 (as a source of unsaturated f a t t y a c i d ) . At the same time, growth ensued w e l l (10 c e l l s / m l from 10 c e l l s / m l i n 3 days) when ergosterol (5 mg/liter of medium) had been emulsified i n the Tween-80 and then dispersed i n the medium. Our i n i t i a l plan was to make a systematic i n v e s t i g a t i o n of the s t r u c t u r a l features of ergosterol by replacing the ergosterol with other s t e r o l s which either lacked a p a r t i c u l a r feature, e_.£., the Δ -unsaturation, or had some other i n t e r e s t i n g aspect to them, e.^., a l t e r e d stereochemistry. The c o n t r i b u t i o n of the feature was then to be assessed by evaluation of the number of c e l l s a f t e r three days. The c e l l population could be measured both v i s u a l l y and by means of a 16-channel Coulter counter which also permits determina t i o n of the d i s t r i b u t i o n of c e l l sizes and volumes. In a d d i t i o n , e x t r a c t i o n of the c e l l s , or s u b c e l l u l a r f r a c t i o n s , would permit us to examine the amount, d i s t r i b u t i o n , and metabolism of the added s t e r o l . This experimental design (27,28) proved adequate to show a great deal about what i s and what i s not important about the s t e r o l mole cule. However, some growth and i t should be added v a r i a b l e growth i n the presence of c h o l e s t e r o l occurred. By examining the s t e r o l d i s t r i b u t i o n i n the n e u t r a l l i p i d f r a c t i o n of the yeast grown i n the manner described we found that there were s t i l l s u f f i c i e n t traces of oxygen l e f t i n the medium to allow very small but s i g n i f i c a n t amounts of endogenous s t e r o l synthesis to occur. We could i d e n t i f y (28) not only ergosterol but also l a n o s t e r o l , zymosterol, 24-methylene-7-dehydrocholesterol, and 22-dihydroergosterol a l l of which are i n t e r mediates i n the ergosterol pathway (1). Such biosynthesis presumably accounts f o r growth on c h o l e s t e r o l i n the experiments of Hossack and Rose (13) who reported that ergosterol was also present. Ergosterol produced b i o s y n t h e t i c a l l y of course complicates the i n t e r p r e t a t i o n of the r e s u l t s obtained with other s t e r o l s . In order to reduce biosynthesis to an i n s i g n i f i c a n t l e v e l i n the oxygen-deprived yeast we added 2,3-iminosqualene i n our more recent experiments (29). This compound i s a competitive i n h i b i t o r of 2,3-oxidosqualene cyclase (30), and when i t was incorporated into the medium along with c h o l e s t e r o l no growth then occurred although on replacement of c h o l e s t e r o l with ergosterol growth proceeded w e l l . Thus, stepwise we were able to achieve a no-growth condition 6

8

5

5

7


258


without s t e r o l by using prolonged flushing with highly p u r i f i e d nitrogen and then by adding 2,3-iminosqualene we also achieved a nogrowth condition i n the presence of c h o l e s t e r o l . One f i n a l experi mental thing must be mentioned. If we want to have both ergosterol and some other s t e r o l present, i t i s obvious we could add both, and we have done j u s t that. Such a method has the advantage of allowing various r e l a t i v e amounts to be added, but we also found f o r some pur poses generation of ergosterol i n s i t u by the addition of a i r was a simple a l t e r n a t i v e . With 10 ml of a i r added to the headspace (ca. 100 ml) of our growth chamber i n the presence of the iminosqualene but with no s t e r o l we (23) obtained 24 χ 10 c e l l s / m l which i s a quarter or a f i f t h of what we obtained with added ergosterol. The e f f e c t of added s t e r o l could then be determined by measuring the increase i n c e l l count. 6

The P o s i t i o n i n g of C-21 and C-22 i n Space I t has been possible to c o r r e l a t e several physical properties with the configuration at C-20 of s t e r o l s . The configuration at C-20 of cholesterol (Figure 1) i s known to be R from degradation of the analog and c o r r e l a t i o n of the product containing C-20 with D-(+)glyceraldehyde (30,31). Cholesterol i s therefore a primary standard. We prepared 20-epicholesterol and both for i t and f o r c h o l e s t e r o l we determined H- and C-NMR spectra, the melting points, the o p t i c a l r o t a t i o n s , and the chromatographic rates of movement (32-34). In some cases t h i s was also done f o r a series of other s t e r o l s (33-35) such as 20R- and 20S-halosterol (35) which i s the C 6-analog of cholesterol which has one less CH2~group i n the side chain and occurs i n the marine environment (1) . There were several consistent s h i f t s . The 20R-isomers moved slower i n both adsorption and g a s - l i q u i d chromatography, showed a downfield s h i f t i n the C-21 proton NMRs i g n a l , a greater separation of the terminal gem-dimethyl proton s i g n a l s , and a more p o s i t i v e (actually less negative), o p t i c a l r o t a t i o n at the D-line of sodium. There was also a tendency for the S-isomer to have a lower melting point, although t h i s was not so f o r the epimeric c h o l e s t e r o l p a i r . This information along with x-ray d i f f r a c t i o n permitted a survey of the C-20 configuration of natural sterols, , r e f . 32_. With one w e l l documented but curious excep t i o n (36) along with one dubious exception lacking strong p h y s i c a l evidence (37), a l l s t e r o l s i n both the marine and t e r r e s t r i a l environments have the 20R-configuration. Why should t h i s be so? I t i s not necessary from a biosynthetic point of view, since reasonable mechanisms are possible to give both the R- and the S-epimers (32). I believe the answer to why the R-configuration at C-20 i s chosen n a t u r a l l y l i e s i n the mating of the shape and s i z e of the s t e r o l to the dimensions of the monolayer i n the l i p i d l e a f l e t of membranes. In order to keep the smallest group on C-20 adjacent to C-18 which l i e s on the front or 3-face of the nucleus, r o t a t i o n of C-20 about the 17(20)-bond should occur preferably so that the 20-H-atom i s i n front. This means that i n the R-series C-22 i s on the r i g h t ("right-handed") and C-21 on the l e f t ("left-handed") when the s t e r o l i s viewed i n the usual way, I.e., with C-3 to the l e f t and C-18 and C-19 toward the observer. X-ray data f o r many natural s t e r o l s corroborates not only the R-configuration but the existence 1

13

2


16. NES


20R

Ε

20S

_ Ι7(20) Δ

Ζ-Δ

, 7 ( 2 0 )

Figure 1 . Stereochemistry of sterols at C - 2 0 . Structures i l l u s t r a t e ring-D and the cholesterol side chain. The carbon bearing the Η-atom shown i s C - 2 0 . In halosterol the isohexyl moiety [(CH )3-CH-(CH ) ] on C - 2 0 i s replaced by an isopentyl group [ (CH )2 -CH-(CH3)2 ] . Other replacements were made with n-alkyl groups. 2

3

2

2


259

260


of the side chain i n the right-handed conformation. We have also been able to show from x-ray d i f f r a c t i o n with the t e t r a c y c l i c t r i t e r penoids euphol and t i r u c a l l o l that the o r i e n t a t i o n of C-22 r e a l l y i s dependent on the configuration of C-20 and the presence of C-18 (38). The right-handed conformation of s t e r o l s i s probably chosen i n an evolutionary sense because with C-22 on the r i g h t and the side chain i n the staggered conformation the long dimension of c h o l e s t e r o l (and other s t e r o l s with the same chain length) coincides almost exactly with that of the thickness of the monolayer. However, the s t e r o l length i s much shorter than the l a t t e r when C-22 i s on the l e f t as we think i t would be i n the 20S-epimer. Direct evidence f o r the above i n t e r p r e t a t i o n was obtained as follows. When incubated with our oxygen-deprived yeast i n the absence of 2,3-iminosqualene, c h o l e s t e r o l , i t w i l l be r e c a l l e d , permitted some growth. However, when c h o l e s t e r o l was replaced by 20e p i c h o l e s t e r o l no growth at a l l occurred (28). S i m i l a r l y , while cholesterol or h a l o s t e r o l i s metabolized by the protozoan Tetrahymena pyriformis, the 20-epi-analogs are not Ç39,40) and c h o l e s t e r o l but not i t s epimer at C-20 induced oospores i n the fungus Phytophthora cactorum (41). In order to show that t h i s negative e f f e c t of inversion at C-20 correlates with the p o s i t i o n i n g of C-22 on the l e f t , we synthesized E- and Z-17(20)-dehydrocholesterol (42). In the E-isomer C-22 i s r i g i d l y oriented to the r i g h t , and to the l e f t i n the Z-isomer. The Ε-isomer permitted some growth of yeast, but as expected the Z-isomer did not (28). Again i n agreement with r e s u l t s with the yeast system, only the Ε-isomer was metabolized by T. pyriformis (39), the Z-isomer being recovered unchanged from the c e l l s (39). S i m i l a r l y , only the Ε-isomer induced formation of oospores i n P_. cactorum (43). The Length of the Side Chain A d i r e c t test of the importance of the long dimension of the s t e r o l molecule was obtained by our synthesizing androst-5-en-33-ol, which i s a Δ - C i 9 - s t e r o l with no side chain at a l l , and by also making a v a r i e t y of A - s t e r o l s i n which the isohexyl group (C-22 to C-27) on C-20 of c h o l e s t e r o l was replaced e i t h e r with hydrogen (pregn-5-en33-ol) or with longer or shorter arrays of carbon atoms than occur i n c h o l e s t e r o l , ±.e., a series of 20R-n-alkylpregn-5-en-33-ols (34). The epimeric 20S-sterols were also prepared and as already mentioned 20R- and 20S-halosterol were made (35). We also prepared 21-isopentylcholesterol (44) which we c a l l e d "wingsterol , since i t has an isohexyl group on both sides of C-20. Neither the C i g - s t e r o l nor wingsterol permitted growth of yeast (44). The C i g - s t e r o l also pre vented growth of T. pyriformis (44) as w e l l as of the larvae of the insect H e l i o t h i s zea (44), although H. zea larvae grow w e l l on c h o l e s t e r o l . In work which has only been p a r t l y published (45) we also found that as the length of the s t e r o l side chain i s lengthened growth of yeast r i s e s to a maximum at f i v e C-atoms on C-20, , with 20R-n-pentylpregn-5-en-33-ol and f e l l v i r t u a l l y to zero with more than seven C-atoms on C-20. The 20S-analogs were a l l i n a c t i v e . The influence of length of the side chain was also assessed i n oospore induction with cactorum (46), metabolism i n T_. pyriformis (47), e s t e r i f i c a t i o n i n T. pyriformis (44), i n h i b i t i o n of t e t r a 5

5

11


16.

NES


261

hymanol production by T. pyriformis (44), and e s t e r i f i c a t i o n with mammalian ACAT (44). In a l l cases maximal a c t i v i t y occurred either with the 20R-n-pentyl or 20R-n-hexyl derivative of pregn-5-en-33-ol agreeing w e l l with the yeast r e s u l t s . The observed length for maximal a c t i v i t y (5 or 6 C-atoms on C-20) correlates w e l l with the length of natural s t e r o l s (8). Importance of the 243-Methyl Group i n Yeast While the stereochemistry at C-20 and the length of the side chain should and a c t u a l l y do appear to have general s i g n i f i c a n c e i n terms of the s i z e of the l i p i d l e a f l e t i n membranes, i t i s not as easy a p r i o r i to assign a r o l e to the 243-methyl group. This i s e s p e c i a l l y d i f f i c u l t , since from data on occurrence (1) the methyl group c e r t a i n l y does not seem to have general importance. Geometrically i t should thicken the side chain and probably make i t more r i g i d . This sort of phenomenon might be interpreted to imply that i t might protect a membrane against too high a f l u i d i t y at elevated temperatures. However, yeast, with an optimum temperature of 28-36° and a maximum temperature of 40-42°, does not grow very w e l l at temperatures much above those which are normal f o r mammals which have c h o l e s t e r o l , and there i s no c o r r e l a t i o n between the optimum temperature f o r a fungus and the s t e r o l i t makes. Nevertheless, the early work of ourselves (27,28) and of Andreasen and S t i e r (11) showed that ergosterol i s better f o r J5. c e r e v i s i a e than i s c h o l e s t e r o l . More recently Ramgopal and Bloch (18) concurred i n t h i s using the GL7 mutant which i s auxotrophic f o r s t e r o l a e r o b i c a l l y having l o s t the oxidosqualene cyclase. We went on to show that t h i s difference i n effectiveness i s not due to the A -bond, since neither l a t h o s t e r o l nor 7-dehydrocholesterol were any better than cholesterol i n supporting the growth of our semi-anaerobic w i l d type yeast (28). On the other hand, 243-methyl-22E-dehydrocholesterol (brassicasterol) was v i r t u a l l y as e f f e c t i v e as was e r g o s t e r o l , and when the double bond was removed to give 243-methylcholesterol the compound was s t i l l 75% as a c t i v e as ergosterol (28), although under the same conditions cholesterol was only 23% as a c t i v e . The mystery deepened when we showed recently (48) that growth on cholesterol or on other s t e r o l s lacking a 243-methyl group (Table I I I ) can be brought to zero by using 2,3-iminosqualene even though the yeast retains i t s a b i l i t y to grow w e l l on ergosterol, b r a s s i c a s t e r o l , 243-methylcholesterol, and desmosterol which we were able to show was converted i n high y i e l d i n the yeast to 243-methylcholest e r o l (48). I n t e r e s t i n g l y , neither 24a-methylcholesterol nor 243e t h y l c h o l e s t e r o l supported growth i n d i c a t i n g that both the exact s i z e and configuration of the substituent at C-24 are important. Thus, the 243-methyl group i t s e l f i s e s s e n t i a l and the idea that ergosterol i s j u s t better than cholesterol i s not quite r i g h t . In some v i t a l way cholesterol can not replace ergosterol at a l l . Yet, i t appeared that i n some way i t could, because we (27,_28) and others (11,13) had gotten yeast to grow i n the absence of 2,3-iminosqualene to v a r i a b l e degrees on c h o l e s t e r o l . Since i n our system without the i n h i b i t o r there was some ergosterol present, though not enough by i t s e l f to l e t growth occur (28), i t appeared that i t must be this small amount of endogenous s t e r o l which was responsible f o r the a b i l i t y of choies7



24a-Alkylsterols 24a-Methylcholesterol 24a-Ethylcholesterol

243-Alkylsterols 243-Methylcholesterol 243-Methyl-22E-dehydrocholesterol 243-Methyl-7,22E-bisdehydrocholesterol (ergosterol) 24 3-Ethylcholes t e r o l

C.

D.

b.

0.2 0.2

0.1 0.2 0.5 99 0.1 0.2 0.1

0.1 0.2

air

95 108 114 0.2

No

a

103

107 89

80 16

-85

112 97 100

21 61

Added a i r

b

C e l l Count ( M i l l i o n s of Cells/ml)

After 3 days, average of 3 experiments. C e l l s adapted to oxygen-deprivation were used. 10 ml of a i r added to cultures which had not grown a f t e r i n i t i a l three days and c e l l count obtained a f t e r another 3 days. Without s t e r o l , the c e l l count was 24 m i l l i o n . Data are averages f o r 3 experiments.

24-Desalkylsterols Cholesterol Lathosterol 7-Dehydrocholes t e r o l 24-Dehydrocholesterol (desmosterol) 5a-Cholestanol E-17(20)-Dehydrocholesterol Z-17(20)-Dehydrocholesterol

B.

a.

Sterols lacking an H0-group Choiest-5-ene 5a-choles tan-3-one

Sterol (5 mg/liter)

Growth Response of Oxygen-Deprived S_. cerevisiae to Sterols with 2,3-Iminosqualene (50 μΜ) Present (From r e f s . 23, 2A> and 48)

A.

Table I I I .

16.

NES

Structure-Function Relationships for Sterob

263

t e r o l to be a c t i v e . This i n turn suggested the existence of a dual (or perhaps multiple) r o l e f o r s t e r o l i n yeast. In one r o l e choles t e r o l could replace ergosterol, but i n the other, r e q u i r i n g less s t e r o l , c h o l e s t e r o l must be i n e f f e c t u a l . Evidence f o r a Dual Role of S t e r o l The supposition discussed above that 243-methylsterol i n an amount too small to support growth by i t s e l f would permit s t e r o l s lacking the methyl group to induce growth was demonstrated d i r e c t l y i n two ways. In the f i r s t place, i n the presence of 2,3-iminosqualene a f t e r three days following i n o c u l a t i o n , cultures containing the s t e r o l s l i s t e d i n Table I I I which had not grown were oxygenated with 1 0 ml of a i r to generate ergosterol i n s i t u 0 2 3 , 2 4 ) . A f t e r an a d d i t i o n a l three days the only cultures which had not now grown more than the control (lacking s t e r o l ) were the ones which contained cholest-5-ene and Z - 1 7 ( 2 0 ) - d e h y d r o c h o l e s t e r o l ( 2 3 , 2 4 ) . Except f o r 5 a - c h o l e s t a n - 3 one which was reduced to 5 a - c h o l e s t a n - 3 3 - o l ( 4 9 ) , none of the s t e r o l s was metabolized, or more p r e c i s e l y a l l except the ketone were recovered unchanged i n s u b s t a n t i a l quantity, and no metabolites were observed ( 2 3 ) . The growth data a f t e r a d d i t i o n of a i r (Table I I I ) show that quite a v a r i e t y of s t e r o l s w i l l support growth i f a small amount of 2 4 3 - m e t h y l s t e r o l i s present. This kind of e f f e c t was f i r s t found i n insects by Clark and Bloch ( 5 0 ) f o r c h o l e s t e r o l and choles tanol, and cholestanol was said to "spare (partly replace) the c h o l e s t e r o l . We ( 2 3 ) have adopted t h i s term, 1.e., cholesterol, for instance, w i l l spare ergosterol. Table I I I reveals that sparing a c t i v i t y occurs with s t e r o l s not only lacking a substituent at C - 2 4 but also with those having 24a-methyl, 2 4 a - e t h y l , or 2 4 3 - e t h y l groups. Unsaturation i n r i n g Β i s unnecessary, as i s also the case i n insects ( 5 0 ) , e i t h e r Δ -, Δ -, or Δ > -unsaturation can be present and C - 2 2 must be oriented to the r i g h t i n the usual view of the molecule. We have also constructed growth curves using mixtures of choles t e r o l and ergosterol i n various r a t i o s from 0 - 1 0 0 % of each ( 2 3 ) instead of generating ergosterol with a i r . The r e s u l t s are shown i n Table IV i n an abbreviated form. I t w i l l be seen that neither a l o t (5 mg/1) of c h o l e s t e r o l alone nor a l i t t l e (< 0 . 5 mg/1) ergosterol alone was supportive of growth, yet maximal growth could be obtained when the s t e r o l s were combined at these l e v e l s , e^g.. » 4 . 5 mg/1 of c h o l e s t e r o l and 0 . 5 0 mg/1 of ergosterol. Since i t takes 1 . 5 0 mg/1 of ergosterol alone f o r the yeast to grow maximally, c h o l e s t e r o l i s c l e a r l y sparing most (about two-thirds) but not a l l of the ergos t e r o l . Under these circumstances, the c h o l e s t e r o l i s presumably f u l f i l l i n g the bulk membrane function. Since the ergosterol (or other 243-methylsterol) i s required i n only a small amount, so long as another s t e r o l i s present i n large amount, we think 243-methylsterols play some regulatory function. This may have something to do with c o n t r o l l i n g r a t i o s of saturated and unsaturated f a t t y acid ( 5 0 , 5 1 ) , although the exact nature of the function needs more exploration. 11

5

7

5

7


264

ECOLOGY AND METABOLISM OF PLANT LIPIDS Table IV. Sparing A c t i v i t y of Cholesterol for Ergosterol i n _S. cerevisiae with 50 μΜ 2,3-Iminosqualene Present (From r e f . 23) C e l l Count ( M i l l i o n s of Cells/ml)

S t e r o l Cone, (mg/1) Ergosterol Cholesterol o.oo

a

5.00

0.05

a

4.95

o.io

a

i.oo

12

a

4.80

48

4.70

71

C

4.50

107

0.50 a) b) c) d)

0.2

4.90

b

0.20 0.30

0.2

d

108

4.00

This This This This

cone. cone. cone. cone.

without without without without

cholesterol cholesterol cholesterol cholesterol

gave gave gave gave

6

0.2 χ 10 0.3 χ 10 0.8 χ 10 54.( 3 χ 10 6 6

6

cells/ml. cells/ml. cells/ml. cells/ml.

Use of mutants. Ramgopal and Bloch (18) have come to conclusions s i m i l a r to ours using the yeast mutant GL7, but for reasons not r e a l l y clear neither they nor others (25,53) have been able to obtain a no-growth-condition with GL7 on c h o l e s t e r o l . However, Ramgopal and Bloch (18) f i n d that the mutant grows better on ergos t e r o l than on c h o l e s t e r o l , and a small amount of ergosterol substan t i a l l y enhances growth on cholesterol which these authors refer to as a s y n e r g i s t i c e f f e c t . Bloch and hie associates have also found a related synergism f o r cholesterol and lanosterol i n mycoplasmas (26). S i m i l a r l y , with GL7 and a mutant (FY3) with much the same defects Parks and h i s associates (25) f i n d ergosterol i s necessary i n small amount i n order for 5a-cholestan-33-ol to support growth. However, Parks and h i s group (25,54) interpret the r e s u l t s only to mean a A -grouping i s being supplied by ergosterol for what they c a l l the sparking function, and they f e e l a 243-methyl group i s not necessary (54) i n view of the a b i l i t y of cholesterol alone to permit growth of GL7 and FY3. The reason for t h i s growth remains unclear. Since the mutants are grown a e r o b i c a l l y and require much less s t e r o l than our w i l d type, e_.£., 0.3 vs. 1.5 mg/1 of ergosterol to give maximum growth for GL7 (18) and w i l d type (23), respectively, s l i g h t l e a k i ness through the block of s t e r o l biosynthesis i n the mutants might account for the apparent lack of an absolute requirement for a 243methylsterol. That i s , a minute amount of regulatory s t e r o l may acually be biosynthesized. I t of course i s also possible that the mutants a c t u a l l y do have a less stringent requirement for 243-methylsterol. 5

S t r u c t u r a l Effects on Feedback I n h i b i t i o n of Sterol

Biosynthesis

Another way to gain evidence on whether the structure of ergosterol i s e s p e c i a l l y important to yeast, would be to measure the e f f e c t of ergosterol and other s t e r o l s on the a b i l i t y of the organism to


16.

NES


265

biosynthesize s t e r o l s . Under oxygen-deprived conditions squalene accumulates i n yeast, because i t can not proceed on to squalene oxide. The squalene l e v e l , therefore, should be a measure of the flow of carbon into the s t e r o l pathway. Ergosterol i s already known by other parameters, to depress s t e r o l biosynthesis i n yeast, and i n agreement we found squalene accumulation was increasingly depressed as we raised the concentration of ergosterol i n the medium (55). However, c h o l e s t e r o l was much less e f f e c t i v e (55). Since c h o l e s t e r o l might have been less e f f e c t i v e l y absorbed, we also measured uptake (and e s t e r i f i c a t i o n ) . Based on the amount of free s t e r o l i n the c e l l s , ergosterol was about four times as e f f e c t i v e as c h o l e s t e r o l i n depressing squalene accumulation (55) . This agrees with the supposit i o n that the choices made i n the biosynthetic process are determined by a mating of the 243-methyl structure with function (23,48). Sterol Uptake Since the concentration and d i s t r i b u t i o n of s t e r o l w i t h i n the c e l l i s important to an understanding of function, we have been measuring among other things the r e l a t i o n s h i p of structure to uptake from the medium. Many d i f f e r e n t steroids were absorbed (56). They included not only the 33-hydroxysterols i n Table I I I which support growth with added a i r but also steroids such as 5a-cholestan-3-one which do not have an HO-group at C-3 (49). E s p e c i a l l y i n t e r e s t i n g was the observation that when a small amount of growth was induced by addit i o n of a i r (10 ml), the s t e r o l s with the wrong, left-handed o r i e n t a t i o n of C-22 (20-epicholesterol and Z-17(20)-dehydrocholesterol) were not absorbed a t a l l (56) even though s t e r o l s with the natural r i g h t handed o r i e n t a t i o n (cholesterol and E-17(20)-dehydrocholesterol were absorbed w e l l under the same conditions (23,56)). This means that the reason why the left-handed s t e r o l s f a i l e d to support growth (23, 28) was that they a c t u a l l y never even entered the c e l l s . This i n turn implies the existence of a s t r u c t u r a l l y s p e c i f i c gate f o r absorption, and t h i s gate seems to be s t e r e o c h e m i c a l ^ r e l a t e d to function, since we can r e l a t e r i g h t - and left-handedness to membrane geometry (see e a r l i e r ) . The existence of a gate i s also manifested i n the f a c t that yeast c e l l s can turn absorption on and o f f . For instance, while ergosterol i s r e a d i l y absorbed anaerobically, i t i s not absorbed a e r o b i c a l l y (56,57). This means that absorption can simply not be a passive entry of s t e r o l into the plasma membrane (56). A c a r r i e r protein may conceivably be involved (56). Such a protein i s w e l l known to e x i s t i n mammals (58) and evidence also e x i s t s i n yeast (58). The t r i g g e r i n g of the entry mechanism by oxygen-deprivation i s believed to occur v i a e l i m i n a t i o n of an oxygenrequiring step i n heme biosynthesis (59). The loss of heme then sets i n motion events leading to s t e r o l auxotrophy (59). Acknowledgmen t s I should l i k e to thank the National I n s t i t u t e s of Health f o r support of t h i s work through Research Grant AM-12172.


266


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Nes, W. R., McKean, M. L. "The Biochemistry of Steroids and Other Isopentenoids"; University Park Press: Baltimore, 1977. Nes, W. R. Adv. Lipid Res. 1977, 15, 233. Goad, L. J.; Goodwin, T. W. In "Progress in Phytochemistry"; Reinhold, L.; Liwschitz, Y., Eds.; Interscience: New York, 1972; Vol. III, p. 113. Garg, V. K.; Nes, W. R. Phytochemistry 1984, 23, 2919. Garg, V. K.; Nes, W. R. Phytochemistry 1984, 23, 2925. Djerassi, C. Pure and Appl. Chem. 1981, 53, 873. Barbier, M. In "Marine Natural Products"; Scheuer, P. J., Ed.; Academic Press: New York, 1981; Vol. IV, p. 147. Nes, W. R. In "Isopentenoids in Plants: Biochemistry and Function"; Nes, W. D.; Fuller, G.; Tsai, L-S., Eds.; Marcel Dekker, Inc.: New York, 1984; p. 325. Adler, J. H.; Young, M.; Nes, W. R. Lipids 1977, 12, 364. Lederer, E. Quart Rev. 1969, 23, 453. Andreasen, Α. Α.; Stier, T. J. B. J. Cell Comp. Physiol. 1953, 41, 23. Proudlock, J. W.; Wheeldon, L. W.; Jollow, D. J.; Linnane, A. W. Biochim. Biophys. Acta 1968, 152, 434. Hossack, J. Α.; Rose, A. J. J. Bacteriol. 1976, 127, 67. Morpurgo, G.; Serlupi-Crescenzi, G.; Tecce, G.; Valente, F.; Venettacci, D. Nature (London) 1964, 201, 897. Andreasen, Α. Α.; Stier, T. J. B. J. Cell. Comp. Physiol. 1954, 43, 271. Nes, W. R. Lipids 1974, 9, 596. Lange, Y.; Ramos, Β. V. J. Biol. Chem. 1983, 258, 15130. Ramgopal, M.; Bloch, K. Proc. Nat'l Acad. Sci., U.S.A. 1983, 80, 712. Huang, C.; Mason, J. T. Proc. Nat'l Acad. Sci., U.S.A. 1978, 75, 308. Dalton, A. J.; Haguenau, F. "The Membranes"; Academic Press: New York, 1968. Bottema, C. K.; Parks, L. W. Lipids 1980, 15, 987. Dahl, J. S.; Dahl, C. E.; Bloch, K. J. Biol. Chem. 1981, 256, 87. Pinto, W. J.; Lozano, R.; Sekula, B. C.; Nes, W. R. Biochem. Biophys. Res. Commun. 1983, 112, 47. Pinto, W. J. Ph.D. Dissertation, Drexel University, Philadel phia, 1982. Rodriquez, R. J.; Taylor, F. R.; Parks, L. W. Biochem. Biophys. Res. Commun. 1982, 106, 435. Dahl, J. S.; Dahl, C. E.; Bloch, K. Biochemistry 1980, 19, 1467. Nes, W. R.; Adler, J. H.; Sekula, B. C.; Krevitz, K. Biochem. Biophys. Res. Commun. 1976, 71, 1296. Nes. W. R.; Sekula, B. C.; Nes, W. D.; Adler, J. H. J. Biol. Chem. 1978, 253, 6218. Corey, E. J.; de Montellano, P. R. O.; Lin, K.; Dean, P. D. G. J. Am. Chem. Soc. 1967, 89, 2797. Riniker, B.; Arigoni, D.; Jeger, O. Helv. Chim. Acta 1954, 37, 546.


16.

31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

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Received September 10, 1986


Ecology and Metabolism of Plant Lipids - American Chemical Society

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