Control of ergosterol biosynthesis in yeast

BIOCHEMICAL

Vol. 33, No. 3, 1968

CONTROL

OF

EFGOSTEROL

Kawaguchi,

A.

Department

of

September

It ion

28,

has been

to MVA

in

ed to be the Rodwell,

organisms produces of

is

sterol. in

ergosterol

by

acidic

not

activity

cholesterol were

to produce

which

are

the

yeast formed

presum-

these

yeast

about

the

which control

metabolism

activity

and

Mycobacterium

With

further

from

are

However,

reported

undergoes

reduct-

(Fimognari

with

1965a).

the

that

of

which

any sterol.

and about

synthesis

acids

reported

has been

reports

of HMG-CoA*

metabolism

and Rodwell,

nothing

paper

lipids

Japan

by bile

results

synthesis

This

in

(Fimognari

ergosterol,

the

controlled

Similar

tiown

ergosterol

of Science

Kyoto,

that

end products

are

Katsuki

Faculty

University,

YEAST

1968

animal

and Pseudomonas

IN

and H.

Chemistry,

established

1965).

RESEARCH COMMUNICATIONS

BIOSYNTBESIS

H. Hatanaka

Kyoto

Received

AND BIOPHYSICAL

of

ergo-

of HMG-CoA reduction a feed-back

inhibition

ergosterol.

MATERIALS AND METHODS Sacoharomyces anaerobic

or

depending

on the

of

Klein

lected,

and

+

the

of

yeast

cells

(20 mg wet for

150 min.

out

by the

method

mixture

The harvested of

Abbreviations acid; WA,

Carborundum

was grown

conditions

(Meyer

experiments.

shaken

modifications: the

(ATCC 12341)

anaerobic

purpose

(1955),

was carried

glutaric

strictly

resuspended

lution

of

cerevisiae

grown

the of

Katsuki

and Bloch

were

and Celite

the

the

ground 535

(1:l)

1963) method

were

col-

glucose-salts

Preparation

cells

to

semi-anaerobically in

semi-

and Bloch,

According

weight/ml)

of

under

with

so-

cell-free (1967)

extracts with

an equal and

the

some weight

mixture

used are as follows: HMG, 3-hydroxy-3=methylmevalonLc acid; NSF, nonsaponifiable fraction.

463

Vol. 33, No. 3, 1968

was treated

with

cells).

It

natant

obtained

the

incubation l-l4

were

carefully

mixture

C-Acetate

according

14 C-Ergosterol

yeast

were

was prepared

cells

followed

80,000

the

x g for

with

the

cell-free

shaking.

After

by the

conventional

extracts the

reaction, method.

of Hilz

by incubating

and

3- 14 C-HMG-CoA

commercially.

method

After was used

14 C-MVA (5 yCi/pmole)

obtained

by purification

super-

1 hr.

supernatant

2-

to the

1 hr and

Q of wet

the

1 hr with

(25 @i/~mole),

(1 ml/l

removed,

was saponified

3-14 C-RMG (2 ~Ci/~ole) was synthesized

at

Incubation

37°C for

at

x g for

centrifuged

extracts.

out

5,000

RESEARCH COMMUNICATIONS

pH 7.0

buffer,

at

was again

cell-free

AND BIOPHYSICAL

g phosphate

lipids

was carried the

0.1

was centrifuged

floating

as the

BIOCHEMICAL

et al.

(1958).

l- 14C-acetate

on thin-layer

with

plates

the

(Bloch,

1957).

RESULTS AND DISCUSSION Conversion conditions, growth of

to NSF with --e

S.

oerevisiae

and the

the

control

various

amount

of

oleio

mechanism

of

ergosterol

of

Fig.

1 shows

decreased

with

increasing

conversion

to

fatty

Conversion

tate.

is

suggest

that

Therefore,,

were

done

to

from

l4 C-aoetate

ergosterol

might

and-from

and

1).

In

the

ergosterol

under

in order

the

cells

strictly

of

for the

to

presence

investigat

conversion

whioh

of

anaerobic

were

of

grown

anaerobic

14C - acetate

ergosterol,

at oon-

to NSF while

the

not. cell-free

the

(Fig.

conversion

NSF with

further

examine

did

strictly

by ergosterol

ooncentrations

acids

to ---

with

the

acid

synthesis,

ergosterol

that

Under

limited acid

to NSF was examined

concentrations

cells oleio

amount

ditions.

above

requires

growth

an adequate

14 C-aoetate

growing

experiments incorporation 14C-MVA.

464

extracts

The results

control

its

with

the

synthesis

Unexpeotedly,

from

cell-free

of radioactivity

obtained ace-

extracts into

ergosterol

NSF itself

Vol. 33, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL

__e-

a.--

---____

RESEARCH COMMUNICATIONS

----__

*/-

1-

Qrowth

. Fatty acids 0

Ergosterol

%ed

loo

(pQ/ml)

Fig. 1. Effect of ergosterol on the conversion of 14 C-acetate to NSF with growing cells. The medium contained in a total volume of 25 ml, 2.5 g of glucose, 170 mg of Difco yeast-nitrogen base, 8.0 mg of potassium oleate, 25 mg of Tween 80, 0.03 pmole of 14C-acetate and (1,400,OOO c.p.m.), 1.25 mmoles of succinate buffer (pH g.O), After inoculation it was incubated indicated amounts of ergosterol. at 25°C for 48 hr under strictly anaerobic conditions. showed

no action

On the

other

the

hand,

disrupted

bition that

of

the

yeast

was shaken neutral with

of they

I,

the

were

precursor

which (2:1),

were

but

into

NSF.

extracted

showed

l4 C-acetate

results,

it

acidic

from

a marked

inhi-

no action

evident

between

Of authentic oholate

in

separate

on

acetate compounds

in

ether

the

acidic

and

from the

lipids

the

solution from

the

the

NaHCO solution was acidified 3 with ether. Each lipid fraction ether

the

solutions.

acidic

inhibited

14 C-acetate that

substance(s)

dissolved

the

lipids

NSF from is

inhibitory

was shaken

was found the

into

oxycholate,

either

lipids,

from

by evaporating

activity

a etep

yeast

1 g NaHCO to 3 After separation,

6 E H2S04 and it

Table

of

chloroform-MeOH

separation

with

activity

at

with

lipids,

was obtained

in

crude

14 C incorporation

ones.

bitory

incorporation

14C-MVA.

For crude

the

cells the

from

on the

but

the

acidic

Most

lipid

fraction.

the

incorporation

not

from

lipids

of

the

As shown of radio-

14C - MVA. exerted

inhi-

From

their

these

action

and MVA. tested

and glyoocholate,

465

so far, at

bile 0.1

e

acids,

such

as de-

showed

a strong

in-

Vol. 33, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I Inhibition Radioactive

of Acidic

substrate

Conversion

to NSF by Acidic

lipids

(1.0

Lipids

Total

Inhibition

incorporation

mg)

c.p.m. 1-14C-Acetate

%

+

52,900 7,600

85.6

3-14C - iMG - CoA " 224 C-MVA

+

79,400 10 ( 400

86.9

n

+

77,400 70,100

9.5

Reaction mixture (1.0 ml) contained 0.3 ml of cell-free extracts (7 mg protein); ATP, 3 mg; GSH, 2 mg; NADP, 0.5 mg; glucose-6-phosphate, 3 mg; crude coenxyme A (Sigma, grade II), 1.5 mg; MgS04*7H20, 0.4 mg; MnSO l 4H 0, 0.3 mg; Been 80, 1 mg; 0.08 &i potassium phosphate buffer! ~~7.0; and l550,000 0.p.m. HMG-CoA (2,100,OOO c.p.m.) or "-eSetate 2C-MVA 200,000 c.p.m. as indicated.

hibition 25 $,

of

the

conversion

respectively).

detergents,

Unlike

such

conversion

not

of

as Triton

only

Inhibition effect

acidic

and the

lipids

that

the

Purification low

value

to

authentic

of

other

nated

of

inhibition

bile

acids

lipids

by column

is

chromatography

the

lipids

in

inhibition of

the

step,

14C-I-DiG-CoA

acidic

them. on silica

of

These

from

of

yeast lipids

The acidic gel.

the

the NSF

to

to NSF,

conversion,

results

reduction

to be due to

466

artificial

inhibited

$ inhibition to NSF.

was found

the

14 C-HMG-CoA was converted

an 86.9

by the

contaminated

the

35 $ and

14C-MVA .

of

conversion

I,

acidic

of

sulfate,

also

to decide

step

of the -I_the

but

converted

inhibition

some kinds

and lauryl

Table

showed

to NSF (48 $,

acids,

on the

in

14 C-HMG was not

free

suggest

order

lipids

As shown

but

bile X-100

In

was examined. acidic

14C-acetate

14 C-acetate

of

step

the

of

strongly

HMG-CoA

to MVA.

The apparently from

the

yeast

compared

interfering

action

lipids

were

The inhibitor(s)

fraotiowas

Vol. 33, No. 3, 1968

BIOCHEMICAL

eluted

(75:25)

by ether-MeOH

with

hexane-ether

tographed acetic

acid

revealed 0.30,

wet

0.40),

Of the

which

cells,

The other

were

determined the

methane

gave

four

acetate

(30:20)

strafe

that

spots

of

the 14

also

in

of the

values%

conversion

of

Ergoeterol

(10 pmoles,

--to from

C-ergosterol 10,000

to

0.65,

of

e

0.45, mg of

3.0

0.40 third

14

are

the

inhibitors

C-acetate which them.

diazo-

of n-hexane-ethyl and 0.09).

inhibitors

yeast

and

500 g of

in with

1963)

one

activities,

components

0.18

al.,

mg from of

inhibitory

system

0.27,

c.p.m.)

benzene-dioxane-

conversion

these

the

was ahroma-

some contamination

solvent

0.73,

ergostero?.

inhibitors

to

it

0.1

yield the

by eluting

(Kritchevsky

were

had

owing

esterification

(Rf

Conversion

the of

acids

G with

components, in

components exactly

gel spray

90 $ inhibition

to NSF.

Furthermore,

silica

four

fatty

purification,

of which

was obtained

showed

not

of

Rf values

respectively.

(Rf,

further

Anisaldehyde

spots,

RESEARCH COMMUNICATIONS

removing

For plate

(60:30:2).

four

after

(5Or50).

on thin-layer

AND BIOPHYSICAL

normal

was added

In

order

to demon-

metabolites, was examined. to

the

the 14,-

growing

Rf value

Fig. 2. Thin-layer chromatogram of the inhibitors derived from C-ergosterol. Inhibitors 500 c.p.m.) were chromatographed with benzene-dioxane-acetic acid I 60~30~2) as described in the text. Radioactivity in the 1 cm segments of the developed plate was counted. 14

467

Vol.33,No.3,

culture

1968

of yeast

hibitors

were

described Fig.

2 shows at

These

results

olites

which

the

AND BIOPHYSICAL

mixture from

the

the

oells

which

strongly are in

to

radioactivity

spots

derived

from

thin-layer

revealed that

for

according

on the

were

support

RESEARCH COMMUNICATIONS

was incubated

and was subjected

that

The details will

the

extraoted

ergosterol

bitors

and

above

located

of

BIOCHEMICAL

the

ergosterol

48 hr. to

the

The inprocedure

chromatography. thin-layer

with

plate

anisaldehyde

inhibitors and control

are

was spray.

normal

the

metab-

biosynthesis

yeast. of

the

be reported

experiments in

and the

a subsequent

properties

of

the

inhi-

paper.

ACKNOWLEDGEMENTS The authors discussion

and

are

indebted

to

Prof.

Shoso

Tanaka

for

his

helpful

encouragement. REFERENCES

Blooh, K., in S.P. Colowick and N.O. Kaplan (Eds) Methods & Enzymolx, Vol. IV, Academic Press Inc., New York, 1957, p.739 Fimognari, G.M., and Rodwell, V.W., Science, 147, 1038 (1965) and Rodwell, V.W., Biochemistry, A, 2086 (lp65a) Fimognari, G.M., Hilz, H., Knappe, J., Ringelmann, E., and Lynen, F., Biochem. z., 329, 496 (1958) K., J. Biol. Chem., 242, 222 (1967) Katsuki, H., and Bloch, Klein, H.P., J. Bacterial., Q,(1955) Kritchevsky, is., Martak, D.S., and Rothblat, G.H., Anal. Biochem., 2, 388 (1963) Meyer, F., and Bloch, K., 2. u. ., 2~8, 2654 (1963)