Localization of the central and peripheral SH-groups on the same polypeptide chain of yeast fatty acid synthetase

BIOCHEMICAL

Vol. 69, No. 4, 1976

LOCALIZATION OF THE ON THE SAME PDLYPEPTIDE Georg-8. lnstitut

Kresze, fi.ir Helga

lnstitut

Received

fDr

February

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CENTRAL AND PERIPHERAL CHAIN OF YEAST FATTY

Dieter Biochemie

Oesterhelt and der UniversitBt

SH-GROUPS ACID SYNTHETASE

Feodor Lynen MUnchen

Castorph and Eckhart Schweizer Biochemie der Universitat

WDrzburg

18,1976

SUMMARY:

Purified fatty acid synthetase isolated from wild type yeast as well as from two different fas-mutant ,etrains was reacted with ce’bi (lC-)iodoacetamide. Tryptic digestsof the C-carboxamidomethylated enzymes were fractionated on Sephadex G-50. Hereby, essentially only one radioactively labeled peptide was eluted from the column. From this it is concluded that under the experimental conditions employed only the “peripheral” SH-group of yeast fatty acid synthetase becomes alkylafed. By sodium dodecylsulfate-polyacrylamide gel electrophoresis of the C-carboxamidomethylated fatty acid synthetase it was shown that in all three enzyme preparations studied the inhibitor is bound to the larger one of the two fatty acid synthetase subunits. These findings indicate that the larger fatty acid synthetase subunit accomodates not only the “central” but also the “peripheral” SH-group of the multienzyme complex. INTRODUCTION Acetate, is

the

covalently

acid

bound

synthetase

the

as

as

(

are form S

P

and

G.-B. to

assumed

to

indicates

as

Kresze

be

sites

These

sites

two

different

as

SH-group,

belongs

to

a cysteine

in

preparation

al.,

in

the

),

the

the

the been

fatty

groups,

desiq-

(2,3). of

the

the

Both

malonate

While

enzyme

“central”

pro-

one thiol

and

following

“central”

yeast chemically

thiol

residue

of

to S

have

biosynthesis,

respectively

condensation

and

of

(5,6).

according

“peripheral”

acid

is

groups

acetate

equation,

to where

SH-group:

C

Inhibition that

Copyright All rights

fatty

4’-phosphopantetheine

-COCH2COO-

enzymeSc-

tase

(1). and

et

involved

enzymeSc

ly

chain distinct

“peripheral”

acetoacetate the

long

chemically complex

enzyme-bound

enzymebound

of

OH-group

SH-group

4,15,

attributed

several

a serine

“central”

“peripheral”

tein

to

substrate

multienzyme

characterized nated

priming

the with

studies B-ketoacyl the

complex

CO-CH,-CO-CH3

performed

( 15,

under

synthetase

alkylation

of G.-B.

+ CO2

Kresze

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

cysteine et

3

+

carefully

component three

.

+ enzymesp-COCH

enzymes

893

P

controlled enzyme

in

conditions

was

SH-groups

al.,

.

preparation

inactivated of

the ).

indicate3 concomitant-

fatty

acid

These

results

synthe--

Vol.

69, No. 4, 1976

indicated

that,

binds

to

the

appropriate

conditions,

W-group

prosthetic

biosynthesis

sodium

AND

“peripheral”

pantetheine the

under

BIOCHEMICAL

group

of

the

of

yeast

may

quently

it

be

the

was

found

priate

into

acid

that

the

corresponding

two

subunits.

acid

synthetase

larger

to

For

“peripheral” identify

the

one (7).

synthetase

these

Thus,

or

by specifically 14 C-iodoacetamide

subunits

of

the

complex.

be

reported

in

this

study.

MATERIALS

AND

inhibitor

component

Experiments

pertinent

on that

(7).

Subse-

contained studies

should

the

with allow

binding

sites

it enzyme this

approthe

labeling

to

by

multienzyme

subunits

subunits similar

instance,

B-ketoacylsynthetase

showed the

protein of

“central” studies

complex

electrophoresis

compounds

with

specifically the

genetic

substrate

SH-group

COMMUNICATIONS

with

Recently,

enzyme-inhibitor

of of

protein

or

react

nonidentical

group

the

either

possible

the

prosthetic

enzyme-substrate

calisation

two

not

complex.

fatty

RESEARCH

iodoacetamide

does

gel

separated

phosphopantetheine

will

but

dodecylsulfate-polyacrylamide

complex

BIOPHYSICAL

loon

the

fatty

should among

be the

question

METHODS

The Saccharomyces cerevisiae haploid wild type strain X2180-lB, a-mating type, was originally obtained from Dr. R.K. Mortimer. The fatty &id synthetase deficient mutants fasl-266, fas2-348 and fas2-288 were derived from this strain as described previously(8).or enzyme isolation wild type as well as -fas-mutant strains were grown at 30°C in 40 liters of yeast extract-pepton sucrose medium, each. After 36 hours incubation without aeration, the cultures were aerated with compressed air for additional 24 to 36 hours. Under these conditions between 200 and 300 grams of wet yeast cells were obtained. The harvested cells were screened for genetic homogeneity as described earlier (9). The procedure used for enzyme purification was essentially that descri bed by Lynen (10). Aftei4the second lOO.OOOxg sedimentation step the enzyme was reacted with lC-iodoacetamide as described below and subsequently subjected to a final purification by sedimentation in a 5 -30 % sucrose density gradient for 15 hr at 4’C and lOO.OOOxg. After the run, 20 dropfractions were collected and analyzed for radioactivity and optical density at 280 nm. The enzyme-containing fractions were pooled and, if not used immediately, kept at -15’C. Aliquots of this solution were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis as described below. Protein samples were denatured by incubation with 1 % sodium dodecyl sulfate in the presence of 0.5 % mercaptoethanol for 3 hr at 25OC. After the addition of 30 % (w/v) solid sucrose the samples were placed on top of urea-sodium dodecylsulfate-polyacrylamide gels as described previously (7) Small cylindrical gels (15 x 0.5 cm) were run for 12 hr at 20 volts and 22’C, slab gels were run in a DESAGA slab electrophoresis chamber for about 60 hr at 23 volts and 22’C. Subsequently the gels were stained with 0.05 % Coomassie Blue G 250 (7). For radioactivity determination the destained slab gels were frozen in a dry ice/methanol bath and cut into slices of 0.5 - R1 mm width. Eachoof the slices was soaked overnight with 1 ml of Sol uene (Packard) at 60 C under slight agitation. For subsequent counting, 16 ml of a I:1 mixture of PPO/POPOP-scintillation fluid (7) and Triton X 100 (Serva) were added together with 0.1 ml of glacial acetic acid to each sample.

894

Vol. 69, No. 4,1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

FRACTIONS Fig. 1: Sephadex G-50 chromatography of the tryptic hydrolysate of 14C carboxamidomethyl-synthetase. For copr) itions of digestion, see Materials and Methods. The hydrolysate of the C-carboxymethylated fas2-349 fatty acid synthetase was dissolved in 1 ml 50 % (v/v) acetic acid and subjected to chromatography on Sephadex G-50. Column: 1.65 x 87 cm; fraction volume: 0.72 ml; eluant: 50 % (v/v) acetic acid. 0.2 ml of every second fraction were tested for radioactivity.

About 20 mg of the enzyme were reacted with I mM (l-l4 C) iodoacetamide (18.4 m$i/mmole, Commissariat d 1’Energie Atomique, Gif-sur-Yvette, France) at y4C in 0.1 M potassium phosphate buffer pH 6.5 for 120 min. Unreacted (1 C) iodoacetamide was removed by filtration of the mixture14 Cover a Sephadex G-25 column (6 x 0.5 cm) at O’C. Tryptic digestion of carboxamidomethyl synthetase was conducted at 25’C for 20 hours (0.5 mg of N-tosyl-L-phenylalanyl-chloromethan-treated trypsin per ml 0.1 M tris.HCl, pH 8.1). Then, the mixture was dried on a rotatory evaporator, the residue dissolved in 1 ml of 50 % (v/v) acetic acid and subjected to chromatography on Sephadex G-50 (column 87 x 1.6 cm) in the same solvent. RESULTS 1.

Chromatography

synthetase. in

As

will

preparation)

acid

the

G-50

these

of

two

the

were

analyzed

pattern is

shown.

peptide

TA

also digests

by

Though

of

detail 14

by

in

for

the

of

the

the

G-50

with

one

representative

the

amount

of

on

the

Kresze

et

digestion

varies.

yeast

fatty

895

separable

by used

was

there

concluded is

synthetase.

only To

synthetase

used

Fig.

small

1,

the from in

one

conin

mutant

isolated very

it

that

acid

In

TA was

this

and

enzyme

TB

conditions

From

al.,

fatty

TA and

digestion

chromatography.

peptide

(G.-B.

peptides,

mutant fatty acid 14 C-carboxamidomethylated

Sephadex

C-carboxamidomethyl

C-carboxamidomethyl-labeled

TA

further

14

of

elsewhere

Depending

site

tryptic

obtained

in

radioactive

larger

from

findings

the

reported

hydrolysate

digestion

iodoacetamide-sensitive

study

tryptic

chromatography.

TB originates

firm

the

produces

proportion

that

be

tryptic

synthetase

Sephadex

of

this

enzymes

radioactivity mutant this

fasl-345

experiment

BIOCHEMICAL

Vol. 69, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

Fig. 2: fied wild Experimental

it

is

obtained

with The

parations

that

essentially

wild

type

of

purified

the

mutants

yeast

cells

dium

dodecylsulfate-polyacrylamide analytical

synthetase the

radioactivity

gels subunits

were

revealed A and associated

same

fatty protein

14

and fasl-266

and to

acid

fas2-349 analytical

of

B described

of earlier

the

896

individual

are

yeast

fatty

acid

synthetase

pre-

as

well

as

from

as

well

as

preparative

electrophoresis.

presence

puri-

synthetases.

component

gel the

of synthetase.

peptides

C-carboxamidomethylated

subject

with

electrophoresis fatty acid (7).

carboxamidomethylated

-fas-mutant binding

The

studied

the

and

iodoacetamide

synthetase.

the

c

Sodium dodecylsulfate-polyacrylamide gel type (a), fas2-349 (b) and fasl-266 (c) conditions were as described earlier

evident

2.

b

After bands

so-

As

shown

in

the

two

fatty

exclusively (7).

wildtype

slicing was

the

determined.

Fig. acid gels AS

2

0

70

20

B A 1111111111

(+I -

0

10

GEL

20

(+j -

c

30

0

BA

(+)

-

10

20

L

Ij SLICES

n J.

3.

0

10

BA

III III

20

(+) i--

14 14 Fig. 3: Protein and radioactivity banding patterns of C-carboxamidomethylated (A-C) and C-pantothenatelabeled (D) fatty acid synthetases upon sodium dodecylsulfate-polyacrylamide gel electrophoresih. The enzymes studied were isolated from wild tvoe (A). fasl-266 (B) and fas2-349 (C) cells. The oattern of ’ ‘C-oantothenatelabeled fas2-288 synthetase (D) was adapted from Schweizer et((7)D.Y ml contaiLing 1.500 - 2.bOO cpm radioactively labeled sodium dodecylsulfate-denatured fatty acid synthetase (0.2 - 0.4 mg protein) were applied to each gel. Radioactivity determinations were averaged from 5-6 IO-min-countings. The black bars below the tracings indicate relative staining intensities of the gel slices at the positions of the fatty acid synthetase subunits A and B. For other experimental details see Materials and Methods.

)-

)-

)_

I-

/ 4.

Vol.

69, No. 4, 1976

it

is

shown

to

the

BIOCHEMICAL

in

Fig.

larger

banding

subunit

pattern

synthetase

shown

boxamidomethyl acid

all

with

in

a

Fig.

residues

fatty

the

A of

observed

is

yeast

3 A-C,

AND

BIOPHYSICAL

radioactive

inhibitor

three 14

3 D.

are

enzymes

is

studied.

C-pantothenic

to

both

the

COMMUNICATIONS

exclusively For

the

bound

comparison,

acid-labeled

Obviously,

bound

synthetase

RESEARCH

the

fatty

acid

pantetheine

same

polypeptide

this

laboratory

and

chain

of

carthe

complex.

DISCUSSION Genetic yeast

evidence

fatty

acid

with

the

larger

This

view

has

the

one now

this

reported

ty

acid

fatty

three

to

different

protein,

Correspondingly, ties,

i.e.

tyl ponent

B,

cated as

the

by well

various as

with

the

by

suggest complex.

with six

the

by

the

three complex

synthetase

assumption

acid

synthetase

genetic

(12)

AbBb

stoichiometry

of

by

conclusion

Kresze

et

carboxymethyl

that

with

apparent

yeast

(for

a review

the

complete

acid as see

14).

898

are

somewhat

Although

et

one

is

is

may endowed with

a reaction

indi(11)

together fatty fatty

at

acid acid

variance that

molecule of

palmicom-

al.

indicating to

however,

observed

it

yeast

inhibition

synthetase

they

the

with as

yeast

bound

acyl

activi-

findings

preparation) are

the

and

Tauro

of

be

at

reductase.

malonyl

intact

to

synthetase

i.e.

associated

content

discrepancy,

fatty

characteristics enzymes

(in

residues

concomitant This

al.

comprising

the

by

fatthe

synthetase

These

seems

chain

subunit,

press). (13)

acid

be

obtained

(in

pantetheine

this

to protein

evidence

Schweizer

and

hand,

assumed

this

fatty

acid

i.e. to

namely

B-ketoacyl

fatty

as

data that

complex,

dehydratase, are

and

activity.

site-reactivity” multimeric

of

the

enzymes

fatty

obtained

only

multienzyme

reductase,

an

other

results but

fasl-encoded

known

well chain,

suggest

the

of

results

as

genetic

encoded

and

the

function,

multienzyme

four

FMN

synthetase

the

the

lines

synthetase

of

to

polypeptide

polypeptide

synthetase

Knobling

known

On

a multifunctional

B-ketoacyl

second

fas2

(7).

labeling

SH-group

a third

in

associated

complex

According

preparation)

the

is

the

Furthermore,

even

Thus,

component the

in

that

specific

a single

A.

the

enoyl

transferase

subunit

functions

of by

pantetheine within

accomodates

be

activity

subunits

“central”

combined

activity.

suggested

synthetase

protein

Schweizer,

subunit

A appears

carrier

the

synthetase

reductase

subunit

B-ketoacyl

confirmed chemically 14 C-iodoacetamide.

are

(E.

in

known

with both

elsewhere

B-ketoacyl

two

further

acid

synthetase

least

the

SH-group

larger

be

of been

study

the”periphera1” the

the

SH-group

in

earlier

synthetase

“peripheral”

reported

obtained

of

fatty

acid

be

resolved

with numerous

“halfother

mechanism

not the

Vol.

BIOCHEMICAL

69, No. 4, 1976

of

the

fatty

acid

“peripheral” until ment

synthetase

SH-groups more

of

is

the

known

various

AND

complex

may about

easily the

active

by

BIOPHYSICAL

involving

half-site-reactivity

envisaged

it

molecular

centers

RESEARCH

interaction

within

the

must and

yeast

COMMUNICATIONS

of

remain the

multienzyme

the

speculative spatial

arrangecomplex.

ACKNOWLEDGEMENTS This

work

was

supported

by

the

Deutsche

Forschungsgemeinschaft

REFERENCES 1. 2.

3. 4. 5. 6. 7. a.

Lynen, F. (1967) Biochem. J. 102, 381-400 Ziegenhorn, J., Niedermeyer, R., Niissler, C. and Lynen, F. (1972) Eur. J. Biochem. 30, 285-300 Proc. of the R.A. Welch Foundation Conference Lynen, F. (1961) in: on Chemical Research, Vol.f5, pp. 293-329 Oesterhelt, D. (1967) Doctoral thesis, Universitst Miinchen Schweizer, E., Piccinini, F., Duba, C., GLinther, S., Ritter, E. and Lynen, F. (1970) Eur. J. Biochem. 15, 483-499 Wells, W.W., Schultz, J. and Lynen, F. (1967) Biochem. Z. 346, 474-490 Schweizer, E, Kniep, B., Castorph, H. and Holzner, U. (1973) Eur. J. Biochem. 39, 353, 362 Schweizer, E. and Bolling, H. (1970) Proc. Natl. Acad. Sci. U.S.A.

67, 9.

KDhn,

660-666 L.,

Castorph,

H.

and

Schweizer,

E.

(1972)

Eur.

J.

Biochem.

24, 492-497 10. 11. 12.

13. 14. 15.

Lynen, F. (1969) Methods Enzymol. 14, 17-33 Tauro, P., Holzner,U., Castorph, H., Hill, F. and Schweizer, E. Molec. gen. Genet. 129, 131-148 Oesterhelt, D., Bauer, H. and Lynen, F. (1969) Proc.Nat1.Acad.Sci.U.S.A. 63, 1377-1382 Schweizer, E., Willecke, K., Winnewiser, W. and Lynen, F. (1970) Vitam. Horm. 28, 329-343 Lazdunski, M. (1974) Progr. Bioorg. Chem. 3, al-140 Lynen, F. (1964) in: New Perspectives in Biology (Sela, M. ed.) Elsevier Publ. Comp., Amsterdam, pp. 132-146

899

(1974)