Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae

Vol. 60, No. 1,1974

ABSENCE

BIOCHEMICAL

nF

INVOLVEMENT

GLUTAMATE

flF

AND BIOPHYSICAL

GLUTAMINE

DEHYORBGENASE

REPRESSION

OF ARGINASE

E,L.

Laboratoire de

de

Bruxelles

Belgium.*

and

Bubois*

~~crobiolo~ie,

and

SYNTHETASE

IN

SACCHARQMYCES

THE

NITROGEN

AND

OTHER

de

ANB

ilF

NAD-LINKED

CATABOLITE ENZYMES

IN

CEREVISIAE.

M,

Grenson

des ScienCesI UniversitG

Facultd

Institut

RESEARCH COMMUNICATIONS

Recherches,

C.E.R.I.A.,

R-107n

Libre

Bruxelles.

*

Received

July

15,1974

SUMMARY

The escape of several enzymes from “ammonia catabolite repression" in gdhA [NA~P-linked glutamate-dehydro~enase-less1 mutants, as well as in gdhCR mutants of Saccharonyces cerevisiae, does not involve glutamine synthetase, either as a positive or as-a negative control element, A glutamine-synthetase-less mutant (gin I was used in this d~onstrat~on, In addition to its derepressing effect on the NAB-linked glutamate dehydrogenase, the gdhCR mutation releases “nitrogen catabolite repression" on arginase and allantoinase, as well as glutamine repression on glutamine synthetase, A gdhCS mutation was used to demonstrate that these effects are not mediated through the NAWlinked glutamate dehydrogenase, The be

an

NAnP-linked

important

element

Saccharomyces main in

**

support

of

activity

Aspirant

Address C.E.R.I.A.,

in

this

idea

in

cells

grown

du

Fonds

National

the

enzymes

request

as

is

some

Avenue

regulation

(l-3)

of

for

dehydrogenase

the

cerevisiae

is observed *

glutamate

and in

Gryzon,

Copyright 0 1974 by Academic Press, Inc. AI1 righis of repr~~ction in any form reserved.

fact

in

other

that

,

whereas

systems

presence

the de

of nitrogen as

uptake

of reprints E.

wall

la : 1 -

(NABP-GBUase?

Recherche Institut P-107?

150

fungi

NH4’,

metabolism

in

(4-61.

The

a strong decrease nitrogen

using of

appears to

this

compounds

“ammonia

Scientifique. de Recherches, Rruxelles, Belgium.

effect”

Vol. 60, No. 1, 1974

is

largely

BIOCHEMICAL

released

NADP-GDHasel. through

in

Whether

molecule

clarified

cerevisiae,

the

NADP-GDHase

activity

Howaver,

lost

have Involved

in

more

activity

the this

directly

or an inhibitory

possibility

second

of

regulation

as a regulatory

binding

protein)

in the

case of

is

favoured

by

this

direct

Magasanik

(lnl

showing

that is

view

of

questions, gdhA-

of

glutamine

is

not

Saccharomyces

the

presently

available

data,

First,

is

mutants

effect

enzymes

of

of

following

the

it

lack

mutation

sulfonate

of

effect

of

to of

mutagenesis.

Delco

and in

Its

of

the

hut

us to answer

the

follo-

by

the

cerevisiae level

lack

of

through

mediated of

NAOP-GDHase

glutamine

? This

gluta-

synthetase

investigation

regulatory

behaviour

have

led

us

of

other

catabolism. AND

METHODS

are

used

have

the

NADP-linked

ure-2

(11,121, MG935

to the

isogenic

cerevisiae.

This

a con-

typhimurium.

to

caused

about,the

nitrogen

strain

Tyler,

transcription

Salmonella

number

A

molecule,

NAOP-GDHase

information

strains

by

seemed crucial

the

mutations

allelic

SdhCR,

DNA

(71. Recently,

[8,91.

the

of

the

in

gene

activate

Valentine

and

genes

glutsmine-synthetase

as

Saccharomyces

structural

presented

can a high

the

C1278b

been

to

MATERIALS

All

has

? Secondly,

Involved

Strains,

obtained

Saccharomyces

of

additional

obtain

been

ammonium

in

Gurney

fixation

nitrogen

the

synthetase

Strelcher,

by

have

operon

these

synthetase same

the

able

utilization)

In

the

on idea

form,

(histidine

role

demonstration

non-adenylated

mine

paper

favouring

vincing

the

a key

was proposed

remarkable

data

wing

hand,

in bacteria

in their

&

is

or rather

as yet.

other

repression

to

(which

RESEARCH COMMUNICATIONS

[31. On the

in

mutants

as a repressor

(such

completely

of

the

catalytic

its

data

gdhA-

AND BIOPHYSICAL

The

glutamate

has

gdhCS.

allelic

been

obtained

provokes

mutation

151

strain

and

characteristics

gdhA;,

affecting

dehydrogenase

Cl,

isolation

described

been

wild-type

:

to

ng13

after a specific

(11,

the

111, 121.

The

ethyl-methanerequirement

for

Vol. 60, No. 1,1974

tl!OCtiEMKAt

glutamine,

which

asparagine,

and

citrulline,

argininel.

that

no

glutamine

even

in

the

all

this

compared

to

throughout, in

as

Sephadex

in

the

in

the

mutation in

nitrogen

other

NH4+

of

be

detected

which

is

+ glutamate,

proline,

Table

strains

by in

per

1 mg

were

the

paper

gin-

a to

(Table

ornithine,

present

in able

G-25

the

mlJ

on

debris

are

strain

dramatically

1,

glutamate

(synthetase

assay)

These NHqf

experiment

medium

in

French

amino

press

acids

13

of

are

[141, and

added

to

O.GZ

4+

* a final

M.

exponentially dialyzed

measured

as

alfantoinase

(161,

NH

supernatant

activities

used was

or

remains

the

M was

medium,

were

extracts and

arginase

this

glutamine,

concentration

dehydrogenases (17,

NH4’

glutamate,

Enzyme :

(131,

is

centrifuged

references

NADP-linked

which

I,

(Pharmacial.

following

described

either

Table

performed

The

been

source,

experiments

of

on

gdhCR

glutamate, faspartate,

can

have

indicated

concentration

cells.

a

activity

the

some

glutamate,

growing

seen

activity

of

NH,+.

tested

be

conditions

but

essays

can

synthetase

enzyme

by

substances It

growth

Enzyme

replaced

RESEARCH CO~NICATIONS

111,

and

replaced

be

other

presence

increase

Medium

cannot

AND BIOPHYSICAL

described

1151,

glutamine

NAG-

and

synthetase

181.

RESULTS Glutamine

synthetase

It is the

not

can

be

seen

changed

in

allantoinase

(experiments strain

activity in

on

due

to

a gin-

the

ammonia

other

mutant

2,

(experiment

high

level

on

of

the

arginase

the

glutamine-synthetase

increased and

7,

to

glutamine

not

of prevent

synthesis

152

41. low

activity

where

compared

a

absence

:

conditions

11,

does

mutation, effect

to

mutants

under

are

NH4+

hand,

gdhA-

? that

activities

to a relatively On the

Table

a gdhA-

6 compared grown

in

to

In

addition,

level

of

the the

arginase wild-type

in arginase

strain

wild-type

the is

associatsd

synthetase. glutamine-synthetase the

gdhA*

(experimsnt

activity, mutation 17

versus

to

and

release 71.

Vol. 60, No. 1, 1974

TARLE

1:

BIOCHEMICAL

Enzyme

(xl

activities

AND BIOPHYSICAL

in

the

wild-tyne

RESEARCH COMMUNlCATlONS

strain

and

in

mutants.

C’

Strains

Pditrogen

and in

source

meclium

growth

: z ti E 4

Exn. no C1278b 1 l

rdH4

(wild

+ glutamate

3,glutamate 4,glutamine 4324~

.z2

I & s

E 5; z

+ glutamate

n.7

1,2

45-SF

4,5-5,l

n,35

1

37

2,9

50

1,n

47

35

14,4-15,fl
If,9

35


2 3,5

1,3

3

29

12.597a

CO,1

(gdhCR1 ?m-Rn + glutamate

95

133-153

lR-22

4,7-5.6

6.6

lU.glutamate

122

185

llrglutsmine

140

80

17

5

(gin-1

12,glutamine

11.412d

9

n,7

18

a,

1

6

0,5

4.3

0,25

15

IgdhCSI

13. NH4+ 14.glutamate

50

12.759a

(gdhA,-

, gdhCR1

15.NH4+ 16, NH4'

4 + glutamate

11.064a

89

13,,336a

36 [gdhCR,

gin-I

IgdhCR,

gdhCS)

18.glutamlns 12.761a

5,fi

gin-I

(gdhA,-,

17, glutamine

19. NH4+

75

165

53

9,7

a, 24

ZO.glutamine

534

(x1 Enzyme per

2

6

40

8.NH4+

MC935

2

8

10-12

7.glutamine

9.NH4+

iy

(u If: -5s

(edhA&

5.NH4+ 6.NH4+

E =1 <

ii a zs (r

type)

+

2*NH4+

a, : L

: Yi 2

activities hour

at

in

urnales

of

product

30°C

153

formed

per

mg

of

protein

1

Vol. 60, No. 1,1974

Can

level

a high

lack

NADP-GDHase

It

was

In

the

a factor presented

wild-type

gdhCR

on

case

this

activity

is

It

is

grown

on

is

strain

wild-type higher

in

the

also

compared

111,

the

level

glutamine

source

is

is

at

the is

The data

low

in

in

the

SC-fold

higher

conditions,

[experiment

111,

responsible

suppressed

by

synthetase

high

for

when

by a gl<,mutation.

1).

growth

same

as

higher

to

least

very

NH4+

is reduced

vsry

were

be

on

hundredfold

of

Under

should

than

0 compared

it

gdhCR mutant

removed

on

in

to

experiment

of arginase

glutamine

the

However.

same

mutant.

the

gdhCR 2).

the

in

was measured

(experiment

that

(experiment

high

the

glutamine-

this

is

not

1I

, whereas

the

arginase

arginase

and

the

They

mutant

grown

are

on

even

gin-

activity

glutamine-synthetase gdhCR

mutant

a single

121.

the

The

in

(experiment

NH4

higher

9)

as

activity

cells

in

the

is

allantoinase

tenfold

activities

(experiment

+ glutamate than

of

in

the

gdhA-

activity

in

a

and

4)

9

mutant

61.

Effect

of

gdhCR

mutant It

whereas

level

the

gdhCR

(experiment

as

grown

NADP-GDUase

activity

41,

effect

striking

NH4+

effect

Ml.

grown

also

more

derepression

the

(MG935)

same

mutants

experiment

nitrogen

in the

arginase.

is

as sole

glutamine-synthetase

a control,

mutant

RESEARCH COMMUNICATIONS

the

gdhCR

the

the

(experiment

(experiment

As

are

glutamine

activity

synthetase

have

in

while

111 see also

mutant

the

effect

activity

(experiment

arginase If

NAD-GtlHasa

? show that

strain

the

that

strain,

Table on

synthatase

previously

(10,

two

grown

the

the

in

AND BIOPHYSICAL

?

wild-type

of

Cell6

glutanine

observed

source

than

the

of

of

nitrogen

in

BIOCHEMICAL

gin-

a

can

the

introduction

high

level

on the

mutation

level

of

NAU-GDHase

: be

seen of

of

in

Table

a gin-

NAD-GDHese,

1

[experiment

16

in

a gdhCR

mutation showing

that

154

the

effect

compared

to

mutant

does

of

the

11

not gdhCR

reduce mutation

that

the is

Vol. 60, No. 1,1974

not

mediated

Effect

through

of

a gdhCR

glutamine

a gdhCS mutant

The

on

mutation

in

level

the

provokes

a gdhCR

glutamine

to

19 on

8).

catabolite

to

two

that

This is

in

not

as

or

a positive

alone

The

is

combination

central

regulation

of

integration

role

the

synthetase

of

arginase the

gdhCR

NAD-GDHase.

glutamate

the

which

The

most

direct

evidence

the

gin-

mutation

assimilation in

enzyme

with

either

in efficient

endowed

with

these

regulatory

fungi

difference in

the

a very

in

the

operates

of either

dehydrogenase

keeping

“ammonia

synthetass.

results

That

from

mutation,

ammonia

systems

assimilation

ammonia

effect

of

in

of

mutants

glutamine

enzymes

is

level

the

of the

gdhCR

or a gdhCR

metabolism.

bacteria

in

a gdhA-

NADP-linked

effect

enzymes

element,

of

catabolic

in

of

key

nitrogen

is

mechanism

of

involve

absence

the

experi-

111 and

through

as

control

with

nitrogen of

properties

the

that

of

lower

not

to

several

well

not

a negative

conclusion

or in

mine

as

does

does

level

CONCLUSIONS. of

as

11~ see also

mediated

escape

gdhA-

(10,

compared

shows

AND

the

repression” cerevisiae

this

in

in the

HN4+

20,

81.

enzymes

Saccharomyces

for

synthetase

decrease

mutation

(experiment

clear

seems

on this

DISCUSSION

It

glutamine

of

strong

grown

However,

compared

these

a

mutant

synthetase

[experiment mutation

RESEARCH COMMUNICATIONS

synthetasa.

mutation

19 compared

ment

AND BIOPHYSICAL

:

gdhCS

NAD-GDHase

of

BIOCHEMICAL

in two

gluta-

and the

groups

of

organisms. The ved

in

fact

that

gdhCR

a high

mutants

under

wild-type

strain

glutamate

dehydrogenase

lity

can

NH 4 + ,the NAD-GDHase

be

ruled

activity

level

might

of

conditions

be

taken

of

since, arginase

an

a double

is

these

indication

still

is negligible.

155

arginase

levels

are the

synthesis.

[gdhCR. high,

and

that

arginase

activate in

NAO-DDHase where

as

could

out

both

whereas

the

low

obser in

the

NAD-linked This

gdhCS1

is

mutant

level

possibi-

grown of

on

Vol. 60, No. lI1.974

%hC8 gdhCR it

th8

mutant

quences

compared

of

case

the produce

same for

all

(IQ-22

that

the

the

and 41,

arglnase.

is

the

in

activity

of

45-50

mutations I,

the

data

NADP-GDHase

is too

units1

But

in

small

15 likely

to

to be the

the

gdhCR

the

from

of

not

this

mutation

by the

in

regulator

might

those

regulation

element

a common

only

reprassion"

to nitrogen

to add that gdhCR

affects

“atenonia

sensitive

existence

the

mutation

were

tested

regulation.

this

a gene

mutation,

gdhA-

which

is none

affect

enzymes

of

the

enzymes

for

coding

The

data studied

a general

repressor',

“nitrogen

A serious

on the

possible

work

Recherche

and comparison

discussion

regulation

differences

in are

of glutamine the

present

was supported Sclentifique

synthetase state

perceptible

of our data in

Candida

by a research

grant Collective,

those

obtained

117-19'1

utilis

our investigation,

of

and interesting

Fondamentale

with

although

some

problems

are raised.

from

Fonds

the

contrat

de

2a

no 985,

REFERENCES

1,

the

in

(Table

the

situation

The

gdhA*

of all of

conse-

excluded

repression" basis

in a

paper),

are

mutation

since

On the

Instead

of two

and this

This

“ammonia

reduction

units

gdhCR

synthetase,

2

11,

(ID.

activity.

to

of

enzymes

allow

presented

This

of

from

liberated

also

to

here,

effacts NADP-GDHase

the

by a factor

strain

enzymes

derepression

fact

are

points

(31,

reduced

RESEARCH COMMUNICATIONS

allantoinase.

The which

now

mutant any

of

compared

until

gdhCR

the

two

is

wild-type

and glutamine

6.7,

obtained

not

whether

relfeve~these

experiments

the

lowering

this

AND BIOPHYSICAL

activity

to

of NAD-GDWese

not

but

NA~-GDHa58

be asked

might

do

BIOCHEMICAL

Grenson, [19721,

M,

and

Hou.

C.:

Biochem,

Biophys.

I.56

Res.

Comm*

2,

749

is

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2. 3.

Dubois, Comm. Dubois,

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Grenson. (19731. Grenson,

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PI.

and

Wiame,

J.M.

PI.

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J.M.:

:

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Siophys.

Res.

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5. Kinghorn, J.R. and Pateman, J.A.: J. gen. Microbial. 2, 39 [19731, 6. Hynes, M.J.: J. gen. f-Xcrobiol. &, 165 (19741. 7. Streicher, S.L., nurney, E.G. and Valentine, R.C.: Nature -239, 495 (1972). 6. Prival, M.J., Brenchley, J.E. and Magasanik, B.: J. Biol. Chem. 246, 4334 (19731. 9. Enchley, J.E., Prival, M.J. and Magasanik, R.: J. Eiol.Chem. 246, 6122 (1973) 0.: Proc. Natl. Acad. 10 .Tyler, 8.. Deleo, A.B. and Wagasanik S&i. USA 71, 225 C19741, ll.Grenson, M., Dubois, E., Piotrowska, M., Dril?ien,R. and Aigle, W.: Molec. gen. Genet. 126, 73 (19741, 12,Drillien, R., Aigle,, and Lacroute, F.: Biochem. Siophys. Res. Corm. 2, 367 C1973). 13,Grenson, M., Mousset, N., Wiame, J.M. and BBchet. J.: Biochim. Biophys. Acta.127, 325 (19661. 14,Messenguy, F.,%nninckx, M. and Wiame, .J.M. : Eur. J. Biochem. 22, 277 (19711. lS.van de Poll, K.W., Verwey, A.A.G. and Koningsberger, V.V.: Proc.

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16,EJoehringer. C.F.: Biochemic~atalogue (Vannheiml, 17.Ferguson, A.R. and Sims, A.P. : J. Fen. picrobiol. le.Ferguson, A.R. and Sims, A.P. : J. gen. Microbial. 19,Ferguson, A.R. and Sims, A.P. : J. pen. ricrohiol.

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