Glutamine and ammonia in nitrogen catabolite repression of saccharomyces cerrevisiae

BIOCHEMICAL

Vol. 75, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

GLUTAMINE AND AMMONIA IN NITROGEN CATAEOLITE REPRESSION OF SACCHARONYCES CEREVISIAE E. DUBOIS, S. VISSERS,

M. GRENSON and J.-M.

Laboratoire de Nicrobiologie, Faculte Bruxelles and Institut de Recherches B-1070 Brussels, Belgium

Received

January

des Sciences, du C.E.R.I.A.,

WIANE

Universite Libre de Avenue E. Gryson, 1,

24,1977

Summary The nitrogen catabolite repression of catabolic (NAO) glutamate dehydrogenase [GDHaseCl, allantoinase and urea amido-lyase is relieved in gin-,gnrR-double mutants. The gin- mutation leads to glutamineauxotrophy; alone it is devoid of regulatory effect. The new gnrR- mutation alone is detectable by a partial derepression of allantoinase only. Considering these facts together with the derepressive effect of the gdhA- mutation on allantoinase, urea amido-lyase and arginase (11, and the absence of gin-,gnrReffect on arginase, one may define three types of nitrogen catabolic enzymes. 1'1 arginase, submitted to NH+ effect relieved in a gdhA- mutant. 2'1 GDHaseC, derepressed by t8e gin-,gnrRcombination. 3'1 allantoinase and urea amido-lyase under both gdhA- and gin-.gnrRderepressive actions. It+is suggested that the gln-gnrR system transmits a signal distinct from NH4 such as glutamine or a close derivative. INTRODUCTION The syntheses the catabolic subject

of arginase.

(NADI glutamate

to nitrogen

repressed

in

occurs

a chemostat

with

with

limiting

Arginase

has been

studied

operator

mutations

allow

gdhA-

catabolite mutations

presence mutation

activity

Copyright All rights

0 1977

is

received

by Academic in any

(41 release added

[21.

by the

Press, Inc.

form reserved.

for

the

to compensate similar

It

induction

as in

state to the

has been proposed

[NADPI glutamate

(31.

and

effects

[Zl.

anabolic

[NADPI

NCR of arginase

The derepression

are

or constitutive indirect

gene

are

of nitrogen.

as well

(21 or in gdhCR mutants

occasional

structural

quantitatively above

nutrient

case to differentiate from

and of

of -S. cerevisiae in 11. Cells as sources

nitrogen

Non inducible

in detail.

or glutamine

mentioned

of reproduction

nutrient

of GOHaseA.

on arginase signal

nitrogen this

[GDHaseCl

or asparagine

as only

[GDHaseAl

of glutamate

derepression metabolic

glutamine

in

of allantoinase

(NCR) (ref.

glutamate

in the

glutamatedehydrogenase catalytic

repression

repression located

amido-lyase,

dehydrogenase

catabolite

when grown

Derepression

nitrogen

of urea

for

even in the the

caused

loss

of the

by the gdhA-

physiological that

dehydrogenase

NH: is

the

which

233 ISSN

0006-291X

Vol. 75, No. 2, 1977

transmits

the

issued

signal

from The

under

gene

GDHaseC

is

control

nature

of

equivalent

for

is

not

effect

the

so

for

ammonium,

a

In

this

presented

for some

the way

glutamine of

input

MATERIALS Strains

participation

of

and and

Hynes

due

we show as

to

is

GOHCR

as

being

and

NCR from

the

The

input

the

glutamine

system by

under

the

signal

regulated

are

amido-lyase

"glutamine

second distinct

is

the of

of a

NH;.

this

"whether

(51.

GDHaseC

urea

and

is

nidulans

question

by

is

are

mechanisms"

differ

synthetase.

enzyme question

manifestations

system", They

confirmed

Allantoinase

the all

occurrence

the

this

Aspergillus

different

"glutamine

;

asparagine

opens

the

control

the

system".

METHODS

: All strains derive from wild type NG1967 are auxotrophic for glutamine and a glutamine auxotroph described previously mutant gdhA-6. Other strains are presented

Medium

a molecule

first

A

In

are

system".

glutamine

[II

and

glutamine

or are

system"

mutants mechanism.

repression.

enzymes

system

gdhA-

glutamine

arginase

of

involves

AND

MG1951. (gin-I, nonsense

and

"NH;-gdhA

"NH:-gdhA

in

NH;,

designated

system.

both

signal.

glutamate

gdhA

the

regulatory

distinct

communication

previously

in

genewith

RESEARCH COMMUNICATIONS

(31.

mechanism

usefully

which

a

number

mechanism,

AND BIOPHYSICAL

derepressed

not

GOHaseC

of

the

gdhCR

of

regulatory

same

to

the

the

the

BIOCHEMICAL

and

growth conditions nitrogen nutrients as 1 mg x ml-l

the carbon source, experiment, usually Growth in chemostat When

limiting,

NH;

given pmoles

Enzyme assays in (31, urea of product

was described is

10'3

M and

formed

Strains NG1958, as strain NG935 Strain ref.

12430~

is

hour-l

are in ref.6. x mg protein-l

measured Activities

is

references

following

are at

a

3.

been described in ref.1. Glucose are specifically mentioned in each as L-isomer ; NH: as 0.01 N (NH412S04. in ref.2 ; doubling time is 6 hours. glutamine 0.5 x IO-3N.

as x

selected (31. in

have

activities

: enzyme amido-lyase

C’l278b.

expressed

as

3O'C.

RESULTS

Ammonia The grown Glutamine

and level

under

glutamine of

four

several and

allantoinase

which of

cause is

as

NH;

measured

in

of

nitrogen

nutrition

repression

repressed signal and

repression

was

similar

less

of one

a

primary

the

interconvertibility glutamine

enzymes

for

signals

conditions

NH:

Identification

as

by for

glutamine.

of

NH;

234

wild

C'l278b

exp.

1 to

except

glutamine.

difficult case

strain 1,

enzymes by

is

type (Table

all

than

NCR The

signal.

the

due of

allantoinase

to

the favors

7).

BIOCHEMICAL

Vol. 75, No. 2, 1977

Table

1 : Activities

Exp.

of

Strains

and

nitrogen

catabolic

enzymes

Genotype

in

a

[wild

12430~

in

Nitrogen

Nr.

Z1278b

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

type1

nitrogen

catabolic

repression

Nutrients

Growth

Arginase

7

1

0.7

1

a

1

0.35

1

NH;

+ glutamine

7

1

0.1

1

Glutamine

9

0.9

0.1

1

Glutamate

50

3.5

94

Chemostat

NH:

Chemostat

glutamine

limited limited

a

52

16

+ glutamate

45

+ glutamine

31

10

Glutamine

11

Chemastatglutamine Chemostat [gdhCR-I

NH;

15

Chemostat 0754G

NH:

limited

NH;

14

16

limited

[gln-ll)

NH;

limited

17

Chemostatglutamine

18

MG195a

excess (0.02 + NH4 + glutamine

19

MG1950

(gln=S,gnrR=51

NH;

+ glutamine

20

PIG1949

[gin-41

NH;

+ glutamine

Tetrad

fromMG195ax3962c

[wild

limited

3

1.3

115

3.6 1.2

37

41

0.5

1

48

a

4.3

322

47

12

3.6

158

45

150

a.5

270

+ glutamine

Chemostatglutamine

[gin-ll,gnrR=11l

50

35

NH:

12597a

Urea amide lyase x 10T3

+ glutamate

NH;

13

Allantoinase

NH:

9

12

Catabolic (NAOI GOHasa

Medium

NH;

[gdhA=Gl

mutants

39

120

IO

300

47

120

4

260

42

62

limited, 9

Ml NH;

138

2

50-100

27

7.6

156

1.5

1.2

33

0.6

0.1

1

type)

21

0746a

[gin-11,gnrR=111

NH;

+ glutamine

IO

8.3

95

22

074ab

[gln+.gnrR+l

NH;

+ glutamine

IO

50-100 1.7

0.1

1

23

0746~

[gin-ll,gnrR+l

NH;

+ glutamine

IO

1.7

0.1

1

24

0746d

(gln+,gnrR=lll

NH;

+ glutamine

3

0.9

0.43

1

25

076ab

[gin-ll,gnrR~l1l

NH;

+ glutamine

50-100

7

108

26

0794d

[gln=4,gnrR=lll

NH;

+ glutamine

50-100

6.6

117

27

0777d

(g1"~5,gnrR=111

NH;

+ glutamine

50-100

6.3

122

28

0863b

(gdhA=6,gl"=11,gnrRz111

NH;

+ glutamine

7.5

45

29

Oa33b

[gdhA=6,gl"=ll,gnrR=l1I

NH:

+ glutamine

[gdhCR-,gdhA=l)

25

a

+ glutamate

176

30

12.759a

NH;

+ glutamine

46

9.9

270

31

0874~

(gdhCR-,gdhA=6.gnrR~11.g1n~~11

NH;

+ glutamine

175

8.6

158

32

Oa42d

[gdhCR-,gl"~ll,gnrR~lll

NH+4 + glutamine

200

9.8

144

glnmutants activity NH;

; +

they

can

glutamine.

A gin-

In

mutant

repression limited

have

the

if

so,

glutamine.

to NH:

They

isolated.

grow

allows

; by

been with

glutamine

latter see will Experiment

case,

as

all needs

if

NH:

be

inert 17

lack

when compared

235

glutamine

sole

synthetase

nitrogen

four conversion added to

or repressed

source

are

enzymes into to

a

16

shows

glutamine

culture

Cexp.231. to

in that

with

a NH;

exert

chemostat

represses

Vol.

75,

No.

2, 1977

arginase

completely

arginase,

result

that

its

GDHase

the

suggests

NH;

is

does

signal

which

into

glutamine.

In

urea

17)

derepressing the

are

fact

(exp.111 is

i

is

is

also

likely

to

be

an

in

mutation

of

indication

an

of

Nitrogen

Exp.

+ glutamate

from

Some

mutants

of

circuit. below.

given

requiring

synthetase

in

these

is

recessive,

it

to

a mutation

allelic

glutamine

character,

and

it

previously

described

allantoinase,

urea

amido-lyase

and

catabolic

NH;

+ glutamine.

The

derepression

when

grown

on

(31.

a result

Some

of

the

is

chemostat Allantoinase

therefore

aboveglutarrine

show

that of

mutation-

glutamine

the

gdhA-

by

be

step

the

in

with

of

have tNAO1

is

a

mutation.

growth

haVcz

is

undetectable.

the

best

in the

cornaination -

as

NCR,

the

auxotrophyl

segregates

mutants

signal would

A first

mutants

for

due

as

for

Auxotrophy

is

IO

gin-[glutamine

glutamine

activity

view

or 9 vs 31 and

mentioned

conditioned and

a rK

in

of arginase.

9 and

gdhA-

is

mutation

this

mutant

to be a primary

glutamine

derepression

NH;

cexp.61.

appears

-distinct

a receptor

type

new

NH:

for

2

already

as

synthesized

individual

such

new IgnrR-I

glutamine

but

excess

o:her bo-ch

a gdhA-

8 vs

requires

with

in

evidence in

(exp.

signal.

a derivative)

catabolite

15

(11

by NH;

that

behavior

simple

mutation

receptor

for

recognition

occurs

the

a signal.

than

suggests

17

also

chemostat

This

[PIAD

experiment

bu-;

be

derepressed Further

the

of

repression

;

of

rjt!h!/:lr.oge,ii3se

and

might

derepression

on

(or

NH:

repression.

effector,

grown

its

less

allantoinase

cells

be

c:sse

previo.:s

glutamate

different

itself

or 61.

5

additional

glutamine

is

a glutamine-limited

with

gdhA‘-

represses

mutation

The

the

a possible

partially As

of

to

the

Insensitivity

mechanism

molecule

II In

the

[NADPI

another

seem

in

strengthens the

COMM’:N’CA”O’\‘S

(21

GOHase

protein.

for

is

contrasts

by

NCR,

glutamine

(exp.

additional

this

affected

NH;

grown

signals

that

not

RESEARCU

(NADI

indeed

of

receptor

amido-lyase

conditions

glutamine

and

signal

so

strain

a gin-

(exp.

repress

not

calls

the

conversion

BlOPHYSlCAL

as a regulatory

only

metabolic

NH:

the

mutation

not

AND

expected

acting

gdhA-

that

does

was

receptor

to

it

but

this

proposition being

BIOCHEMICAL

been

obtained.

a monogenic gin--

mutation

a derepressed glutamate

variable

level

of

dehydrogenase, : high

in

mutant

Beside the present problem, it must be mentioned that an additional mechanism is involved in (NAOI GOHase regulation. This is obvious when one examines the derepression in a chemostat (exp. 6 and 71, which is much lower than in cells grown on glutamate alone in batch cultLlre (exp. 5). No such discrepancy occurs with other enzymes, A simple process of induction of (NADI GDHase is not suggested because of the lack of effect of addition of glutamate to ammonium medium (exp. 2 vs 11. The existence of very different levels of derepression from 6 to 160 units, such as in exp. 6, 7, 13, 16 remains an open question.

236

Vol. 75, No. 2, 1977

MG1956

[exp.

PIG1949

lexp.20).

and

wild

example

It

can

(0748al.

that

mutants

recall

its

gin-

is

and

mutations MG1958

0768b

Iexp.

level

similar We

reach

of

those

sion

with

NH;

the

case

by

gdhA-

mechanism. genes.

For

effect

through

a gdhA-

the toinase,

it

reduction

of by

is the

as

well

a

gdhA-

a stronger

with

reverse,

the

by

shown

partial

by

derepression

repression

and is

by

by by

cells

NH:

and

dry

of

an

of

gln-gnrR of

NH;

by

an a

exp.17. is

clearly

NH:

in

the

NH:

in

NH:

linked

in

the

-5

NH;-gdhA

derepression

of

and,

weight

amido-lyase,

the

of

than

0765~1

indication

mechanism

glutamine

the

no derepres-

absence

limitation

glutamine

237

x mg

urea

also

in

(strain

effect

the

are

NCR

glutamine

only

glutamine-gln,gnrR

effect,

of

provoke

prevented. for

the

is

enzymes

to

participation

there

arginase

combination

is

by

to the

some

unable

signal

nomles

allantoinase

the

is

pool

conclude

mechanism as

of gin--gnrR-

may

For

NH4-gdhA

a participation the

one glutamine

GDHase.

the

mutation

by

by

NH;

glutamine

172

an

gin--gnrR-

a

gnrR>l

recombinant has

of

repression

which

with or

combination

identical

glnr-5

or I).

22

the

The

MG1956,

combined MG1958,

vs

as

gl
in

occur,

(NADPI of

the

are

caused

arginase.

participation

indicated

is

into

presence

the

and

(0746a)

that

glutamine

to

the

repression

shown

mutation

mutations.

of

combination.

(NADlGOHase

mutation NCR

has

these

that

glnY4

when

by

only

mutation

of

mutants

with

21

241.

in of

new

initial

derepression

require

in

the

MG1950,

catabolite

type

show

individual

(exp.

mutant

conditionedby

271

transformation

+ glutamine of

selection

designate

23,

observed

for

[exp.

type

17

and

is

the

gin-

22,

double

reason

gnrR-

and

the

21,

gnrR-

are derepresseo

gin-,gnrR-

0754~1

In

However,

genes.

which

the

(strain

growing

nitrogen

gln

its

when

by

gin=II

the

the

recombinant

wild

[exp.

The

in

the

of

experiment

enzymes

derepressed

the

in

effect

in MG1958

gnrR-. 1

recombinations

0777d

level

to

allantoinase

to

gln~ll.gnrR1ll

which

which

repression

full

and

enzymes

for

those

Hence,

to

to that gnrR

used

MG1949

high The

three

The

by and

251.

that

the

The

observed

mutants

the

18,

is

associated

only.

derepressive

dictated

from

of

undetectable between

Table

obtained

gin-,gnrR-

(exp.261

conclude

involves

are

are

issued

from

gnrR-

derepression

0749d

in

mutation

MG’l950.

mutant

is

cross

mutation

given

derepression gnrR-

it a

always

additional

is

catabolite

MG1949

Recombinants

an is

Different of

(exp.191, from

derepression

unknown.

levels

MG1950

of

the

nitrogen

MG1950

is

[tetratype)

the

mutation.

different

presence

high

in

in

tetradsresulting derepression

bearing

present

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

a partial

0748d

also

medium of

tetrad

a

However,

double

of

needsthe

seen

segregant

11,

indicates

of be

Table Analysis

but

An

is

16,

type

mutation

BIOCHEMICAL

effect, case

the

by of

wild

allantype.

.

Vol. 75, No. 2, 1977

BIOCHEMICAL

A recombination increase the

ofgdhA-.

derepression

derepression

cexp.291

(exp.261.

high

enzymes

(I31

and 14 vs 31. is

an obligatory

is

The presence

of

is

13,

mutations

of gdhAindeed

known to cause

14) imposes

expected

participant

RESEARCH COMMUNICATIONS

does not

mutation

addition

if

the

acting

"1.

a high

a maximal

GDHCR molecule

concomitantly

may lower

of glutamate

[NAD) GDHase (see footnote

which

and exp.

This

and gnrRz11

in glutamate,

levels

The gdhCR'- mutation four

gln='ll

by shortage

causes

AND BIOPHYSICAL

derepression

of all

effect

(exp.

30 vs 9

issued

from

gdhCR gene

in both

NCR mechanisms.

CONCLUSION AND DISCUSSION The nitrogen considered

in

mechanisms

acting

arginase.

It

NCR of the glutamine

catabolite

this

alone

mechanism

under lxx)

An element

issued

gnrR"

regulatory

participant

In enteric

bacteria,

enzymes inhibits

the

the

for the here

the

expression

degradation

is

under

is

likely

"NH:-gdhA"

NCR of the

to transmit

a

and urea

common

catabolite

stimulates acids

dehydrogenase

the

and "glutamine-gln,

an obligate

of amino

enzymes

governs

Allantoinase

of nitrogen

synthetase

governs

mechanism

signal.

synthetase

of glutamate glutamine

it

NH;)

gene gdhCR is

glutamine

catabolic

of two distinct

The gln-gnrR

of both

the

the

control

The gdhA mechanism

from

control

from the

synthesis

presented

the

dehydrogenase,

distinct

for

responsible

the way by which circuit

glutamate

derivative

are

(NCR1 of the four

under

an NH; signal.

catabolic

amido-lyase

is

or together.

transmits

(or

repression

publication

(6,

participates

the

repression. synthesis

to glutamate

of and

91.

In -S. cerevisiae, to the glutamine

study.

Acknowledgements This work was supported by grant Recherche Fondamentale Collective. The skilful technical assistance gratefully acknowledged.

nr.2.4542.75

from

the Fonds

of F. Muyldermans-Verhaegen

de la is

REFERENCES 1. Dubois, E., Grenson, M. and Wiame, Res. Commun. 50, 967-972. 2. Dubois. E., GreGon, PI. and Wiame, -46, 603-616.

J.M.

I19731

Biochem.

J.M.

(19741

Eur.

Biophys.

.I. Biochem.

(XXI The double control of allantoinase and urea amido-lyase is, in part, at the origin of the opinion that the anabolic glutamate dehydrogenase is not involved in the regulation of these enzymes T71. lack of isogenicity among strains used by Bossinger and Cooper increased the confusion (Oubois. Vissers and Wiame, unpublished data).

238

Vol. 75, No. 2, 1977

3.

Dubois,

60, 4. Grzson, 5. 6.

7.

E.

and

BIOCHEMICAL

Grenson,

PI.

[I9741

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Eiochem.

Biophys.

Res.

Commun.

150-157. M., Oubois,

E., Piotrowska, M., Drillien, R. and Aigle, M. (19731 Molec. Gen. Genet. 128, 73-85. Hynes, M.J. (19741 3. Bacterial. 120, 115-123. Roon, R.J. and Levenberg, 8. [1970)Methods in Enzymology 17A. 317. Bossinger. J. and Cooper, T.G. [I9751 Biochem. Biophys. RerCommun.

66, 889-892. 8. Przal, M.J., Brenchley, 240, 4334-4344. 9. BrGhley, J.E., Prival. -248, 6122-6128.

J.E.

and Magasanik,

8. (19731

J.

Biol.

Chem.

M.J.

and

8.

J.

Sol.

Chem.

Magasanik,

239

(1973)