Effect of ammonia and glutamine on macromolecule synthesis and breakdown during sporulation of saccharomyces cerevisiae

BIOCHEMICAL

Vol. 79, No. 2, 1977

EFFECT

OF AMMONIA

AND BREAKDOWN Odile

AND GLUTAMINE

DURING

September

ON MACROMOLECULE

SPORULATION

Durieu-Trautmann

Institut Universite

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

de Recherche Paris VII.

Colette

en Biologie Moleculaire 2 Place Jussieu 75005

is

well

glutamine

synthesis ;

specific

- 3).

and

some,

observed

to

cells

- 6).

(5 and and

the

Some

sporogenesis

they

differ

same

glutamine nonsporulating

the on

in

strains

- 6)

such

not

such

as premeiotic

are

specific (51,

both the

since

they

we

describe biochemical

Saccharomyces

and the

(1) DNA

sporu-

partially

sporulating protein

sporulating

above of

in

ammonia for

are

as RNA,

specific

study,

of

by

quantitatively

are

present

inhibited

breakdown

others,

extent some

(5

protein

only

'; while

sporulation in yeast, events during sporogeand non sporulating on the increase in dry suppression of the after 4 hours. The inhiand protein syntheses is so for sporulation.

events

breakdown

necessary, In

is

biochemical

as RNA and

cells though

synthesis,

that

glycogen

such

because

sporulating

tine;

known

(2 (4)

lation

nia

du C.N.R.S. Paris.

13,1977

It

non

CEREVISIAE

Delavier-Klutchko

SUMMARY. The effect of two known inhibitors of ammonia and glutamine, on certain biochemical nesis have been studied using sporulating a/# eC/oC cells. Both strains gave similar results cell weight, protein and RNA breakdown and the intensive RNA and protein syntheses occurring bitory effect of ammonia and glutamine on RNA reversible under the same conditions which do

and

SYNTHESIS

OF SACCHAROMYCES

and and

have

glycogen been

nonsporulating effects events

of in

ammo-

sporula-

cerevisiae.

METHODS Two diploid strains, isogenic except for the mating type, Cultures were grown in a prespo532 a/oc and 533 or/k were used (7). rulation medium containing yeast nitrogen base, 6.5 g, ammonium sulacetate, 10 g, per liter of 0.5 mM potassium fate, 5 gT and potassium phtalate pH 5.7. After 6 generations,cells in exponential phase were transferred to a sporulation medium of potassium acetate and raffinose these conditions, the first asci appeared at about T16 (8) ; under and the final yield attained approximately 60% (3)(Fig. 2). When required 5mM ammonia or 10 mM glutamine were added. They were removed by washing and placing cells in fresh sporulation medium. DNA was extracted according to the procedure of Roth and Silva-Lopez reported by Haber and Halvorson (9) and essayed colorimetrically using Burton modification of the diphenylamine reaction (DI. RNA and protein synthesis were measured by pulse labelling with p3H] adenine, 2 pc/ml, andp4C11eucine, 0.2 p&ml, as described by Hopper and al. (5). cells were grown with and protein breakdown, leucine and transferred to unlabelled sporuadenine or

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

438 ISSN

0006-291X

BIOCHEMICAL

Vol. 79, No. 2, 1977

AND BlOPHYSlCAL

i5&cLz

co 0 2 40 .F

tl a

; 20 z A 0

2

4

6 tn sporulation

Hours

0

8

s CI

10 medium

0,

1

2

Fig.

1.

Fig.

Increase of At To, weight cells (Ad) with inhibitor

2.

Effect cells Without

dry of was

ccl 10 8 2.3 (w,

RESEARCH COMMUNICATIONS

Hours

weight a/4 cells me;. Without u).

in sporulation

(0-O) was inhibitor

of inhibitor on the kinetics during sporulation. inhibitor (O-01, with inhibitor

of

2,l

medium

mg, cf 108 0(/w (O-0, u) and

DNA synthesis (0-O

in

a/

).

RESULTS As only

one

identical

curve Cell

results

will number

experiments

in

all

In

the

be

were

obtained

presented

did

not

cases

for

change

(a/oc

with

both

ammonia

inhibitors

appreciably

and

q/q

in during

cells,

with

and

glutamine,

each

figure.

the and

course

of

without

inhi-

bitors). Dry

weight.

cells

increased

tion

medium

weight

by contained

was

higher

of

dry

presence

of

inhibitors

in

the

synthesis in

will

Fig.

both

2 for the rate

Protein synthesis

synthesis. was

In

weight of

the

the

initial

value

or

glutamine,

cells was

(70%)

The effect be reported

inhibitor,

the

ammonia

cell

presence

DNA synthesis.

of

over

(70%).

increase a/oc

absence 50%

and

same

was

rise

to

sporulate

in

the

similar

of

sporulating

When

sporula-

of

dry

cell

((* /a~ ) the

absence

tothat

and

observed

in

the

with

inhibitor. of ammonia in detail

of glutamine elsewhere

the sake of comparison. of DNA synthesis as well In observed

weight

(Fig.1). the

unable the

dry

In the presence of as the quantitywere

the absence of inhibitor * around T 1 ' followed hours

after

439

on premeiotic DNA (3) and is indicated

a clear by

transfer

peak

a rise of

inhibitor, reduced.

at the

of

protein

T4 which cells

to

BIOCHEMICAL

Vol. 79, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

60 I

Hours

Fig.

3.

Fig.

to

RNA synthesis Same legend

maintained

incorporation both

and T1, TO.5 ne was added prevented depressed.

of

incorporation a second

5 and 6, incorporation bitor, by

or

of

protein

show

whatever a decline

To

added

at

Addition

T2,50,

q/q

time

of

3, one

this

the

(Fig.

adenine

rate

T4.

When

glutamine

of c 14c I were obtained

440

was

incorporation, after This

and

showed

peak

two

between of

RNA synthesis

glutamiwas

immediately was

. Results

later.

varied

ammonia

Inhibitor

removal. hours

4)

: a sharp

after

C leucine [ RNA 1 fractions

three

lo6

second increaof the inhi-

results

medium

f4espectively-

of or

or

, RNA synthesis

RNA synthesis.

T7

4 x

3). andq/cq

sporulation of

and

Similar

(Fig. a/q

and

an increase into protein the

ammonia

T2 . 5 the second period of at T4 or T RNA synthesis 7

and

approximately

of

synthesis rapidly.

broad

arrows.

T2 . 3. prevented the into protein. Addition

strains in

the

(broken

or

protein

and

both

; when

removed

To

declined

and

Reversibility and

7

a/q

at

at

stopped

incubation

periods

Ti2.

incorporation

In

throughout main

medium

strains

synthesis.

least

inhibitor

cells.

in a/q or o( /o( in Fig. 3.

at

the

leucine with

as

until

sporulation lb C leucine se of [ 3 bitors at T4 or T

RNA

medium

Protein synthesis in a/q or o(/o( cells. Without inhibitor (solid lines) and with lines). Inhibitor was added at the time shown by cells were used for counting.

4.

was

in sporulotion

added

at

illustrated the

increase

To

in Fig. 3 or H adenine removal of inhi-

I 3

was

follewed

BIOCHEMICAL

Vol. 79, No. 2, 1977

0

0 2 Hours in

5

Fig.

5.

Fig.

6. Effect

In

4 6 sporu~al~on

8

2 in

4 6 sporuiation

- -a 8 10 medrum

0 6

Effect of inhibitor removal on protein synthesis in a/q cells. or q/w Control cultures without inhibitor (-1 and with inhibitor (o--o). Inhibitor was removed at the times indicated by arrows. 4 x 106 cells were used for counting.

and

cells

removal Fig.

RNA breakdown.

RNA breakdown

tely

0 Hours

10 medtum

of inhibitor cells. * /o( Same legend as in

and

RESEARCH COMMUNICATIONS

+ ---O--5”*

i-i”--$-p--o

I?

Protein

AND BlOPHYSlCAL

submitted

to

three-fold

in

all

of

this

described

more

the

in

a/q

cells

or

5.

As

occurred

on RNA synthesis

cells

by

inhibitors,

cases

Hopper

extensively

and

in

a/q

breakdown

(data

not

al. than

(51, in

increased

protein

d /W cells.

approxima-

shown).

DISCUSSION The and

aim

glutamine,

involved

which

in

at

least

two

RNA

ses

occurring

with rulation.

our

determine

the

sporogenesis,

it.

previously

of

(3)

action,

on that

one

functioning

and

a later

The

however

inhibitors

premeiotic DNA synthesis work, we have observed and

to

DNA synthesis,

to to

inhibit

shown

sites

related

related

was

effect

some

of

of the

ammonia

main

events

sporulation.

We have bly

work

syntheses

which after

strains, Moreover

T4. inhibitors when

place inhibitor

occur

before

Thus,

it

seems

T4

have

no

around

between affect

glutamine T4

the

have

T4, and

probaTIR

not

initiation

of

before T4. In the present acts on the protein

; but that,

effect

added

and

early one

do not

which takes that neither

inhibitors

ammonia

at

both

prevent

the

in our conditions on the initiation To

are

removed

syntheand of spo-

later

than

Vol. 79, No. 2, 1977

Tq,sporogenesis protein

recommences

syntheses,

which

protein to not

BIOCHEMICAL

the

and

action

endowed

obtained

of

synthesis

RNA syntheses

and

of

in 3

RNA and

ACKNOWLEDGMENTS : manuscript.

We thank

Dr.

parallely

RNA and

again. after

T4

events since

and

; but

similar

sensitive are

probably

results

are

cells. are

untreated

protein

and

occurring

d /o(

adenine

(3) increase

specificity

c 1precursors these H

T4

necessary

RNA breakdown

than

of

are

a certain

and

about blocked,

nonsporulating

Protein the inhibitors 14 I: C 3 leucine utilisation

were

inhibitors

with with

at

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

higher

cells. into and which

The

for

medium

may

also

help

cells

intensive

the follows

A. Kovox-

in

may be explain

the

submitted release due the

removal

in preparing

to of

to

the

non

brief of

inhibitors.

the

REFERENCES I. MILLER, J.J. (1963) Can. J. Microb. 9, 259-277. 2. PIfiON, R. (1977) Exp. Cell. Res. 105, 367-378. 3. DELAVIER-KLUTCHKO, C., DURIEU-TRAUTMANN, O., ALLEMAND, P., and TAVLITZKI, (in preparation). 4. ROTH, R., and LUSNACK, K. (1970) Science 168, 493-494. 5. HOPPER, A.K., MAGGE, P.T., WELCH, S., FRIEDMAN, M., and HALL, B.D. (1974) J. BacterioLll9, 619-628. 6. KANE, S.M., and ROTH, R. (1974) J. Bacterial. 118, 6-14. 7. PELLECUER, M. (1973) ThPse de spGcialit6 U.S.T.L. Montpellier. 8. ROTH, R., HALVORSON, H.O. (1969) J. BacterioL 98, 831-832. 9. HABER, J.E., and HALVORSON, H.0, (1975) Methods in cell biology, vol. XI, 46-67. IO. BURTON, K. (1956) Biochem. J. 62, 315-323.

442

J.,