Decrease of the plasma membrane H+-ATPase activity during late exponential growth of Saccharomyces cerevisiae

Vol.

133,

No. 3, 1985

December31,

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS Pages

1985

DECREASE DURING LATE Philippe

OF THE PLASRA MEMBRANE H+-ATPase EXPONENTIAL GROWTH OF Saccharomyces

Tuduri,

Laboratoire

NSO,

Jean-Pierre

October

31,

ACTIVITY

cerevisiae

Dufour

d'Enzymologie and Laboratoire des de Louvain, 1348 Louvain-la-Neuve,

Universite Received

Emmanuel

917-922

and

Sciences

Andre

Goffeau*

Brassicoles, Belgium

1985

During the last cell division of exponential growth, the H+-ATPase activity from the yeast plasma membrane decreases by a factor of two to three. This "arrest growth control" of ATPase activity is not accompanied by modification of the sensitivity to vanadate. Q 1985 Academic Press, Inc.

Upon

addition

cation

of

pump

located

process

the

has

pump

is

of

medium

in

plasma

the

identified

discovered

by

reconstituted

proposed

that

Ht

of

K+

from

(II),

the

pyrimidic ATPase in

bases from

the

tightly

the

uptake (13),

cell.

yeast

H+pump

intracellular recently

It

can

been

whan all

incubation

correspondence

(IO) (12)

of

amino of

commands

of of

washed

should

(IO)

of

yeast

and Ca

cells

2+

not

only

movement puric

and

The

Ht-

functions its

activity

the

is

fungal pH

(16).

and been

(14).

internal

with

(41,

has

cellular

physiological

effecters

Ht

(6)

related

acids

that

or

It

sugars,

expected

external ATP

acids,

vital

Indge

9).

to the

a Ht

This

Goffeau

is

organic

thus be

proposed:

also

an

and

membrane

but

physiological

concentration that

plasma

therefore

Several have

yeast

(3).

Eddy

8,

by

elect rogenic

crassa

(7,

acidifi-

ated

similar

Dufour

vesicles

production

membrane

A

by by

This med

Neurospora

purified

CaZt the

(2).

in

(II.

partially

postulated

cells

of and

plasma

Slayman

from

acids

least

at

proteolipid

yeast

secrete

membrane

(S),

H+-ATPase

regulated.

membrane

-0

et al.

intact

is

ATPase

in

the

efflux

by

membrane

Matile

recently

to

yeast

plasma

cells

yeast

extracellular

been

the

glucose,

Serrano glucose

plasma (IO,

IS),

reported activates

be addressed. 0006-291X/85

917

All

Copyright 0 I985 rights of reproduction

$1.50

by Academic Press, Inc. in any form reserved.

Vol.

133,

remarkably

the

were

recently

tion

of

ATPase

the

suggested

by

not

activation

report

here

During

the

ATPase

activity

is

(18)

glucose

not

cell

of

regulation

cell

generation

of

from

different

with

results a stimula-

by

other

sugars.

protoplasts,

some

peripheral

wall

with

snail-gut

of

plasma

it

was

proteins

membrane growth

strains

These

observed

also

but

exponential

yeast

(17). who

observed

requires the

type

membranes

Kotyk

by

of

another

last

only

activation

removal

yeast and

glucose

by

ATPase

during

from

Sychrova

activity,

that

lost

activity

confirmed

ATPase

Because

are

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

No. 3, 1985

decrease

which

juice

(18).

ATPase

activity.

the

plasma

by

a factor

We

membrane of

2 to

3. Materials

and

methods

The haploid wild type strain Saccharomyces cerevisiae 21278b (19) was used. One hundred ml of 1% yeast extract (Difso), 1% Bacto peptone (Difco) and 2% glucose were inoculated with 1.25 x 10 cells and agitated at 30°C from 15 to 30h. The cells were washed twice with 30 ml of distilled water and homogenized as follows. 450 mg wet weight of washed cells were suspended in 1 ml of grinding medium containing 250 mM sucrose, 10 mM Tris-HCL pH 7.5 and 1 mM phenyl methane sulfonyl fluorid (100 mM stock solution in dimethyl sulfoxid). To 900 ~1 of this suspension, 900 mg of glass beads (0 0.45 mm) were added and the mixture was agitated 9 times during 17 s with an interval of 3 set using the CO2 refrigerated MSK Braun homogenizer with a micro adapter. The homogenate was centrifuged 5 min at 1000 x g and the pellet was rinsed at 1000 x g for 5 min with 500 ~1 of grinding medium. The combined supernatants were centrifuged again 5 min at 1000 x g. The resulting supernatant was centrifuged 40 min at 15000 x g. The pellet was suspended in 300 JJ~ of 250 mM sucrose, IO mM Tris-HCC pH 7.5. A maximum of 5 ~1 of this final suspension called "crude membrane fraction" was incubated for 8 min at 38°C in 100 ~1 of 6 mM ATP, 9 mM MgCL 50 mM MES-KOH pH 6.0, 10 mM NaN . The reaction was stopped by 300 ~1 o f ' 1% sodium dodecyl sulfate, 250 p? molybdate reagent and 250 ~1 of Elan reducer (20). The phosphate released was estimated by absorbance at 600 nm. Protein were determined by the Folin procedure (21) using bovine serum albumin as standard. Results The ned

by

membrane

assays

centrifugation This

min.

all

ATPase

were of

crude

the

membrane

ATPase

measurements

efficient

inhibitor

conditions

the

carried

fraction

activity

is

were

carried

ATPase

of

total

out

crude

membrane

cell

homogenate

with

contains

heavily out

in

the

918

at

by

presence

ATPase

measured

at

fractions

15000

of

is

all

the

mitochondria.

activity

pH 6.0

obtai-

x g during

virtually

contaminated

mitochondrial

activity

in

more

40 plasma

However,

NaN3

which

is

(22).

Under

these

than

80%

inhibi-

an

Vol.

133,

No. 3, 1985

ted

by

10

membrane

PM

after

our

20

from

by

a

h of

to

of

20th

the

20fh

medium

it

is

the

at

not

was

w

for

a

the

secreted by

of

with

dropped

raise

a

of

by

marked 1.2

15th of

pH

in

the

ATPase

achieved

the

pmol

and

return

part

yeast

ethanol.

the

by

in

the

from

between

large

followed growth

acids

accompanied

300

medium

exponential

was

-1

to

the

metabolized

followed

is

medium effect activity".

other

the

for

sugars when

during

the

culture

still cells

here This

effect

rebound the

industrial

showed

a

similar

drop

activity

seems growth

in

strains

the

thus

of

sugars been

it

has

available

(around

2X

weight/weight)

might

called

the

be

seems

919

to

be

different

in

very

(temperature,

strain

and

be

since

grown

decrease

growth

of to

yeast

industrial.

stopped

ATPase

for

conditions

to

of

growth

industrial growth

CE18

and

exponential

related

were

described

ATPase

of

different

the

the

of

and

specifically

since

strains

end

laboratory

under

exponential

decrease

to

in

decrease

brewer

The

related

not

similar

end of

the

shown).

and

fermentable

ATPase

x

was

with

that

Four

both

medium,

growth

drop

which

shown

CE18.

It

The of

is

observed

media

growth that

it

observed

aeration).

This

-1

of

utilization

activity

x min

concomitantly

phenomena

different

Pi

value

h,

2

(data

a general

I).

original

strain

activity

umol

were

glucose

ATPase

of

the

up

hour

organic

diauxic

in

slowly

change

end

glucose,

known

a

obtained

I).

was

brewer

plasma

During

twenty-fifth

the

glucose

growth

0.6

at

of

well

the

was

cells/ml. raise

by

and pH

IO6 to

accompanied

of

membrane

to

24th

Fig.

activity

the

growth

x

continued

of

the

(Fig.

its

(Fig. In

-1

m9

to

raise

exponential

culture to

largely

exponential 200

twentieth

fermentation

plasma

x

h of

activity

of

was

exhaustion

during)

the

-1

x min

the the

(or end

changes

The

of

density

the

5.0.

during

The

end

growth

between

after

before

the

reflects

approached

cell

slow

pH

that

cells

therefore

density the

5.8

to

medium

cell

the culture,

return

and

conditions,

This

4.9

indicates

Pi

culture

cells/ml.

x IO6 pH

orthovanadate activity.

h when

10

the

of

ATPase

In

next

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

pH, in

the

verified in

to floculate.

began "growth

from

arrest "activation

control of

Vol.

133,

BIOCHEMICAL

No. 3. 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Growth

I

I 50

01

TimeIhoursl

Time course for cell division, plasma membrane ATPase activity siae E1278b. Conditions for measurements are described in

Fig.

2. Time course of cellular division and plasma membrane ATPase activity in the brewer strain 5'. curZsbergensis CE 18. The cells were grown at 10°C in a non aerated fermentor (height 1.2 m - e 6 cm) containing 3 1 of uort at 11.87 g of fermentable sugars/100 g of medium. Agitation of the medium was carried out by the CO2 produced during the fermentation. Aliquots for ATPase activity (measured at 30°C) and cell counts measurements were taken from the uppp~ part of the fermentor. The decrease of cell counts after the 100 h of culture is due to floculation.

by

glucose"

Sychrova

cells

5

in

min

was

plasma

membrane

30°C

and

Kotyk

from

early

6.4,

glucose

the

the

concentration PM,

the

the

activated

ATPase

In

contrast,

"the

decrease exponential

of

the

to

growth.

It

pmol

Pi was

of

for

half

arrest by

a

observed

to controL

factor in

920

of water

incubation the -1

x

reversed

ATPase m9

-1

in

25

UM of

of

ATPase"

2

to

specific in

enriched

about

5 min

shifted was

1.2

mM to

oligomycin

at

cells

from

5.7

decreased Cl.3

mM (17)

(18).

described

3 observed

washed

about

modifications

pH was

from

in

for

inhibition

decreased

sugars

described

qualitative

optimal

sensitive

other

was

x min

the

ATP

for

galactose

Remarkable

vanadate

confirmed

After

activation:

Km for

is

1.3

glucose.

growth

activity

or

stimulation

became

ATPase

phase.

maltose

0.2

and

activation"

growth

This

out

(17)

"glucose

stationary

fractions.

to-3-5

The

from

during

17-18

Serrano

trehalose,

washing

occured

by (18).

stimulated

after

from

described

2% glucose,

activity

and

pH of the extracellular medium and in the laboratory strain S. cereuiculture and for growth ATPase and pH Materials and Methods.

1.

washed

to

150

Fig.

ATPase by

100 Time (hours)

here at

which

the

is end

were

a of

not

Vol. 133, No. 3, 1985

Table

1. siue

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vanadate Z1278b.

number cells (IO6

sensitivity Growth and described

of

time of culture

x ml-')

0.92

0.3

100

17

1.04

0.2

200

20

0.54

0.3

230

22

0.78

0.2

accompanied

by

a modification

for

50%

which

the

data

plasma

membrane

which

occur and

Table

the

establish

the

ATPase during C.

activity the

Gancedo,

last personal

glucose

or

sensitivity

1 that low

decreased to

with of

remains

markedly

Francois

incubation

in

inhibition

interesting

(g4)

16

a short

the

CO.2

ATPase

to

0.5

PM)

specific

causal

the

cell

sugars.

vanadate.

under

of

vanadate

the

growth

division

It

between

numerous of

It

Indeed

activity.

relations

and

other

concentration

to

S. cemuiout as

50% ATPase inhibition vanadate

85

by

from

ATPase specific activity Curnot x min -1 x w -5

(hours)

"activated"

seen

of the plasma membrane ATPase from ATPase measurements were carried in Materials and Methods.

is

it

can

be

required conditions

should this

not

be

very

decrease

metabolic

modifications

exponential

growth

of

CJ.

communication).

Acknowledgements Thanks are due to Nieuwenhuis and A. Schlesser This work was supported

Etienne for technical assistance. Dr. B. are thanked for help in computer analysis. by the Services de Programnation de la PoliBeige and the Fords NationaZ de la Recherche Scientiof the Direction BioZogie de la is publication no 2291

tique Scientifique fique Beige. This Commission oks Comnaut&

P.

Europdennes.

References

1. 2. 3. 4. 5. 6. 7.

8.

Lavoisier, Trait& 6LCmentaire de chimie present& dans un nouveau et d'apres les decouvertes modernes (1789). Conway, E.J., Brady, T.G. (1947) Nature 159, 137-138. Slayman, C.L. (1965) Gen. Physiot. 49, 69-92. Eddy, A.A., Indge, K. (1962) Biochem. J. 82, 15-16. Matile, P., Moor, H., Muhlenthaler, K. (1967) Arch. Mikrobiol. 201-211. Goffeau, A. J. Biol. Chem. (1978) 253, 7026-7032. Dufour, J.P., Villalobo, A., Boutry, M., Goffeau, A. (1981) J. Biol. Chem. 12091-12087. Dufour, J.P., Goffeau, A., Tsong, T.Y. (1982) J. Biol. Chem. 9365-9371.

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AND

BIOPHYSICALRESEARCH

COMMUNICATIONS

Malpartida, F., Serrano, R. (1981) FEBS Lett. 131, 351-354. Pena, A., Cinco, G., Gomez-Puyou, A., Tuena, M. (1972) Arch. Biochem. Biophys. 153, 413-425. Pena, A. (1975) Arch. Biochem. Biophys. 167, 397-409. Boutry, M., Foury, F., Goffeau, A. (1977) Biochem. Biophys. Acta 464, 602-612. Foury, F., Goffeau, A. (1975) J. Biol. Chem. 250, 2354-2362. Duro, A.F., Serrano, R. (1981) Current Microbial. 6, 111-114. Sigler, K., Knotknova, A., Kotyk, A. (1981) Biochem. Biophys. Acta 643, 572-582. Brooker, R.J., Slayman, C.W. (1983) J. Biol. Chem. 258, 8827-8832. Serrano, R. (1983) FEBS Lett. 156, 11-14. Sychrova, H., Kotyk, A. (1985) FEBS Lett. 183, 21-24. Bechet, J., Grenson, M., Wiame, J.M. (1970) Eur. J. Biochem. 12, 31-39. Dufour, J-P., Goffeau, A. (1986) Methods in Enzymol. (in press). Lowry, O.H., Rosebrough, N.J,, Farr, A.L., Randall, R.J., (1951) J. Biol. Chem. 193, 265-275. Goffeau, A., Slayman, C.W., (1981) Biochem. Biophys. Acta 639, 197-223.

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