BIOCHEMICAL

Vol. 178, No. 3, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1359-1364

August15,1991

PROTEIN PHOSPHORYLATION Lucia

Department

Received

BY INORGANIC PYROPHOSPHATE IN YEAST MITOCHONDRIA

Pereira

of Biochemistry,

June

28,

Marika Lindahl, Baltscheffsky

da Silva*, and Herrick

Maria

Arrhenius Laboratories, S-106 91 Stockholm, Sweden

Lundin#

University

of Stockholm,

1991

SUMMARY. Inorganic pyrophosphate can function as phosphate donor in protein phosphorylation reactions in yeast mitochondria. It was shown that, when PPi substitutes for ATP as inhibitor of the pyruvate dehydrogenase reaction, maximal activity is reached after a lag-period of 30-60 minutes. 32P-labeling of peptides shows that [32P]PPi gives about 25% of the labeling obtained by [v-~~P]ATP in the protein kinase reaction. The PPi dependent phosphorylation is increased several fold by the presence of cold ATP. 0 1991 Academic Press, Inc.

The covalent sidues

reversible

in many enzymes

lation.

This

process

in eukaryotic protein

[l]

including

acting

together

cellular

with

The activity

of

and plant

tissues

cycles

of phosphorylation

the

complex

the

covalently

hydrogenase tion

kinase

of this

activities

from

agreement

with

All

reports

these

*Present #Present medical

address:

their

Neurospora

phosphatase

(PDH)

the

with

kinase

during

Saccharomyces

de Bioqu?mica,

complex

found

from

different

through inactivates by removing

no pyruvate

de-

and purifica-

on PDH complex crassa

[2],

carlsbergensis isolated

UNICAMP,

Institute for Cancer Research, S-751 23 Uppsala, Sweden.

kinase but

in

[17]. PDH comp-

Campinas-SP-Brazil.

Uppsala

Branch,

Bio-

0006-291X/91

1359

in

and they

A kinase

of the IB,

been

regulated

isolation

reports

activities

by

the

have

cerevisiae

and Neurospora

obtained

catalyzed

activity

lactis

results

address: Ludwig Center, Box 599,

its

with

concerned

Departamento

is

in contrast [12]

is

regu-

to occur

[4-61

[12-141.

reported

re-

[7-Ill.

crassa

regenerates

has been

[15-161,

kinases

activity

In Saccharomyces

phosphate.

It

mitochondria

and dephosphorylation

Kluyveromyces the

including

acid

reported

to perform

These

dehydrogenase

and from

activity

complex

[3],

the pyruvate

[2].

phosphatases

on CAMP for

and a specific bound

crassa

amino

of metabolic

and has been

reactions.

may or may not be dependent

to certain

known mechanisms

Neurospora

compartments

animal

best

in nature

phosphorylation-dephosphorylation different

of phosphate

one of the

is widespread

cells

kinases,

is

binding

$1.50

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 178,

lex.

On the

reported

other

were

complex

lost

of the

incubated

in the

reactions

that

PPi can also

no significant proteins MATERIALS Yeast a galactose

PDH complex

in spinach

kinase

[18].

and/or

isolated

as ATP acts

becomes

has been

phosphatase

we investigated

mitochondria

shown

inhibited

to act

thylakoids

from

when

the

activithe

PDH

Saccharomyces

as phosphate

donor

was observed with

PPi

as a phosphate

[19,20].

be used as phosphate

labelled

pea mitochondria

in situ

the

RESEARCH COMMUNICATIONS

donor

and that

mitochondria

are

of PPi or ATP.

difference

were

whole

PPi has been

nase

intact

procedure,

PPi as well

presence

Recently,

that

purification

the

that

of

phosphorylated

the

within

AND BIOPHYSICAL

PDH complex

possibility

during

We show

activity

the

reversibly the

activity

cerevisiae. the

hand

to become Considering

ties

BIOCHEMICAL

No. 3, 1991

donor

in protein

Here we show for in yeast

the

mitochondria.

in the autoradiogram

first

kitime

Moreover,

patterns

when

or ATP.

AND METHODS cells (Saccharomyces medium as described

cerevisiae in

strain,

NCYC 1075)

were

grown

in

Cells were harvested in the beginning of the stationary phase and mitochondria were isolated using the zymolyase method as reported previously in [21] with some modifications. The lysis buffer contained 1 mM PMSF, 0.1% BSA, 2 mM DTT and 4 mM MgC12, PMSF and BSA were excluded from the second washing. The mitochondrial suspension was stored in the same It could be used up to the second washing and kept on ice. significant loss of the phosphorylating capacity. Protein performed using a BIO-RAD colour reagent with bovine serum dard.

buffer as used in seven days without determination was albumin as stan-

Studies on the mitochondrial PDH complex were performed tochondria, incubated at 25°C in 500 pl of 0.6 M mannitol/20 phosphate buffer pH 7.0, containing 2 mM MgC12, 2 mM DTT and mitochondrial protein. At different times, aliquots of 20 to transferred to a cuvette containing the medium for the assay hydrogenase complex activity, which was performed as described al. in [lS].

with intact mimM potassium 150 to 250 pg of 50 ul were of pyruvate deby Uhlinger et

Protein phosphorylation experiments with [32P]PPi or [Y-32P]ATP were performed with intact mitochondria in a 0.6 M mannitol/2D mM Tris-HCl buffer pH 7.2, in the presence of 1 mM DTT and 5 mM MgC12, at a protein concentration of 200 ug/ml (100 ul final volume). 10 mM NaF was present in order to abolish any phosphatase and pyrophosphatase reactions. Other additions to the reaction media are described in the legends of the figures. The mitochondria were incubated for 30 min at 25°C with 0.4 mM [32P]PPi or [Y-32P]ATP in amounts corresponding to 24 pCi per mg of mitochondrial protein. Additional MgC12 was added to maintain a constant final concentration of 5 mM free Mg2+ ions. The reaction was stopped by the addition of an equal volume of the same buffer used for the incubation, containing 20 mM EDTA. The mitochondria were then spun down in an Eppendorf centrifuge for 5 min and resuspended in the same buffer without EDTA. An equal amount of Laetmili solubilizing buffer containing 10% B-mercaptoethanol was added and the samples were heated at 80°C for 5 min before being submitted to SDS-PAGE. The gels were prepared according to Laemnli [22], using a linear 12 to 22.5% polyacrylamide gradient and run overnight at 30 mA and -4.5"C. Bromophenol blue was used as a run indica1360

Vol.

178,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

tor. The gels were stained with Comassie brilliant blue, destained, radiographed. The relative phosphate incorporation was quantified densitometry scanning of autoradiograms. Pyrophosphate hydrolysis red according to [23].

and autoby laser was measu-

RESULTS AND DISCUSSION When intact complex (Fig.

yeast

activity

it

increases

1) and reaches

ding

on the

activation

ence

of the If

process action

this

incubation

due to a kinase its

activity.

the

terns

be the pears

at about

band

at -40

kDa is

the

incubation

of a phosphate rate,

and so inhibiting

corresponding is

donor,

probably

known

decrease to inhibit

is able to act as a phosphate

[32P]PPi proteins

obtained

decreased

201

;

15-

% z

lo-

of 0.4 appears

ATP is 3).

mitochondria.

[y-32P]ATP

present

40 kDa.

strongly

extremely and the

are

phosphate

band aplow

label

concentof the

to this

Z

E 2

50 ~,,,,‘,,,,[,,.,‘,,,,‘,,,,I,,,,‘,,,,[,*,,’ 0

5

10

15

Time Fig.

1.

20

25

30

35

40

(min)

PDH-complex activation is inhibited by ATP and PPi. Control without additions (O), 1 mM ATP present (@, 1 mM PPi (A), 10 mM NaF (a). 1361

do-

may well

labeled

(i.e.

explanation

The pat-

This

kDa appears

One possible

donor

or [32P]PPi

mM of any of these at around

A second

band at 5-10 (Fig.

c -ii

either

[ll].

PDH complex

a strong

in yeast

with

band

If no cold

28 kDa.

of [v-~~P]ATP

the

activation

which

inhibits

as a consequ-

1).

labeled

of the

is

10 mM NaF,

In the presence

same.

during

PDH complex

interpretation

of several

strongly

a-subunit

ration

(Fig.

acting

the

The additi-

progressively results

presence

in the the

with

2 we show that

the

in the

depento get

shown).

these

PDH

manner

required

(not

medium,

phosphatase,

a decrease

this

autoradiograms

the most

incubation

We interpreted

performed

obtained

phosphorylation

essentially nors

with

rate

activity

of the

is

ageing

phosphorylating

In line

In Figure

1).

of the

of incubation,

The time

mitochondrial

we observed

activity,

activation

phosphatase

for

(Fig.

measurement

and time-dependent

and age.

of a PDH complex

such as ATP or PPi,

in the

with

for

30 to 60 minutes

preparation

decreases

incubated

in a significant

to 1 mM PPi or ATP to the

this period.

are

a maximum within

mitochondrial

maximum activity on of 0.2

mitochondria

phe-

Vol.

178, No. 3, 1991

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

kDa 94 94

67

67

43

43 30 30 20

20 14 14

03 Fig.

2.

Phosphorylation of yeast mitochondrial phosphate donors respectively.

Fio.

3.

Incorooration A, enhogenous cold ATP.

nomenon

could

be that

trigger

a cascade

of rv-32PlATP ATPl,oncentration

this

results

of Bandlow

mitochondria.

But when

41 kDa and 35 kDa. their

higher

[v-~~P]ATP.

They

is

the

ensure

an artefact [32P]PPi

we obtained obtained glucose

[32P]PPi

FCCP does only

is

grown

about

not decrease

to a very

and a result

of phosphorylation could less

low extent.

we see with

and pyrophosphatases that

proteins. of 0.4

somewhat

bands

Lane mM

and

strong

from

the

in glycerol

was performed

in

bands

could

grown

isolated appeared

at

be explained

25% of that

with

increases

the

of NADH (5 mM) or succinate

labeling

showed

differs

show a 10 kDa band which

the apparent

to 32 Pi which

ATP as

leve 1s of ATP,

2 major

phosphorylation were

with

donor.

however,

ion

and

by

of ATP.

achieved

phosphate

phosphatases

experiments

cells

concentration

does, that

when

the

The presence

oligomycin

both

[ll].

They do not

The labeling PPi

which

et al.

increased

PPi

reactions.

pattern

at 28 kDa and 30 kDa,

with

Yeast mitochondrial only. Lane B, addit

can sense

of phosphorylation

The autoradiogram cells

peptide

into

proteins

phosphorylation

of endogenous

20 nmoles 1362

for

when

but

10 mM NaF was present

PPi as phosphate

and thereby

be substrate than

the

labeling

donor

is not

ADP. NaF inhibits

prevents

the

ADP phosphorylation. of PPi were

to

hydrolyzed

hydrolysis

of

Control under

the

Vol.

178, No. 3, 1991

BIOCHEMICALANDBIOPHYSICAL

RESEARCH COMMUNICATIONS

I

0’ 030

092

036

094

078

190

mM ATP Fig.

4.

PPi-dependent phosphorylation upon titration constant PPi-concentration of 0.4 mM.

phosphorylation

conditions.

concentration

used and could

horylation

with

in

to what these

self

would

This

energy

cold

cold higher

concentration

site,

one would

amounts

expect

of the

kinase, the

the

competes

residues

(24)

that

in thylakoids

ATP would

for

same phosphorylation

the

the

labeling

case when

low

phosphorylation

Increased sites,

cause

If

same active

the

by an entirely

and hence

[24].

to the not

at

at

by an autophosphoryla-

activity.

activity

of

no ATP was ad-

diminish

is

be explained its

kinase

proteins,

when thylakoids

and bind This

con-

and decreases

spinach

protein

PPi

protein reaction

possibly

utilizing

a constant

of PPi dependent

enhances

itmi-

concentrations

one achieved

site.

donor

PPi

the

concentration ATP dependent

i.e.

the

observed

same amino

decrease

in

yeast

mi-

phosphorylation.

To sumnarize, tochondrial

of cold active

that

phosphorylation

with

At low

same kinasefs)

this

phosphate

kinases

intensified

with

The stimulation

of certain

PPi-dependent

kinase

(25)

to the

results

of ATP could

kinase

that

close

addition

for

added.

of ATP may favour acid

that

by competing

concentration

tion

is

in con-

succinate.

was performed

40 kDa protein

by the

phosp-

of yeast

of PPi dependent donor.

similar

be used

of ATP are

curve

real

indicate

associated

as phosphate

to a value obtained

PPi and ATP could

low

of the

observed

reactions

of NADH or

The phosphorylation

labeling

clearly

membrane

oxidation

mM [32P]PPi

the ATP

is observed

ATP was the

in phosphorylation with

the

no 10 kOa band

results

4 we show a titration

We recently

by [32P]PPi

the

5% of

endogenous our

occur

from

to approximately for

at a

observed.

donor

if

donor

may well

of 0.4

ATP the

ded.

as phosphate

cold ATP,

not account

donor

Consequently

ATP added.

centration

consequently

be expected

liberated

In Figure with

used

as a phosphate

tochondria. the

is

experiments.

can act

corresponds

PPi as phosphate

When PPi trast

This

with

are

can substitute

kinase,

and the

remarkably obtained

for effects

similar from

ATP as a substrate of cold

to those

spinach 1363

which

chloroplasts.

for

ATP on the we recently

PPi-dependent observed

BIOCHEMICAL

Vol. 178, No. 3, 1991

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS This supported Estado

work

was supported

by a travel de Sao Paulo,

excellent

advice

grant Brazil).

and strong

by a NYRP grant from

to H.B.

FAPESP (Fundacao We thank

Professor

and M.L.

de Amparo Bertil

L.P.daS.

a Pesquisa

Andersson

for

was do his

support.

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