Protein phosphorylation by inorganic pyrophosphate in yeast mitochondria

Vol. 178, No. 3, 1991

August15,1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1359-1364

PROTEIN PHOSPHORYLATION BY INORGANIC PYROPHOSPHATE IN YEAST MITOCHONDRIA

Lucia Pereira da Silva*, Marika Lindahl, Maria Lundin# and Herrick Baltscheffsky

Department of Biochemistry, Arrhenius Laboratories, University of Stockholm, S-106 91 Stockholm, Sweden

Received June 28, 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, maxi- mal 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 reversible binding of phosphate to certain amino acid re-

sidues in many enzymes is one of the best known mechanisms of metabolic regu-

lation. This process is widespread in nature and has been reported to occur

in eukaryotic cells [l] including Neurospora crassa [2]. It is catalyzed by

protein kinases, acting together with phosphatases to perform the

phosphorylation-dephosphorylation reactions. These kinases have been found in

different cellular compartments [3], including mitochondria [4-61 and they

may or may not be dependent on CAMP for their activity [7-Ill.

The activity of the pyruvate dehydrogenase (PDH) complex from different

animal and plant tissues and from Neurospora crassa is regulated through

cycles of phosphorylation and dephosphorylation [12-141. A kinase inactivates

the complex and a specific phosphatase regenerates its activity by removing

the covalently bound phosphate. In Saccharomyces cerevisiae no pyruvate de-

hydrogenase kinase activity has been reported during isolation and purifica-

tion of this complex [15-161, in contrast with reports on PDH complex kinase

activities from Kluyveromyces lactis [12] and Neurospora crassa [2], but in

agreement with the results obtained with Saccharomyces carlsbergensis [17].

All these reports concerned the kinase activities of the isolated PDH comp-

*Present address: Departamento de Bioqu?mica, IB, UNICAMP, Campinas-SP-Brazil.

#Present address: Ludwig Institute for Cancer Research, Uppsala Branch, Bio- medical Center, Box 599, S-751 23 Uppsala, Sweden.

0006-291X/91 $1.50

1359 Copyright 0 1991 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lex. On the other hand the PDH complex of intact pea mitochondria has been

reported to become reversibly phosphorylated in situ [18].

Considering the possibility that the kinase and/or phosphatase activi-

ties were lost during the purification procedure, we investigated the PDH

complex activity within the whole mitochondria isolated from Saccharomyces

cerevisiae. We show that PPi as well as ATP acts as phosphate donor and that

the activity of the PDH complex becomes inhibited when the mitochondria are

incubated in the presence of PPi or ATP.

Recently, PPi has been shown to act as a phosphate donor in protein ki-

nase reactions in spinach thylakoids [19,20]. Here we show for the first time

that PPi can also be used as phosphate donor in yeast mitochondria. Moreover,

no significant difference was observed in the autoradiogram patterns when

proteins were labelled with PPi or ATP.

MATERIALS AND METHODS

Yeast cells (Saccharomyces cerevisiae strain, NCYC 1075) were grown in a galactose medium as described 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 buffer as used in the second washing and kept on ice. It could be used up to seven days without significant loss of the phosphorylating capacity. Protein determination was performed using a BIO-RAD colour reagent with bovine serum albumin as stan- dard.

Studies on the mitochondrial PDH complex were performed with intact mitochondria, incubated at 25°C in 500 pl of 0.6 M mannitol/20 mM potassium phosphate buffer pH 7.0, containing 2 mM MgC12, 2 mM DTT and 150 to 250 pg of mitochondrial protein. At different times, aliquots of 20 to 50 ul were transferred to a cuvette containing the medium for the assay of pyruvate dehydrogenase complex activity, which was performed as described by Uhlinger et al. in [lS].

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 abo- lish 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 indica-

1360


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

RESULTS AND DISCUSSION

When intact yeast mitochondria are incubated for measurement of the PDH

complex activity it increases in a significant and time-dependent manner

(Fig. 1) and reaches a maximum within 30 to 60 minutes of incubation, depen-

ding on the mitochondrial preparation and age. The time required to get the

maximum activity decreases with mitochondrial ageing (not shown). The additi-

on of 0.2 to 1 mM PPi or ATP to the incubation medium, progressively inhibits

this activation process (Fig. 1). We interpreted these results as a consequ-

ence of the action of a PDH complex phosphatase, acting during the incubation

period. If this incubation is performed in the presence of a phosphate donor,

such as ATP or PPi, we observed a decrease in the activation rate, probably

due to a kinase activity, phosphorylating the PDH complex and so inhibiting

its activity. In line with this interpretation is the corresponding decrease

in the activation rate obtained with 10 mM NaF, which is known to inhibit

phosphatase activity (Fig. 1).

In Figure 2 we show that [32P]PPi is able to act as a phosphate donor

for the phosphorylation of several proteins in yeast mitochondria. The pat-

terns of the autoradiograms obtained with either [y-32P]ATP or [32P]PPi are

essentially the same. In the presence of 0.4 mM of any of these phosphate do-

nors the most strongly labeled band appears at around 40 kDa. This may well

be the a-subunit of the PDH complex [ll]. A second strongly labeled band ap-

pears at about 28 kDa. If no cold ATP is present (i.e. extremely low concent-

ration of [v-~~P]ATP a strong band at 5-10 kDa appears and the label of the

band at -40 kDa is decreased (Fig. 3). One possible explanation to this phe-

c -ii

201

; 15-

% z lo-

Z E 2 5-

0 ~,,,,‘,,,,[,,.,‘,,,,‘,,,,I,,,,‘,,,,[,*,,’ 0 5 10 15 20 25 30 35 40

Time (min)

Fig. 1. 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).

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Vol. 178, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS

kDa

94

67

43

30

20

14

0 3

94

67

43

30

20

14

Fig. 2. Phosphorylation of yeast mitochondrial proteins with PPi and ATP as phosphate donors respectively.

Fio. 3. Incorooration of rv-32PlATP into Yeast mitochondrial proteins. Lane ion of 0.4 mM A, enhogenous ATPl,oncentration only. Lane B, addit

cold ATP.

1s of ATP, and nomenon could be that this peptide can sense increased leve

trigger a cascade of phosphorylation reactions.

The autoradiogram pattern which we obtained differs somewhat from the

results of Bandlow et al. [ll]. They obtained 2 major bands in glycerol grown

cells at 28 kDa and 30 kDa, when phosphorylation was performed in isolated

mitochondria. But when the cells were glucose grown strong bands appeared at

41 kDa and 35 kDa. They do not show a 10 kDa band which could be explained by

their higher concentration of ATP.

The labeling achieved with [32P]PPi is about 25% of that with

[v-~~P]ATP. The presence of NADH (5 mM) or succinate increases the labeling when

PPi is the phosphate donor. FCCP does not decrease the phosphorylation but

oligomycin does, however, only to a very low extent. 10 mM NaF was present to

ensure that the apparent labeling we see with PPi as phosphate donor is not

an artefact and a result of phosphorylation of endogenous ADP. NaF inhibits

both phosphatases and pyrophosphatases and thereby prevents the hydrolysis of

[32P]PPi to 32 Pi which could be substrate for ADP phosphorylation. Control

experiments showed that less than 20 nmoles of PPi were hydrolyzed under the

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Vol. 178, No. 3, 1991 BIOCHEMICALANDBIOPHYSICAL RESEARCH COMMUNICATIONS

0’ I

030 092 094 036 078 190

mM ATP

Fig. 4. PPi-dependent phosphorylation upon titration with cold ATP, at a constant PPi-concentration of 0.4 mM.

phosphorylation conditions. This corresponds to approximately 5% of the ATP

concentration used and could consequently not account for the observed phosp-

horylation with PPi as phosphate donor observed.

When PPi is used as phosphate donor no 10 kOa band is observed in con-

trast to what would be expected if endogenous ATP was the real phosphate donor

in these experiments. Consequently our results clearly indicate that PPi it-

self can act as a phosphate donor in phosphorylation reactions of yeast mi-

tochondria. This may well occur with membrane associated kinases utilizing

the energy liberated from the oxidation of NADH or succinate.

In Figure 4 we show a titration curve of PPi dependent phosphorylation

with cold ATP added. The phosphorylation was performed with a constant con-

centration of 0.4 mM [32P]PPi as phosphate donor. At low concentrations of

cold ATP the labeling of the 40 kDa protein is intensified and decreases at

higher concentration to a value close to the one achieved when no ATP was ad-

ded. We recently obtained similar results with spinach thylakoids [24]. I f

PPi and ATP could be used by the same kinasefs) and bind to the same active

site, one would expect that addition of cold ATP would diminish the labeling

by [32P]PPi by competing for this active site. This is not the case when low

amounts of ATP are added. The stimulation of PPi dependent phosphorylation at

low concentration of ATP could possibly be explained by an autophosphoryla-

tion of the kinase (25) that enhances its activity. Increased concentration

of ATP may favour the protein kinase activity by an entirely ATP dependent

kinase, that competes for the same phosphorylation sites, i.e. the same amino

acid residues of certain proteins, and hence cause the observed decrease in

the PPi-dependent phosphorylation.

To sumnarize, PPi can substitute for ATP as a substrate for yeast mi-

tochondrial protein kinase, and the effects of cold ATP on the PPi-dependent

kinase reaction are remarkably similar to those which we recently observed

(24) in thylakoids obtained from spinach chloroplasts.

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ACKNOWLEDGMENTS

This work was supported by a NYRP grant to H.B. and M.L. L.P.daS. was

supported by a travel grant from FAPESP (Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo, Brazil). We thank Professor Bertil Andersson for his

excellent advice and strong support.

REFERENCES

Krebs, E.G. (1985) Biochem. Sot. Trans. 13, 813-820. Wieland, O.H., Hartmann, U. and Siess, E.A. (1972) FEBS Lett. 27, 240-244. Krebs, E.G. and Beavo, J.A. (1979) Ann. Rev. Biochem. 48, 923-959. Kitigawa, Y. and Racker, E. (1982) J. Biol. Chem. 257, 4547-4551. Bradford, A.P. and Yeaman, S.J. (1986) Adv. Prot. Phsophatases 3, 73-106. Miernyk, J.A: and Randall, D.D. (1987) Plant Physiol. 83, 306-310. Hixson, C.S. and Krebs, E.G. (1980) J. Biol. Chem. 255, 2137-2145. Lastick, S.M. and McConkey, E.H. (1981) J. Biol. Chem. 256, 583-585. Pohlig, G. and Holzer, H. (1985) J. Biol. Chem. 260, 1381813823. Delattre, P., Mareck, A. and Foucher, B. (1985) Biochimie 67, 633-636. Miller, G. and Bandlow, W. (1987) Yeast 3, 161-174. Reed, L.J. (1974) Account Chem. Res. 7, 40-46. Denton, R.M., Randle, P.J., Bridges, B.J., Cooper, R.H., Kerbey, A.L., Pask, H.T., Severson, D.L., Stansbie, D. and Whitehouse, S. (1975) Mol. Cell Biochem. 9, 27-53. Wieland, O.H. (1983) Rev. Physiol. Biochem. Pharmacol. 96, 124-170. Kresze, G.B. and Ronft, H. (1981) Eur. J. Biochem. 119, 573-579. Uhlinger, D.J., Yang, C.Y. and Reed, L.J. (1986) Biochemistry 25, 5673-5677. Wais, U., Gillmann, U. and Ullrich, J. (1973) Hoppe-Seyler's Physiol. Chem. 354, 1378-1388. Budde, R.J.A. and Randall, D.D. (1987) Arch. Biochem. Biophys. 258, 600-606. Pramanik, A., Bingsmark, S., Baltscheffsky, H., Baltscheffsky, M. and Andersson, B. (1988) Abstracts, XV Congress of the Scandinavian Society for Plant Physiology, Turku, Finland, Physiol. Plant, 73, A6. Pramanik, A., Bingsmark, S., Baltscheffsky, H., Baltscheffsky, M. and Andersson, B. (1990) Current Research in Photosynthesis (Baltscheffsky, M ed.) L;idin, M

Vol. II, pp. 763-766, Kluwer Academic Press, Dordrecht. ., Pereira da Silva, L. and Baltscheffsky, H. (1987) Biochim.

Biophys. Acta 890, 279-285. Laemnli, U.K. (1970) Nature 227, 680-685. Shatton, J.B., Ward, C., Biochem. 130, 114-119.

Williams, A. and Weinhouse, S. (1982) Anal.

Pramanik, A.M., Bingsmark, S., Lindahl, M., Baltscheffsky, H., Baltscheffsky, M. and Andersson, 186.

B. (1991) Eur. J. Biochem. 198, 183-

Coughlan, S.J. and Hind, G. (1987) Biochemistry 26, 6515-6521.

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Protein phosphorylation by inorganic pyrophosphate in yeast mitochondria