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On the mechanism of mitochondrial phosphorylation of phosvitin
BIOCHIMICA ET BIOPHYSICA ACTA
603
Short Communications ~c 239 °
On the mechanism of mitochondrial phosphorylation of phosvitin It was previously reported 1,2 that phosphorylation of intramitochondrial phosphoproteins is not strictly dependent upon ATP production, but also involves some intermediates preceding ATP in the oxidative phosphorylation reactions. On the other hand mitochondria are known to catalyze the incorporation of E32P~Pt also into extramitochondrial phosphoproteins, such as cytoplasmic proteins 3, casein 4 and phosvitinS, n. In the case of non-mitochondrial phosphoproteins, the mechanism of phosphorylation must be considered as altogether different from that of mitochondrial ones, since molecular size and electric charge make the accessibility of external proteins into mitochondria very unlikely. The results reported in the present paper concerning the E*zP]Pt incorporation into phosvitin in the presence of rat-liver mitochondria, are consistent with the view that phosvitin is phosphorylated by ATP produced in mitochondria and leaking in the external medium. Rat-liver mitochondria, isolated in 0.25 M sucrose containing 2 mM EDTA, were incubated in the presence of E32PIPt and phosvitin, under the conditions described in a previous paper 2. No phosphate acceptor (ADP or AMP) was added. At the end of incubation mitochondria were removed by centrifugation and phosvitin was precipitated from the supernatant by the addition of cold perchloric acid (3 %). The perchloric acid supernatants were combined with the mitochondrial pellets for "mitochondrial ATP" determinations. Determinations of "external ATP" were carried out on the supernatants alone. The amount and radioactivity of ATP were measured as previously described 2. E3~P]Pt incorporation into phosvitin was estimated by measuring the specific radioactivity of phosvitin alkali-labile phosphate (15 min at IOO° in I N NaOH) v. Incorporation of [3~PIPI into phosvitin by rat-liver mitochondria was studied using/~-hydroxybutyrate and ~-ketoglutarate as oxidizable substrates (Table I). Such an incorporation as well as the amount of mitochondrial ATP, in the presence of fl-hydroxybutyrate was strongly reduced by inhibitors of oxidative phosphorylation, such as 2,4-dinitrophenol 9, oligomycin 1°, zl and atractylosidO 2. On the contrary, when a-ketoglutarate was the substrate, the inhibition varied in a very characteristic manner: oligomycin, which is known to be ineffective on the substrate-linked phosphorylationl3,14, accordingly only slightly affected both phosvitin labelling and ATP production. 2,4-Dinitrophenol inhibited completely the labelling of phosvitin and greatly reduced the amount of ATP. Atractyloside abolished the phosphorylation of phosvitin while did not interfere with tile amount and labelling of ATP, thus suggesting its probable ineffectiveness on the substrate-linked phosphorylation. These results show that a constant parallelism between ATP production and 32p incorporation into phosvitin does not exist. Indeed, when ~-ketoglutarate is the substrate, in the presence of atractyloside, and, to a lesser extent, of 2,4-dinitrophenol, Biochim. Biophys. Acta, 82 (1964) 603-605
604
SHORT
COMMUNICATIONS
TABI.E
1
EFFECT OF INHIBITORS ON a2p INCORPORATION INTO PHOSVIT1N AND "MITOCHONDRIAL .'V['] ~'' IN THE" PRESENCE OF fl-HYDROXYBUTYRATE O R O & K E T O G L I 2 T A R A T E I m l m i t o c h o n d r i a s u s p e n d e d i n o . 2 5 M s u c r o s e a n d e q u i v a l e n t t o 27 m g m i t o c h o n d r i a l p r o t e i n s w e r e i n c u b a t e d i n 3 m l of a m e d i u m c o n t a i n i n g 1 2 / , m o l e s MgCI2, t o o l~moles T r i s b u f f e r ( p H 7.4), t o / * C aep, 6 o t t m o l e s s u b s t r a t e ( p o t a s s i u m s a l t ) , I m g p h o s v i t i n , o..~- o.,o e t h a n o l ( f r o m o l i g o m y c i n o r in s u b s t i t u t i o n of o l i g o m y c i n ) , a n d v a r i a b l e q u a n t i t i e s of I(CI in o r d e r t o m a i n t a i n c o n s t a n t t h e t( + c o n c e n t r a t i o n . T e m p e r a t t | r e , 30:'. I n c u b a t i o n t i m e , 30 r a i n . fl H.vdro t yhulyrah' Addition
Phosvitin
:e Kctot~lularafc
.ll itochondrial A T I'
l'hosvithl
M ttochondrea1.4 -Il'
/counts/rain~irE 19
m l,molcs
counts,roD,
(cou* Is~rain~liE I '
m/tmol~
counls,ml,z
| 13
3~
lo 7 52o
19o
8~)
39(7 (73;
Oligomycin (I.5/tmoles per g protein)
5
o
IO 8f) 4
| I,%
7°
295 77~
2,4-Dinitropheno]
2
5
I8 58. t
t
0
51 217
(,
7
t(, 97 °
5
So
333 7 r('
Nil
( l o 4 M)
Atractyloside (20 t t m o l e s p e r g p r o t e i n )
a discrepancy, between the anaount of nlitochondrial ATP and the labelling of phosvitin appears evident. In order to clarify the significance of such a difference, a2I) incorporation int(, phosvitin was again checked, not in relation with the total a m o u n t of mitochondrial ATP, but only of "external A T P " , i.e. ATP leaked during inculmti|m trom mito chondria in the external medium. As shown in Table II phosvitin labelling closely follows the presence of '>xternal ATP". Indeed, whenever there is ATP in the external medium, that is in the absenev of inhibitors or in the presence of oligomycin, the labelling of phosvitin takes place. TABLE
I1
EFFECT OF INHIBITOR.% (IN :PaI) INCORPORATION 1NTO PHI)SVITIN AND ON TIlE AMOITN'I OF "EXTERNAL : \ T ] ~ ' ' ' IN THE PRESENCE OF ~-KETOGLUTAIe.AI'I.I Experinlental
c o n d i t i o n s a s in T a b l e I. M i t o c h o m l r i a
e q u i v a l e | i t t o 29 m g n i i t o c h o n d r i a l p r o t e i n . ~.Acti)glularah"
.IddttI'on
l'hosvitin (c°unls/min/ltg l'~
"l(~tcrmlI .I l'I"' mtonoh'~
~-,,ltJtlw,mt)l
157
0•
3()~ 174,
83
I~1
TS 7 27-"
2 , 4 - 1 ) i n i t r o p h e n o l (IO 4 M)
4
0
2() [
Atractvlosidc (2o/inioles per g protein)
3
o
[ 0S
Nil Oligomyciu (i. 5 F m o l e s p e r g p r o t e i n )
* ATI' leaked from mitochondria
in t h e e x t e r n a l m e d i u m . B ic, c h i m .
Bzoph.vs...tcta,
82 {10o4) 0o 3 0 0 5
SHORT COMMUNICATIONS
605
On the contrary, in the presence of 2,4-dinitrophenol and atractyloside, no "external A T P " is present and no labelling of phosvitin does occur. The mechanisms by which 2,4-dinitrophenol and atractyloside abolish the presence of ATP in the external medium, are, very likely, quite different. In the presence of 2,4-dinitrophenol which also reduces mitochondrial ATP (see Table I), the complete lack of "external ATP" probably depends on 2,4-dinitrophenolstimulated ATPase activity 9. This cannot be the case for atractyloside, which inhibits ATPase activity 12,15 and accordingly does not reduce the amount of intramitochondrial ATP or its labelling (Table I). It seems therefore that atractyloside inhibits, by some unknown mechanism, the process by which intramitochondrial ATP leaks in the external medium, becoming available for the labelling of phosvitin. This hypothesis is supported by the finding that when atractyloside and phosvitin are added after preincubation of mitochondria with a-ketoglutarate and [3~PIPj, the presence of "external ATP", and consequently the labelling of phosvitin, are no more abolished (unpublished data). In conclusion, while the phosphorylation of mitochondrial phosphoproteins, as previously demonstrated 2, is not necessarily dependent on ATP, being also mediated by energy-rich phosphate-bond intermediates in the oxidative phosphorylation chain, the incorporation of E32P]PI into exogenous phosphoproteins, such as phosvitin, appears to be strictly dependent on the availability of [32PIATP outside mitochondria. This work has been supported by grants from the Impresa Enzimologia, Consiglio Nazionale delle Ricerche. We are very grateful to Dr. A. BRUNI for generous supply of atractyloside and to Dr. M. A. COLETTIfor phosvitin preparation. V. MORET
Institute of Biological Chemistry, University of Padova, Padova (Italy)
L. A. PINNA S. S P ER TI
M. LORINI N . SILIPRANDI 1 K. AHMED AND J. D. JUDAH, Biochim. Biophys. Acta, 71 (1963) 295. 2 V. MORET, L. A. PINNA, S. SPERTI, M. LORINI AND N. SILIPRANDI, Biochim. Biophys. Acta, 78 (1963) 547. 3 IV[. B. LIVANOVA, Vopr. Med. Khim., 8 (1962) 429 . 4 G. BURNETT AND E. P. KENNEDY, J. Biol. Chem., 211 (1954) 969. 5 S. P. 1~. ROSE AND P. J. HEALD, Biochem. J., 81 (1961) 339. 6 V. MORET, L. A. PINNA, S. SPERTI AND M. LORINI, Boll. Soc. Ital. Biol. Sper., 38 (1962) 1876. 7 i . RABINOWITZ AND F. LIPMANN, J. Biol. Chem., 235 (196o) lO43. 8 A. G. GORNALL, C. J. BARDAWILL AND IV[. M. DAVID, J. Biol. Chem., 177 (1949) 751. 9 F. E. HUNTER, Symposium on Phosphorus Metabolism, Vol. i, The J o h n s H o p k i n s Press, B a l t i more, 1959, p. 297. 10 H. A. LARDY, D. JOHNSON AND W. C. McMURRAY, Arch. Biochem. Biophys., 78 (1958) 587 . 11 F. HUIJING AND E. C. SLATER, J. Biochem. Tokyo, 49 (1961) 493. 12 A. BRUNI, A . . . . CONTESSA AND S. LUCIANI, Biochim. Biophys. Acta, 60 (1962) 3Ol. 13 j . B. CHAPPEL ~ ND G. D. GREVILLE, Nature, 19o (1961) 502. 14 L. DANIELSON AND L. ERNSTER, Biochem. Biophys. Res. Commun., i o (1963) 85. 15 p. V. VIGNAIS, P. M. VIGNAIS AND E. STANISLAS, Bioehim. Biophys. Acta, 6o (1962) 284.
Received October 29th, 1963 Biochim. Biophys. Acta, 82 (1964) 603-605
603
Short Communications ~c 239 °
On the mechanism of mitochondrial phosphorylation of phosvitin It was previously reported 1,2 that phosphorylation of intramitochondrial phosphoproteins is not strictly dependent upon ATP production, but also involves some intermediates preceding ATP in the oxidative phosphorylation reactions. On the other hand mitochondria are known to catalyze the incorporation of E32P~Pt also into extramitochondrial phosphoproteins, such as cytoplasmic proteins 3, casein 4 and phosvitinS, n. In the case of non-mitochondrial phosphoproteins, the mechanism of phosphorylation must be considered as altogether different from that of mitochondrial ones, since molecular size and electric charge make the accessibility of external proteins into mitochondria very unlikely. The results reported in the present paper concerning the E*zP]Pt incorporation into phosvitin in the presence of rat-liver mitochondria, are consistent with the view that phosvitin is phosphorylated by ATP produced in mitochondria and leaking in the external medium. Rat-liver mitochondria, isolated in 0.25 M sucrose containing 2 mM EDTA, were incubated in the presence of E32PIPt and phosvitin, under the conditions described in a previous paper 2. No phosphate acceptor (ADP or AMP) was added. At the end of incubation mitochondria were removed by centrifugation and phosvitin was precipitated from the supernatant by the addition of cold perchloric acid (3 %). The perchloric acid supernatants were combined with the mitochondrial pellets for "mitochondrial ATP" determinations. Determinations of "external ATP" were carried out on the supernatants alone. The amount and radioactivity of ATP were measured as previously described 2. E3~P]Pt incorporation into phosvitin was estimated by measuring the specific radioactivity of phosvitin alkali-labile phosphate (15 min at IOO° in I N NaOH) v. Incorporation of [3~PIPI into phosvitin by rat-liver mitochondria was studied using/~-hydroxybutyrate and ~-ketoglutarate as oxidizable substrates (Table I). Such an incorporation as well as the amount of mitochondrial ATP, in the presence of fl-hydroxybutyrate was strongly reduced by inhibitors of oxidative phosphorylation, such as 2,4-dinitrophenol 9, oligomycin 1°, zl and atractylosidO 2. On the contrary, when a-ketoglutarate was the substrate, the inhibition varied in a very characteristic manner: oligomycin, which is known to be ineffective on the substrate-linked phosphorylationl3,14, accordingly only slightly affected both phosvitin labelling and ATP production. 2,4-Dinitrophenol inhibited completely the labelling of phosvitin and greatly reduced the amount of ATP. Atractyloside abolished the phosphorylation of phosvitin while did not interfere with tile amount and labelling of ATP, thus suggesting its probable ineffectiveness on the substrate-linked phosphorylation. These results show that a constant parallelism between ATP production and 32p incorporation into phosvitin does not exist. Indeed, when ~-ketoglutarate is the substrate, in the presence of atractyloside, and, to a lesser extent, of 2,4-dinitrophenol, Biochim. Biophys. Acta, 82 (1964) 603-605
604
SHORT
COMMUNICATIONS
TABI.E
1
EFFECT OF INHIBITORS ON a2p INCORPORATION INTO PHOSVIT1N AND "MITOCHONDRIAL .'V['] ~'' IN THE" PRESENCE OF fl-HYDROXYBUTYRATE O R O & K E T O G L I 2 T A R A T E I m l m i t o c h o n d r i a s u s p e n d e d i n o . 2 5 M s u c r o s e a n d e q u i v a l e n t t o 27 m g m i t o c h o n d r i a l p r o t e i n s w e r e i n c u b a t e d i n 3 m l of a m e d i u m c o n t a i n i n g 1 2 / , m o l e s MgCI2, t o o l~moles T r i s b u f f e r ( p H 7.4), t o / * C aep, 6 o t t m o l e s s u b s t r a t e ( p o t a s s i u m s a l t ) , I m g p h o s v i t i n , o..~- o.,o e t h a n o l ( f r o m o l i g o m y c i n o r in s u b s t i t u t i o n of o l i g o m y c i n ) , a n d v a r i a b l e q u a n t i t i e s of I(CI in o r d e r t o m a i n t a i n c o n s t a n t t h e t( + c o n c e n t r a t i o n . T e m p e r a t t | r e , 30:'. I n c u b a t i o n t i m e , 30 r a i n . fl H.vdro t yhulyrah' Addition
Phosvitin
:e Kctot~lularafc
.ll itochondrial A T I'
l'hosvithl
M ttochondrea1.4 -Il'
/counts/rain~irE 19
m l,molcs
counts,roD,
(cou* Is~rain~liE I '
m/tmol~
counls,ml,z
| 13
3~
lo 7 52o
19o
8~)
39(7 (73;
Oligomycin (I.5/tmoles per g protein)
5
o
IO 8f) 4
| I,%
7°
295 77~
2,4-Dinitropheno]
2
5
I8 58. t
t
0
51 217
(,
7
t(, 97 °
5
So
333 7 r('
Nil
( l o 4 M)
Atractyloside (20 t t m o l e s p e r g p r o t e i n )
a discrepancy, between the anaount of nlitochondrial ATP and the labelling of phosvitin appears evident. In order to clarify the significance of such a difference, a2I) incorporation int(, phosvitin was again checked, not in relation with the total a m o u n t of mitochondrial ATP, but only of "external A T P " , i.e. ATP leaked during inculmti|m trom mito chondria in the external medium. As shown in Table II phosvitin labelling closely follows the presence of '>xternal ATP". Indeed, whenever there is ATP in the external medium, that is in the absenev of inhibitors or in the presence of oligomycin, the labelling of phosvitin takes place. TABLE
I1
EFFECT OF INHIBITOR.% (IN :PaI) INCORPORATION 1NTO PHI)SVITIN AND ON TIlE AMOITN'I OF "EXTERNAL : \ T ] ~ ' ' ' IN THE PRESENCE OF ~-KETOGLUTAIe.AI'I.I Experinlental
c o n d i t i o n s a s in T a b l e I. M i t o c h o m l r i a
e q u i v a l e | i t t o 29 m g n i i t o c h o n d r i a l p r o t e i n . ~.Acti)glularah"
.IddttI'on
l'hosvitin (c°unls/min/ltg l'~
"l(~tcrmlI .I l'I"' mtonoh'~
~-,,ltJtlw,mt)l
157
0•
3()~ 174,
83
I~1
TS 7 27-"
2 , 4 - 1 ) i n i t r o p h e n o l (IO 4 M)
4
0
2() [
Atractvlosidc (2o/inioles per g protein)
3
o
[ 0S
Nil Oligomyciu (i. 5 F m o l e s p e r g p r o t e i n )
* ATI' leaked from mitochondria
in t h e e x t e r n a l m e d i u m . B ic, c h i m .
Bzoph.vs...tcta,
82 {10o4) 0o 3 0 0 5
SHORT COMMUNICATIONS
605
On the contrary, in the presence of 2,4-dinitrophenol and atractyloside, no "external A T P " is present and no labelling of phosvitin does occur. The mechanisms by which 2,4-dinitrophenol and atractyloside abolish the presence of ATP in the external medium, are, very likely, quite different. In the presence of 2,4-dinitrophenol which also reduces mitochondrial ATP (see Table I), the complete lack of "external ATP" probably depends on 2,4-dinitrophenolstimulated ATPase activity 9. This cannot be the case for atractyloside, which inhibits ATPase activity 12,15 and accordingly does not reduce the amount of intramitochondrial ATP or its labelling (Table I). It seems therefore that atractyloside inhibits, by some unknown mechanism, the process by which intramitochondrial ATP leaks in the external medium, becoming available for the labelling of phosvitin. This hypothesis is supported by the finding that when atractyloside and phosvitin are added after preincubation of mitochondria with a-ketoglutarate and [3~PIPj, the presence of "external ATP", and consequently the labelling of phosvitin, are no more abolished (unpublished data). In conclusion, while the phosphorylation of mitochondrial phosphoproteins, as previously demonstrated 2, is not necessarily dependent on ATP, being also mediated by energy-rich phosphate-bond intermediates in the oxidative phosphorylation chain, the incorporation of E32P]PI into exogenous phosphoproteins, such as phosvitin, appears to be strictly dependent on the availability of [32PIATP outside mitochondria. This work has been supported by grants from the Impresa Enzimologia, Consiglio Nazionale delle Ricerche. We are very grateful to Dr. A. BRUNI for generous supply of atractyloside and to Dr. M. A. COLETTIfor phosvitin preparation. V. MORET
Institute of Biological Chemistry, University of Padova, Padova (Italy)
L. A. PINNA S. S P ER TI
M. LORINI N . SILIPRANDI 1 K. AHMED AND J. D. JUDAH, Biochim. Biophys. Acta, 71 (1963) 295. 2 V. MORET, L. A. PINNA, S. SPERTI, M. LORINI AND N. SILIPRANDI, Biochim. Biophys. Acta, 78 (1963) 547. 3 IV[. B. LIVANOVA, Vopr. Med. Khim., 8 (1962) 429 . 4 G. BURNETT AND E. P. KENNEDY, J. Biol. Chem., 211 (1954) 969. 5 S. P. 1~. ROSE AND P. J. HEALD, Biochem. J., 81 (1961) 339. 6 V. MORET, L. A. PINNA, S. SPERTI AND M. LORINI, Boll. Soc. Ital. Biol. Sper., 38 (1962) 1876. 7 i . RABINOWITZ AND F. LIPMANN, J. Biol. Chem., 235 (196o) lO43. 8 A. G. GORNALL, C. J. BARDAWILL AND IV[. M. DAVID, J. Biol. Chem., 177 (1949) 751. 9 F. E. HUNTER, Symposium on Phosphorus Metabolism, Vol. i, The J o h n s H o p k i n s Press, B a l t i more, 1959, p. 297. 10 H. A. LARDY, D. JOHNSON AND W. C. McMURRAY, Arch. Biochem. Biophys., 78 (1958) 587 . 11 F. HUIJING AND E. C. SLATER, J. Biochem. Tokyo, 49 (1961) 493. 12 A. BRUNI, A . . . . CONTESSA AND S. LUCIANI, Biochim. Biophys. Acta, 60 (1962) 3Ol. 13 j . B. CHAPPEL ~ ND G. D. GREVILLE, Nature, 19o (1961) 502. 14 L. DANIELSON AND L. ERNSTER, Biochem. Biophys. Res. Commun., i o (1963) 85. 15 p. V. VIGNAIS, P. M. VIGNAIS AND E. STANISLAS, Bioehim. Biophys. Acta, 6o (1962) 284.
Received October 29th, 1963 Biochim. Biophys. Acta, 82 (1964) 603-605