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Occurrence of phosphorylated forms of an acidic protein in the large ribosomal subunit of rat liver
537
Biochimica et Biophysica Acta, 519 (1978) 537--541 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 91472
OCCURRENCE OF PHOSPHORYLATED FORMS OF AN ACIDIC PROTEIN IN THE LARGE RIBOSOMAL SUBUNIT OF RAT LIVER
M O N I Q U E ARPIN, JEAN-JACQUES M A D J A R and JEAN-PAUL R E B O U D Laboratoire de Biochimie M~dicale, U.E.R. Lyon-Nord 43, Boulevard du 11 Novembre 1918, 69621 Villeurbanne (France)
(Received February 28th, 1978)
Summary An acidic protein from rat liver 60-S ribosomal subunits was selectively extracted with 50% ethanol. It was revealed as three different spots by twodimensional gel electrophoresis, two of them being attributable to phosphorylated forms since they disappeared after alkaline phosphatase treatment. The relationship between this protein and similar acidic proteins found in eucaryotic cells is discussed.
In rat liver ribosomes, one protein of the small subunit ($6), and none from the large subunit, has been found to be phosphorylated in vivo [1]. Quantitative variations in $6 phosphorylation were found under several circumstances: hepatic regeneration [1 ], peptidic hormone injections [2--4], inhibition of protein synthesis by cycloheximide [ 5]. Recently, the presence of phosphorylated acidic proteins has been shown in the large ribosomal subunit of several eucaryotic organisms at different evolutionary stages [ 6 - 9 ] . Interestingly, the acidic proteins found in Artemia salina ribosomes have been shown to be implicated in the interaction with elongation factors and to have a similar function to that of L7/L12 in Escherichia coli [8, 10]. In this paper it is shown that rat liver also contains an acidic phosphoprotein in the large ribosomal subunit. This protein was extracted selectively in the presence of 50% ethanol. The presence of different phosphorylated forms was demonstrated by incubating the acidic protein fraction with phosphatase. Ribosomal subunits from rat liver were prepared from free polysomes as previously described [11]. Proteins were extracted from the 60-S subunits by the acetic acid procedure [12], dialyzed against 1 M acetic acid and lyophilized. The acidic proteins were selectively extracted from 60-S subunits suspended in buffer A (50 mM triethanolamine. HCI, pH = 7.4/80 mM KC1/5 mM MgCI2/20 mM 2-mercaptoethanol) by treatment with 1 vol. of ethanol at 4 ° C.
538 The ethanol extract was dialyzed against 1 M acetic acid and lyophilized. For treatment with alkaline phosphatase, acidic proteins were dissolved in 1 mM (NH4)2 CO3 and incubated with the enzyme. The electrophoretogram in Fig. 1A shows the analysis of 60-S ribosomal subunit proteins, both dimensions being performed at an acidic pH. On the anodic side, three spots are visible which will appear as only one spot (see arrows) when migration in the second dimension is carried out in the presence of sodium dodecylsulphate (Fig. 1B). Therefore these spots should represent different forms of one single protein of about 12 500 daltons. Evidence that two of these spots correspond to phosphorylated derivatives was then obtained by the following experiment: a purified fraction of acidic proteins, extracted from the 60-S subunits with ethanol was treated with alkaline phosphatase. Fig. 2 shows the electrophoretograms of control and phosphatasetreated acidic proteins. It is clear that the enzymatic treatment produces a progressive disappearance of two spots and a simultaneously increase of the third one. Besides the spots of the phosphorylated protein, two other faint spots corresponding to slightly less acidic proteins were also visible on the gel slab but they are difficult to see on the photographs. Our results demonstrate that rat liver 60-S ribosomal subunits contain an acidic protein of 12 500 daltons, which exists under at least two distinct phosphorylated forms. This protein (phosphorylated and unphosphorylated forms) was extracted by 50% ethanol in a very specific manner, since only two slight contaminants could be detected. The ionic conditions for specific extraction are critical since, by using other salt conditions, Reyes et al. completely removed other proteins from the subunits [ 16]. The pH value during electrophoresis is also important: phosphorylated forms are well separated because the pH was very near of the protein pH i. It is also worth noticing that this same protein could be phosphorylated in vitro by an endogenous kinase associated with rat liver polysomes [17]. Our finding can be correlated with results obtained in other eucaryotic systems. The electrophoretic behaviour and the molecular weight (12 500) of the acidic phosphorylated protein in rat liver 60-S subunits are very similar to those of analogous proteins found in yeast [6], Krebs II ascite cells [9], Hela cells [7] and Artemia salina cysts [8]. The protein studied in the latter system, eL12(p), has been shown to be involved in the EF-2~lependent GTP h.vdrolysis, and therefore to be analogous to the bacterial protein L7/L12. However, antibodies against LT/L12 did not react with eL12(p) [8]. On the other hand, Stt}ffler et al. [18] have described two acidic proteins, L40/IA1, in the rat liver large subunit, which are immunologically and functionally related to E. coli L7/L12 [19]. None of these proteins was found phosphorylated [1]. Moreover, the molecular weight of both proteins (25 500) [20] is much higher than that determined by all other authors, as well as by us, for the acidic phosphoprotein of the large subunit and also for bacterial protein L7/L12. Therefore, if the determination of L40/L41 molecular weight is correct, the exact relationship between the acidic protein described in this paper and L40/L41 is not quite clear. In any case, our results bring a new argument to assume that the occurrence of an acidic phosphoprotein in the large ribosomal subunit is general among eucaryotic cells. Thus, they are in agree-
539
Fig. 1. E l e e t r o p h o r e t o ~ m m s of proteins e x t r a c t e d from 60-S s ubuni t s of rat liver. Eleetrophorescs were p e r f o r m e d essentially according to the m e t h o d s described b y K nopf e t al. [13] (Fig. 1A) and Mete and Bogorad [14] (Fig. 1B). I n b o t h eases, migrat/on in the first d i m e n s i o n was p e r f o r m e d unde r the same con ditions: pro teins were dissolved at pH 4.2 and m i g r a t i o n was c o n d u c t e d at pH 5.5 t o w a r d s t he cathode a t 150 V for 5 h. Migration in the second d i m e n s i o n was carried o u t i n a water-cooled a ppa ra t us [15] at pH 4.5 in 18% a c r y | a m l d e (A) or in the presence of s odi um d o d e c y l s u l p h a t e (B]. In t he l a t t e r case, the mo lecular weight of the p r o t e i n was d e t e r m i n e d w i t h standard prot e i ns layered at the extrem i t y of the gel slab. The standard p r o t e i n s were bovine serum a l b u m i n (M~ 67 000), aldolasc (M r 40 000), d e o x y r l b o n u e l e a s e ( M r 3 1 000), trypsin (M r 23 900), r i b o n u c i e a i e A (M r 13 700) and t y r o chrome C (M~ 12 500). The 60-S s u b u n i t p r e p a r a t i o n used in this pa rt i c ul a r e x p e r i m e n t was sUghtiy con tamin ate([ with 40-S subunlts. However the acidic proteins, w h i c h are i ndi c a t e d b y t he arrows, beIonlj to the 60-S s u b u n / t s because t h e y are n o t found in the electrophoretog~azns of 40-S eubunlts, w h i c h are easily o b t a i n e d c o m p l e t e l y pure.
540 mm #Is
|
v
v
w
t
u
~vim~0
6
zb,
o
C
o
~
O
W~
Q
Fig. 2. Effect of alkaline p h o s p h a t a s e on acidic p r o t e i n s e x t r a c t e d from 60-S s ubuni t s by 50% ethanol. Acidic pro teins dissolved in 1 mM (NH 4 )2 COs were i n c u b a t e d at 25°C w i t h o u t (A) and w i t h alkaline phosphatase (3.6 U) for different times: 0(B), 5 rain (C), 40 m i n (D). The r e a c t i o n was s t o p p e d by freezing; after lyophiUzation, the samples were analyzed by t w o di me ns i ona l gel electrophoresis according to the m e t h o d described in Fig. 1A. The first 2.5 em of the first-dimensional gels were cut o u t and e m b e d d e d on the same gel slab. The a m o u n t of p r o t e i n in the c o n t r o l (A) is twice as m u c h as in the samples treated w i t h alkaline phosphatase. The spots visible i n t he u p p e r ps.rt of the gel slab corresponds to alkaline phosphatase.
ment with the hypothesis that, during evolution, a protein playing the role of L7/L12 in procaryotic cells has been conserved, but for still unknown reasons, this protein became phosphorylated instead of being N-acetylated*. After submission of this manuscript, we read a report [22] describing the existence of phosphorylated forms of two proteins called Pl and P2 in 60-S ribosomal subunits of rat liver, and the purification of these proteins. It is very likely that the acidic phosphoprotein we described here is the same as one of these two proteins, although the molecular weight we found is smaller than the values determined for PI and P2 (16 100 and 15 200, respectively). This work has been supported by the Centre National de la Recherche Scientifique (ERA No. 399) and the Ddldgation Gdndral ~ la Recherche Scientifique et Technique (ACC 77-7-1748). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gressner, A.M. and Wool, I.G. (1974) J. Biol. Chem. 249, 6 9 1 7 - - 6 9 2 5 Biat, C. and Loeb, J.E. (1971) FEBS Lett. 18, 124--126 Gressner, A.M. and Wool, I.G. (1976) J. Biol. Chem. 251, 1 6 0 0 - - 1 5 0 4 Gressner, A.M. and Wool, I.G. (1976) Nature 259, 1 4 8 - - 1 5 0 Gressner, A.M. and Wool, I.G. (1974) Biochem. Biophys. Res. C o m m u n . 60, 1482--1490 Zinker, S. and Warner, J.R. (1976) J. Biol. Chem. 251, 1799--1807 Horak, I. and Schiffmann, D. (1977) Eur. J. Biochem. 79, 375--380 Van Agthoven, A.J., Maaasen, J.A. and MSller, W. (1977) Biochem. Biophys. Res. Commun. 77, 969--998 Leader, D.P. and Cola, A.A. (1977) Biochem. J. 1 6 2 , 1 9 9 - - 2 0 0 M~ller, W., Slobin, L.I., Amons, R. and Richter, D. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4 7 4 4 - 4748 Madjaz, J.-J., Arpin, M., Marion, M.-J. and R e b o u d , J.-P. (1977) Mol. Biol. Rep. 3 , 2 8 9 - - 2 9 6 Sherton, C.C. and Wool, I.G. (1974) Mol. Gen. Genet. 135, 97--112 Knopf, U.C., S o m m e r , A., Kenny, J. and Traut, R.R. (1975) Mol. Biol. Rep. 2, 35--40 Mets, L.J. and Bogorad, L. (1974) Anal. Biochem. 5 7 , 2 0 0 - - 2 1 0 Madjaz, J.-J., Arpin, M. and R e b o u d , J.-P. (1977) Anal. Blochem. 63, 304--310 *It has b e e n fo und t h a t , at least in A r t e m i a salina, e L 1 2 ( p ) has an u n b l o c k e d m e t h t o n i n e [8] at the N-terminal p osition. On the o t h e r hand, E. coli L7 is the a c e t y l a t e d form of L12 on the Nt e r m i n a l serlne [ 2 1 ] .
541
16 17 19 19 20 21 22
Reyes, R., Vazquez, D. and Ballesta, P.G. (1977) Eur. J. Biochem. 73, 25--31 Genot, A., Reboud, J.-P., Cenatiempo, Y. and Cozzone, A.J. (1978) FEBS Lett. 86,103--107 St6ffler, G., Wool, I.G., Lin, A. and Rak, K.-H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4723-4726 St~ffler, G. (1974) in Ribosomes (Nomttra, M., Tissi~res, A. and Lengye|, P., eds.), pp. 615-667, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Lin, A. and Wool, I.G. (1974) Mol. Gen. Genet. 134, 1---6 Terhorst, C., M61ler, W., Laursen, R. and Wittmann-Liebold, B. (1973) Eur. J. Bioehem. 34, 138--152 Tsu~mgi, K., Col]atz, E., Todokoro, K., Ulbrich, N., Lightfoot, H.N. and Wool, I.G. (1978) J. Biol. Chem. 253, 946--955
Biochimica et Biophysica Acta, 519 (1978) 537--541 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 91472
OCCURRENCE OF PHOSPHORYLATED FORMS OF AN ACIDIC PROTEIN IN THE LARGE RIBOSOMAL SUBUNIT OF RAT LIVER
M O N I Q U E ARPIN, JEAN-JACQUES M A D J A R and JEAN-PAUL R E B O U D Laboratoire de Biochimie M~dicale, U.E.R. Lyon-Nord 43, Boulevard du 11 Novembre 1918, 69621 Villeurbanne (France)
(Received February 28th, 1978)
Summary An acidic protein from rat liver 60-S ribosomal subunits was selectively extracted with 50% ethanol. It was revealed as three different spots by twodimensional gel electrophoresis, two of them being attributable to phosphorylated forms since they disappeared after alkaline phosphatase treatment. The relationship between this protein and similar acidic proteins found in eucaryotic cells is discussed.
In rat liver ribosomes, one protein of the small subunit ($6), and none from the large subunit, has been found to be phosphorylated in vivo [1]. Quantitative variations in $6 phosphorylation were found under several circumstances: hepatic regeneration [1 ], peptidic hormone injections [2--4], inhibition of protein synthesis by cycloheximide [ 5]. Recently, the presence of phosphorylated acidic proteins has been shown in the large ribosomal subunit of several eucaryotic organisms at different evolutionary stages [ 6 - 9 ] . Interestingly, the acidic proteins found in Artemia salina ribosomes have been shown to be implicated in the interaction with elongation factors and to have a similar function to that of L7/L12 in Escherichia coli [8, 10]. In this paper it is shown that rat liver also contains an acidic phosphoprotein in the large ribosomal subunit. This protein was extracted selectively in the presence of 50% ethanol. The presence of different phosphorylated forms was demonstrated by incubating the acidic protein fraction with phosphatase. Ribosomal subunits from rat liver were prepared from free polysomes as previously described [11]. Proteins were extracted from the 60-S subunits by the acetic acid procedure [12], dialyzed against 1 M acetic acid and lyophilized. The acidic proteins were selectively extracted from 60-S subunits suspended in buffer A (50 mM triethanolamine. HCI, pH = 7.4/80 mM KC1/5 mM MgCI2/20 mM 2-mercaptoethanol) by treatment with 1 vol. of ethanol at 4 ° C.
538 The ethanol extract was dialyzed against 1 M acetic acid and lyophilized. For treatment with alkaline phosphatase, acidic proteins were dissolved in 1 mM (NH4)2 CO3 and incubated with the enzyme. The electrophoretogram in Fig. 1A shows the analysis of 60-S ribosomal subunit proteins, both dimensions being performed at an acidic pH. On the anodic side, three spots are visible which will appear as only one spot (see arrows) when migration in the second dimension is carried out in the presence of sodium dodecylsulphate (Fig. 1B). Therefore these spots should represent different forms of one single protein of about 12 500 daltons. Evidence that two of these spots correspond to phosphorylated derivatives was then obtained by the following experiment: a purified fraction of acidic proteins, extracted from the 60-S subunits with ethanol was treated with alkaline phosphatase. Fig. 2 shows the electrophoretograms of control and phosphatasetreated acidic proteins. It is clear that the enzymatic treatment produces a progressive disappearance of two spots and a simultaneously increase of the third one. Besides the spots of the phosphorylated protein, two other faint spots corresponding to slightly less acidic proteins were also visible on the gel slab but they are difficult to see on the photographs. Our results demonstrate that rat liver 60-S ribosomal subunits contain an acidic protein of 12 500 daltons, which exists under at least two distinct phosphorylated forms. This protein (phosphorylated and unphosphorylated forms) was extracted by 50% ethanol in a very specific manner, since only two slight contaminants could be detected. The ionic conditions for specific extraction are critical since, by using other salt conditions, Reyes et al. completely removed other proteins from the subunits [ 16]. The pH value during electrophoresis is also important: phosphorylated forms are well separated because the pH was very near of the protein pH i. It is also worth noticing that this same protein could be phosphorylated in vitro by an endogenous kinase associated with rat liver polysomes [17]. Our finding can be correlated with results obtained in other eucaryotic systems. The electrophoretic behaviour and the molecular weight (12 500) of the acidic phosphorylated protein in rat liver 60-S subunits are very similar to those of analogous proteins found in yeast [6], Krebs II ascite cells [9], Hela cells [7] and Artemia salina cysts [8]. The protein studied in the latter system, eL12(p), has been shown to be involved in the EF-2~lependent GTP h.vdrolysis, and therefore to be analogous to the bacterial protein L7/L12. However, antibodies against LT/L12 did not react with eL12(p) [8]. On the other hand, Stt}ffler et al. [18] have described two acidic proteins, L40/IA1, in the rat liver large subunit, which are immunologically and functionally related to E. coli L7/L12 [19]. None of these proteins was found phosphorylated [1]. Moreover, the molecular weight of both proteins (25 500) [20] is much higher than that determined by all other authors, as well as by us, for the acidic phosphoprotein of the large subunit and also for bacterial protein L7/L12. Therefore, if the determination of L40/L41 molecular weight is correct, the exact relationship between the acidic protein described in this paper and L40/L41 is not quite clear. In any case, our results bring a new argument to assume that the occurrence of an acidic phosphoprotein in the large ribosomal subunit is general among eucaryotic cells. Thus, they are in agree-
539
Fig. 1. E l e e t r o p h o r e t o ~ m m s of proteins e x t r a c t e d from 60-S s ubuni t s of rat liver. Eleetrophorescs were p e r f o r m e d essentially according to the m e t h o d s described b y K nopf e t al. [13] (Fig. 1A) and Mete and Bogorad [14] (Fig. 1B). I n b o t h eases, migrat/on in the first d i m e n s i o n was p e r f o r m e d unde r the same con ditions: pro teins were dissolved at pH 4.2 and m i g r a t i o n was c o n d u c t e d at pH 5.5 t o w a r d s t he cathode a t 150 V for 5 h. Migration in the second d i m e n s i o n was carried o u t i n a water-cooled a ppa ra t us [15] at pH 4.5 in 18% a c r y | a m l d e (A) or in the presence of s odi um d o d e c y l s u l p h a t e (B]. In t he l a t t e r case, the mo lecular weight of the p r o t e i n was d e t e r m i n e d w i t h standard prot e i ns layered at the extrem i t y of the gel slab. The standard p r o t e i n s were bovine serum a l b u m i n (M~ 67 000), aldolasc (M r 40 000), d e o x y r l b o n u e l e a s e ( M r 3 1 000), trypsin (M r 23 900), r i b o n u c i e a i e A (M r 13 700) and t y r o chrome C (M~ 12 500). The 60-S s u b u n i t p r e p a r a t i o n used in this pa rt i c ul a r e x p e r i m e n t was sUghtiy con tamin ate([ with 40-S subunlts. However the acidic proteins, w h i c h are i ndi c a t e d b y t he arrows, beIonlj to the 60-S s u b u n / t s because t h e y are n o t found in the electrophoretog~azns of 40-S eubunlts, w h i c h are easily o b t a i n e d c o m p l e t e l y pure.
540 mm #Is
|
v
v
w
t
u
~vim~0
6
zb,
o
C
o
~
O
W~
Q
Fig. 2. Effect of alkaline p h o s p h a t a s e on acidic p r o t e i n s e x t r a c t e d from 60-S s ubuni t s by 50% ethanol. Acidic pro teins dissolved in 1 mM (NH 4 )2 COs were i n c u b a t e d at 25°C w i t h o u t (A) and w i t h alkaline phosphatase (3.6 U) for different times: 0(B), 5 rain (C), 40 m i n (D). The r e a c t i o n was s t o p p e d by freezing; after lyophiUzation, the samples were analyzed by t w o di me ns i ona l gel electrophoresis according to the m e t h o d described in Fig. 1A. The first 2.5 em of the first-dimensional gels were cut o u t and e m b e d d e d on the same gel slab. The a m o u n t of p r o t e i n in the c o n t r o l (A) is twice as m u c h as in the samples treated w i t h alkaline phosphatase. The spots visible i n t he u p p e r ps.rt of the gel slab corresponds to alkaline phosphatase.
ment with the hypothesis that, during evolution, a protein playing the role of L7/L12 in procaryotic cells has been conserved, but for still unknown reasons, this protein became phosphorylated instead of being N-acetylated*. After submission of this manuscript, we read a report [22] describing the existence of phosphorylated forms of two proteins called Pl and P2 in 60-S ribosomal subunits of rat liver, and the purification of these proteins. It is very likely that the acidic phosphoprotein we described here is the same as one of these two proteins, although the molecular weight we found is smaller than the values determined for PI and P2 (16 100 and 15 200, respectively). This work has been supported by the Centre National de la Recherche Scientifique (ERA No. 399) and the Ddldgation Gdndral ~ la Recherche Scientifique et Technique (ACC 77-7-1748). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gressner, A.M. and Wool, I.G. (1974) J. Biol. Chem. 249, 6 9 1 7 - - 6 9 2 5 Biat, C. and Loeb, J.E. (1971) FEBS Lett. 18, 124--126 Gressner, A.M. and Wool, I.G. (1976) J. Biol. Chem. 251, 1 6 0 0 - - 1 5 0 4 Gressner, A.M. and Wool, I.G. (1976) Nature 259, 1 4 8 - - 1 5 0 Gressner, A.M. and Wool, I.G. (1974) Biochem. Biophys. Res. C o m m u n . 60, 1482--1490 Zinker, S. and Warner, J.R. (1976) J. Biol. Chem. 251, 1799--1807 Horak, I. and Schiffmann, D. (1977) Eur. J. Biochem. 79, 375--380 Van Agthoven, A.J., Maaasen, J.A. and MSller, W. (1977) Biochem. Biophys. Res. Commun. 77, 969--998 Leader, D.P. and Cola, A.A. (1977) Biochem. J. 1 6 2 , 1 9 9 - - 2 0 0 M~ller, W., Slobin, L.I., Amons, R. and Richter, D. (1975) Proc. Natl. Acad. Sci. U.S. 72, 4 7 4 4 - 4748 Madjaz, J.-J., Arpin, M., Marion, M.-J. and R e b o u d , J.-P. (1977) Mol. Biol. Rep. 3 , 2 8 9 - - 2 9 6 Sherton, C.C. and Wool, I.G. (1974) Mol. Gen. Genet. 135, 97--112 Knopf, U.C., S o m m e r , A., Kenny, J. and Traut, R.R. (1975) Mol. Biol. Rep. 2, 35--40 Mets, L.J. and Bogorad, L. (1974) Anal. Biochem. 5 7 , 2 0 0 - - 2 1 0 Madjaz, J.-J., Arpin, M. and R e b o u d , J.-P. (1977) Anal. Blochem. 63, 304--310 *It has b e e n fo und t h a t , at least in A r t e m i a salina, e L 1 2 ( p ) has an u n b l o c k e d m e t h t o n i n e [8] at the N-terminal p osition. On the o t h e r hand, E. coli L7 is the a c e t y l a t e d form of L12 on the Nt e r m i n a l serlne [ 2 1 ] .
541
16 17 19 19 20 21 22
Reyes, R., Vazquez, D. and Ballesta, P.G. (1977) Eur. J. Biochem. 73, 25--31 Genot, A., Reboud, J.-P., Cenatiempo, Y. and Cozzone, A.J. (1978) FEBS Lett. 86,103--107 St6ffler, G., Wool, I.G., Lin, A. and Rak, K.-H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4723-4726 St~ffler, G. (1974) in Ribosomes (Nomttra, M., Tissi~res, A. and Lengye|, P., eds.), pp. 615-667, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Lin, A. and Wool, I.G. (1974) Mol. Gen. Genet. 134, 1---6 Terhorst, C., M61ler, W., Laursen, R. and Wittmann-Liebold, B. (1973) Eur. J. Bioehem. 34, 138--152 Tsu~mgi, K., Col]atz, E., Todokoro, K., Ulbrich, N., Lightfoot, H.N. and Wool, I.G. (1978) J. Biol. Chem. 253, 946--955