BIOCHIMIE, 1975, 57, 167-173.
Eukaryotic ribosomal proteins. Molecular weights of proteins from rabbit liver subunits. Francine CREUSOT, Jean DELAUNAY a n d Georges SCHAPI~AO. lnstitul de Pathologie Mal~culaire (*), 24, rue du faubourg Saint-Jacques 75015 Paris. (23-10-1974). Summary. - - The molecular weights of the proteins from rabbit liver ribosomal 40 S and 60 S subunits were determined after preliminary separation of these proteins by twodimensional electrophoresis : each spot present in the polyacrylamide slab was cut off, eluted and rerun in a SDS one-dimensional polyacrylamide gel. The molecular weights range from 9,000 to 35,000 with a number-average molecular weight of 19,600 for the 40S proteins, and from 9,400 to 52,000 with a number-average molecular weight of 23,600 for 60 S proteins.
INTRODUCTION. Studies on the role of ribosomes d u r i n g p r o t e i n synthesis r e q u i r e the knowledge of physico-chemical characteristics of ribosomal components. Waller and Harris [1] demonstrated that prokaryotic (E. cold ribosomes c o n t a i n e d a large n u m ber of basic proteins. At the p r e s e n t time, the m a i n s t r u c t u r a l features [2, 3] of E. colt ribosomal prot e i n s are k n o w n a n d their p u r i f i c a t i o n [4, 5] has been the starting p o i n t of most extensive investigations. Studies on eukaryotic ribosomal p r o t e i n s have been i n i t i a t e d by Low and Wool [6] and A n h T u a n But a n d S c h a p i r a [7]. They showed that eukaryotic ribosomes were also composed of numerous heterogenous basic proteins. I n eukaryotes, however, p u r i f i c a t i o n of ribosomal p r o t e i n s is still a c h a l l e n g i n g p r o b l e m because of the greater difficu,lty i n o b t a i n i n g pure ribosomal s u b u n i t s a n d of the higher n u m b e r of r i b o s o m a l p r o t e i n s : only very r e c e n t l y did W e s t e r m a n n et al. [8, 9~ a n d Terao and Ogata [10] u n d e r t a k e purification processes. So far, the physico-chemical properties of ribosomal proteins have been studied m a i n l y by a n a l y t i c a l electrophoresis [11-20]. In this work, we have d e t e r m i n e d the molecular weights of the p r o t e i n s from r a b b i t liver 40 S and 60 S r i b o s o m a l s u b u n i t s b y using t w o - d i m e n s i o n a l (2-D) electrophoresis [21] and SDS-electrophoresis To whom all correspondence should be addressed. (~) Groupe U 15 de l'Institut National de la Sant~ et de la Recherche M~dicale, Laboratoire Associ4 au Centre National de la Recherche Scientifique.
[22] i n succession as has been very r e c e n t l y deviced by H o w a r d et al. [18, 19] and Lin a n d Wool [20]. MATERIAL AND METHODS.
Buffers and solutions. Buffer
I : 50 mM Tris-HC1 pH 7.6, 5 mM MgCl 2, 80 mM KC1, Buffer II : 50 mM Tris-HC1 pH 7.6, 5 mM MgC12, 880 mM KC1, Buffer I I I : 2.9 mM NaH2PO 4, 7.2 mM Na2HPO 4, pH 7.2, 20 p. 10,0 glycerol, 0.3 MB-mercaptoethanol, Solution A : 6 M urea, 1 p. 100 SDS, Solution B : 4 volumes of solution A and 1 volume of buffer III.
Preparation of ribosomes. Rabbit liver ribosomes were p r e p a r e d at 4°C a c c o r d i n g to Martin et al. [23], w i t h m i n o r modifrcations. Livers w e r e h o m o g e n i z e d w i t h a PotterElvejhem homogenizer i n 2.5 volumes of buffer I c o n t a i n i n g '0.25 M sucrose. Ribosomes, once pelleted from the p o s t - m i t o c h o n d r i a l s u p e r n a t a n t , were r e s u s p e n d e d in buffer I. After a d d i t i o n of sodium deoxycholate to the final c o n c e n t r a t i o n of 1.2 p. 100, they were f u r t h e r purified by ultracent r i f u g a t i o n t h r o u g h a c u s h i o n of 0.5 M sucrose in buffer I. The pellets w e r e kept at - - 8 0 ° C .
Preparation of ribosomal subunits. The dissociation of ribosomal s u b u n i t s (80120 OD26o/ml) was p e r f o r m e d in buffer II, cont a i n i n g 0.1 mM p u r o m y c i n [13]. The s u b u n i t s were centrifuged t h r o u g h a 10-30 p. 100 l i n e a r su.cros~
F. Creusot, J. D e l a u n a y and G. Schapira.
168
g r a d i e n t i n buffer II, i n a SW 27 rotor at 25,'000 r p m for 5 1/2 hrs at 28°C. F r a c t i o n s corresp o n d i n g to 40 S a n d 60 S s u b u n i t s (fig. 1 a) w e r e collected a n d dialyzed overnight against 5 liters of buffer 1 c o n t a i n i n g 50 mM ~-mercaptoethanol at 4°C a n d then, precipitatect w i t h ,0.5 volume of
60 S subunits
OD
40S subunits
260 nm
After the 2-D electrophoresis of ribosomal proteins, th.e spots were eluted from the slab a c c o r d i n g to H o w a r d a n d T r a u t [18], with some modificat i o n s ; the spots s t a i n e d w i t h a m i d o b l a c k were cut off f r o m the 2-D slab ; gel pieces were crushed a n d m a c e r a t e d for 24 hrs i n 1 m l of solution A at
purified 6OS subunits
OD 260nm
~
2
2_
0
0
Top
Bottom
Bottom
Top Fractions
Fractions
Fro. 1, - - Sedimentation profile of ribosomal subunits in 10-30 p. 100 sucrose gradient. a. Separation of the 40 S and 60 S subunits, b. Purification of the 60 S subunits. ////// : collected fractions.
ethanol [24]. 60 S s u b u n i t s c o n t a m i n a t e d by various amounts of 40 S s u b u n i t s [23] were purified again w i t h the same method, (fig. lb). Thus, 60 S s u b u n i t s were free of 40 S s u b u n i t s as described by Sherton a n d Wool [13].
Extraction of ribosomal proteins. P r o t e i n s w e r e extracted from ribosomes a n d ribosomal s u b u n i t s by 06 p. 100 acetic acid a c c o r d i n g to Waller and Harris [1] i n the presence of 33 mM MgC12 [25].
Two-dimensional phoresis.
polyacrglamide
gel electro-
W e used K a l t s c h m i d t and W i t t m a n n ' s t e c h n i q u e [21], modified as p r e v i o u s l y reported [26]. F u r t h e r modifications ~vere i n t r o d u c e d : the first d i m e n sion was p e r f o r m e d for 15 hrs u n d e r 180 volts a n d the electrode buffer did not c o n t a i n a n y urea. I n some experiments, the voltage was r e d u c e d to 100 volts and 65 volts i n the first and the second d i m e n s i o n s respectively.
Determination of the molecular weights of ribosomal proteins. The molecular weights of r i b o s o m a l p r o t e i n s were d e t e r m i n e d by disc-electrophoresis on polya c r y l a m i d e gel i n the presence of SDS, a c c o r d i n g to W e b e r and Osborn [22].
BIOCHIMIE, 1975,
57, n ° 2.
room temperature. The eluate was dialyzed for 24 hrs i n 2 × 1 liter of 5 p. 100 acetic acid i n order to r e m o v e SDS, urea a n d the amidoblack. After lyophilization, r i b o s o m a l p r o t e i n s were i n c u b a t e d i n 50 i~d of solution B for 10 rain. at 65°C, t h e n l a y e r e d w i t h a m a r k e r dye (5 ~1 of 0.05 p. 100 b r o m o p h e n o l blue) on top of a 10 p. 1,00 c y l i n d r i c a l p o l y a c r y l a m i d e gel, 120 m m long a n d 5.5 m m i n n e r diameter, c o n t a i n i n g 0.1 p. 100 SDS a n d 0.'06 M ,~-mercaptoe[hanol at pH 7.2. Five reference p r o t e i n s of k n o w n m o l e c u l a r weights w e r e i n c u b a t e d i n solution B for 10 rain. at 65°C : b o v i n e s e r u m a l b u m i n (BSA = 08,0.00) ; c r e a t i n e - k i n a s e (CK = 40;000); t r y p s i n (Tr = 23,300) ; r i b o n u c l e a s e A (RA ~ 13,900) and i n s u l i n (In ~ 6,000). 20/~l of this m i x t u r e (1 to 2 ~g of each protein) were layered i n the p r e s e n c e of the m a r k e r dye on top of the gel. Ribosomal a n d reference proteins, on separate gels, w e r e s i m u l t a n e o u s l y submitted to 1 h r electrophoresis w i t h a 4 mA i n t e n s i t y per gel, a n d 2 to 3 hrs at 1.0 mA per gel. The r u n was stopped w h e n the t r a c k i n g dye reached a p o i n t 1 cm from the ~/nodic end of the gel. After s t a i n i n g a n d dest a i n i n g the gels, the electrophoretic mobilities of both the reference and the ribosomal p r o t e i n s were d e t e r m i n e d a c c o r d i n g to the t e c h n i q u e o,f W e b e r a n d Osborn w h i c h had a p r e c i s i o n of 1,0 p. 100 [22] :
Molecular w e i g h t s of proteins f r o m rabbit ribosomes. mobilities =
distance of p r o t e i n m i g r a t i o n length after d e s t a i n i n g length before s t a i n i n g × distance of dye m i g r a t i o n
The reference line was o b t a i n e d by plotting the log (M.W.) versus mobilities of the above reference proteins. F u r t h e r m o r e , the molecular weights of ribosomal p r o t e i n s were also read directly from the reference line [22].
RESULTS. Figures 2 (a, b and c) and 3 (a, b and c) ; show the finger-prints o b t a i n e d by 2-D electrophoresis of 40 S, 61)S and 80 S ribosomal proteins. 40 S ribosomal p r o t e i n s were separated into 31 cationic and 2 a n i o n i c spo{s. 60 S ribosomal p r o t e i n s were separated into 37 cationi.c spots. P r o t e i n s of total ribosomes were separated into 3 a n i o n i c a n d 55 cationic spots. The latter n u m b e r - - lower than the sum of the spots obtained w i t h the s u b u n i t s separatel~y - - can be accounted for by the o v e r l a p p i n g of some p r o t e i n s h a v i n g the same m i g r a t i o n i n both s u b u n i t s (fig. 2c). As the molecular weights of these spots were generally different for the two subunits, they were not the result of a c r o s s - c o n t a m i n a t i o n between the two subunits. Conversely, two n e w a n i o n i c spots, a a n d b, were p r e s e n t i n the 80 S proteins. The molecular weights of the p r o t e i n s from the small s u b u n i t range from 9,00'0 to 35,000 a n d their number-average molecular weight is 19,61)0 ; those of the large s u b u n i t range from 9,400 to 52,000 and their n u m b e r - a v e r a g e molecular weight is 23,600 (table I). After 2-1) electrophoresis, the proteins of the region Se9 w h e r e four close spots were present, as well as L12, L~6 and. L22, were o b t a i n e d in too small amounts, w h i c h made it impossible to determ i n e their molecular weights, t n general, p r o t e i n s migrated along the SDS-polyacrylamide gel i n one single band. 5 spots only : ~ , LI~ , Lz3, L27 a n d Ls~, w e r e separated into 2 b a n d s after SDS-el'ectrophorests (fig. 4).
Fro. 2. - - 2-D electrophoresis of rabbit liver ribosomal proteins performed under standard conditions.
The anode is on the left hand side in the first dimension and at the top in the second dimension : a. 1.2 m.g of 40 S proteins, h. 1 mg of 60. S proteins, c. 1.5 nag of 80 S proteins.
BIOCHIMIE, 1975, 57, n o 2.
169
F. Creusot, J. Delattna g and G. Schapira.
170
4-
1-D
4-
2-C $3
I~ $2
So~
C~ s4
ssO sz(~
0
s~
S9~
3a
$1oc~
Scl~
sd 0
(~S8 0
514
S 11
s~70 d O ~5 S16,18 S2o 0 SCb ~ C] S19 $24
S250 S?8°$2 7 0S26
s2 e ( ~ )
I~$26
Eukaryotic ribosomal proteins. Molecular weights of proteins from rabbit liver subunits. Francine CREUSOT, Jean DELAUNAY a n d Georges SCHAPI~AO. lnstitul de Pathologie Mal~culaire (*), 24, rue du faubourg Saint-Jacques 75015 Paris. (23-10-1974). Summary. - - The molecular weights of the proteins from rabbit liver ribosomal 40 S and 60 S subunits were determined after preliminary separation of these proteins by twodimensional electrophoresis : each spot present in the polyacrylamide slab was cut off, eluted and rerun in a SDS one-dimensional polyacrylamide gel. The molecular weights range from 9,000 to 35,000 with a number-average molecular weight of 19,600 for the 40S proteins, and from 9,400 to 52,000 with a number-average molecular weight of 23,600 for 60 S proteins.
INTRODUCTION. Studies on the role of ribosomes d u r i n g p r o t e i n synthesis r e q u i r e the knowledge of physico-chemical characteristics of ribosomal components. Waller and Harris [1] demonstrated that prokaryotic (E. cold ribosomes c o n t a i n e d a large n u m ber of basic proteins. At the p r e s e n t time, the m a i n s t r u c t u r a l features [2, 3] of E. colt ribosomal prot e i n s are k n o w n a n d their p u r i f i c a t i o n [4, 5] has been the starting p o i n t of most extensive investigations. Studies on eukaryotic ribosomal p r o t e i n s have been i n i t i a t e d by Low and Wool [6] and A n h T u a n But a n d S c h a p i r a [7]. They showed that eukaryotic ribosomes were also composed of numerous heterogenous basic proteins. I n eukaryotes, however, p u r i f i c a t i o n of ribosomal p r o t e i n s is still a c h a l l e n g i n g p r o b l e m because of the greater difficu,lty i n o b t a i n i n g pure ribosomal s u b u n i t s a n d of the higher n u m b e r of r i b o s o m a l p r o t e i n s : only very r e c e n t l y did W e s t e r m a n n et al. [8, 9~ a n d Terao and Ogata [10] u n d e r t a k e purification processes. So far, the physico-chemical properties of ribosomal proteins have been studied m a i n l y by a n a l y t i c a l electrophoresis [11-20]. In this work, we have d e t e r m i n e d the molecular weights of the p r o t e i n s from r a b b i t liver 40 S and 60 S r i b o s o m a l s u b u n i t s b y using t w o - d i m e n s i o n a l (2-D) electrophoresis [21] and SDS-electrophoresis To whom all correspondence should be addressed. (~) Groupe U 15 de l'Institut National de la Sant~ et de la Recherche M~dicale, Laboratoire Associ4 au Centre National de la Recherche Scientifique.
[22] i n succession as has been very r e c e n t l y deviced by H o w a r d et al. [18, 19] and Lin a n d Wool [20]. MATERIAL AND METHODS.
Buffers and solutions. Buffer
I : 50 mM Tris-HC1 pH 7.6, 5 mM MgCl 2, 80 mM KC1, Buffer II : 50 mM Tris-HC1 pH 7.6, 5 mM MgC12, 880 mM KC1, Buffer I I I : 2.9 mM NaH2PO 4, 7.2 mM Na2HPO 4, pH 7.2, 20 p. 10,0 glycerol, 0.3 MB-mercaptoethanol, Solution A : 6 M urea, 1 p. 100 SDS, Solution B : 4 volumes of solution A and 1 volume of buffer III.
Preparation of ribosomes. Rabbit liver ribosomes were p r e p a r e d at 4°C a c c o r d i n g to Martin et al. [23], w i t h m i n o r modifrcations. Livers w e r e h o m o g e n i z e d w i t h a PotterElvejhem homogenizer i n 2.5 volumes of buffer I c o n t a i n i n g '0.25 M sucrose. Ribosomes, once pelleted from the p o s t - m i t o c h o n d r i a l s u p e r n a t a n t , were r e s u s p e n d e d in buffer I. After a d d i t i o n of sodium deoxycholate to the final c o n c e n t r a t i o n of 1.2 p. 100, they were f u r t h e r purified by ultracent r i f u g a t i o n t h r o u g h a c u s h i o n of 0.5 M sucrose in buffer I. The pellets w e r e kept at - - 8 0 ° C .
Preparation of ribosomal subunits. The dissociation of ribosomal s u b u n i t s (80120 OD26o/ml) was p e r f o r m e d in buffer II, cont a i n i n g 0.1 mM p u r o m y c i n [13]. The s u b u n i t s were centrifuged t h r o u g h a 10-30 p. 100 l i n e a r su.cros~
F. Creusot, J. D e l a u n a y and G. Schapira.
168
g r a d i e n t i n buffer II, i n a SW 27 rotor at 25,'000 r p m for 5 1/2 hrs at 28°C. F r a c t i o n s corresp o n d i n g to 40 S a n d 60 S s u b u n i t s (fig. 1 a) w e r e collected a n d dialyzed overnight against 5 liters of buffer 1 c o n t a i n i n g 50 mM ~-mercaptoethanol at 4°C a n d then, precipitatect w i t h ,0.5 volume of
60 S subunits
OD
40S subunits
260 nm
After the 2-D electrophoresis of ribosomal proteins, th.e spots were eluted from the slab a c c o r d i n g to H o w a r d a n d T r a u t [18], with some modificat i o n s ; the spots s t a i n e d w i t h a m i d o b l a c k were cut off f r o m the 2-D slab ; gel pieces were crushed a n d m a c e r a t e d for 24 hrs i n 1 m l of solution A at
purified 6OS subunits
OD 260nm
~
2
2_
0
0
Top
Bottom
Bottom
Top Fractions
Fractions
Fro. 1, - - Sedimentation profile of ribosomal subunits in 10-30 p. 100 sucrose gradient. a. Separation of the 40 S and 60 S subunits, b. Purification of the 60 S subunits. ////// : collected fractions.
ethanol [24]. 60 S s u b u n i t s c o n t a m i n a t e d by various amounts of 40 S s u b u n i t s [23] were purified again w i t h the same method, (fig. lb). Thus, 60 S s u b u n i t s were free of 40 S s u b u n i t s as described by Sherton a n d Wool [13].
Extraction of ribosomal proteins. P r o t e i n s w e r e extracted from ribosomes a n d ribosomal s u b u n i t s by 06 p. 100 acetic acid a c c o r d i n g to Waller and Harris [1] i n the presence of 33 mM MgC12 [25].
Two-dimensional phoresis.
polyacrglamide
gel electro-
W e used K a l t s c h m i d t and W i t t m a n n ' s t e c h n i q u e [21], modified as p r e v i o u s l y reported [26]. F u r t h e r modifications ~vere i n t r o d u c e d : the first d i m e n sion was p e r f o r m e d for 15 hrs u n d e r 180 volts a n d the electrode buffer did not c o n t a i n a n y urea. I n some experiments, the voltage was r e d u c e d to 100 volts and 65 volts i n the first and the second d i m e n s i o n s respectively.
Determination of the molecular weights of ribosomal proteins. The molecular weights of r i b o s o m a l p r o t e i n s were d e t e r m i n e d by disc-electrophoresis on polya c r y l a m i d e gel i n the presence of SDS, a c c o r d i n g to W e b e r and Osborn [22].
BIOCHIMIE, 1975,
57, n ° 2.
room temperature. The eluate was dialyzed for 24 hrs i n 2 × 1 liter of 5 p. 100 acetic acid i n order to r e m o v e SDS, urea a n d the amidoblack. After lyophilization, r i b o s o m a l p r o t e i n s were i n c u b a t e d i n 50 i~d of solution B for 10 rain. at 65°C, t h e n l a y e r e d w i t h a m a r k e r dye (5 ~1 of 0.05 p. 100 b r o m o p h e n o l blue) on top of a 10 p. 1,00 c y l i n d r i c a l p o l y a c r y l a m i d e gel, 120 m m long a n d 5.5 m m i n n e r diameter, c o n t a i n i n g 0.1 p. 100 SDS a n d 0.'06 M ,~-mercaptoe[hanol at pH 7.2. Five reference p r o t e i n s of k n o w n m o l e c u l a r weights w e r e i n c u b a t e d i n solution B for 10 rain. at 65°C : b o v i n e s e r u m a l b u m i n (BSA = 08,0.00) ; c r e a t i n e - k i n a s e (CK = 40;000); t r y p s i n (Tr = 23,300) ; r i b o n u c l e a s e A (RA ~ 13,900) and i n s u l i n (In ~ 6,000). 20/~l of this m i x t u r e (1 to 2 ~g of each protein) were layered i n the p r e s e n c e of the m a r k e r dye on top of the gel. Ribosomal a n d reference proteins, on separate gels, w e r e s i m u l t a n e o u s l y submitted to 1 h r electrophoresis w i t h a 4 mA i n t e n s i t y per gel, a n d 2 to 3 hrs at 1.0 mA per gel. The r u n was stopped w h e n the t r a c k i n g dye reached a p o i n t 1 cm from the ~/nodic end of the gel. After s t a i n i n g a n d dest a i n i n g the gels, the electrophoretic mobilities of both the reference and the ribosomal p r o t e i n s were d e t e r m i n e d a c c o r d i n g to the t e c h n i q u e o,f W e b e r a n d Osborn w h i c h had a p r e c i s i o n of 1,0 p. 100 [22] :
Molecular w e i g h t s of proteins f r o m rabbit ribosomes. mobilities =
distance of p r o t e i n m i g r a t i o n length after d e s t a i n i n g length before s t a i n i n g × distance of dye m i g r a t i o n
The reference line was o b t a i n e d by plotting the log (M.W.) versus mobilities of the above reference proteins. F u r t h e r m o r e , the molecular weights of ribosomal p r o t e i n s were also read directly from the reference line [22].
RESULTS. Figures 2 (a, b and c) and 3 (a, b and c) ; show the finger-prints o b t a i n e d by 2-D electrophoresis of 40 S, 61)S and 80 S ribosomal proteins. 40 S ribosomal p r o t e i n s were separated into 31 cationic and 2 a n i o n i c spo{s. 60 S ribosomal p r o t e i n s were separated into 37 cationi.c spots. P r o t e i n s of total ribosomes were separated into 3 a n i o n i c a n d 55 cationic spots. The latter n u m b e r - - lower than the sum of the spots obtained w i t h the s u b u n i t s separatel~y - - can be accounted for by the o v e r l a p p i n g of some p r o t e i n s h a v i n g the same m i g r a t i o n i n both s u b u n i t s (fig. 2c). As the molecular weights of these spots were generally different for the two subunits, they were not the result of a c r o s s - c o n t a m i n a t i o n between the two subunits. Conversely, two n e w a n i o n i c spots, a a n d b, were p r e s e n t i n the 80 S proteins. The molecular weights of the p r o t e i n s from the small s u b u n i t range from 9,00'0 to 35,000 a n d their number-average molecular weight is 19,61)0 ; those of the large s u b u n i t range from 9,400 to 52,000 and their n u m b e r - a v e r a g e molecular weight is 23,600 (table I). After 2-1) electrophoresis, the proteins of the region Se9 w h e r e four close spots were present, as well as L12, L~6 and. L22, were o b t a i n e d in too small amounts, w h i c h made it impossible to determ i n e their molecular weights, t n general, p r o t e i n s migrated along the SDS-polyacrylamide gel i n one single band. 5 spots only : ~ , LI~ , Lz3, L27 a n d Ls~, w e r e separated into 2 b a n d s after SDS-el'ectrophorests (fig. 4).
Fro. 2. - - 2-D electrophoresis of rabbit liver ribosomal proteins performed under standard conditions.
The anode is on the left hand side in the first dimension and at the top in the second dimension : a. 1.2 m.g of 40 S proteins, h. 1 mg of 60. S proteins, c. 1.5 nag of 80 S proteins.
BIOCHIMIE, 1975, 57, n o 2.
169
F. Creusot, J. Delattna g and G. Schapira.
170
4-
1-D
4-
2-C $3
I~ $2
So~
C~ s4
ssO sz(~
0
s~
S9~
3a
$1oc~
Scl~
sd 0
(~S8 0
514
S 11
s~70 d O ~5 S16,18 S2o 0 SCb ~ C] S19 $24
S250 S?8°$2 7 0S26
s2 e ( ~ )
I~$26
