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Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits
Biochimica et Biophysica Acta, 697 (1982) 101-112
101
Elsevier Biomedical Press BBA 91051
EFFECTS OF ETHIONINE TREATMENT ON PROTEIN-SYNTHESIZING APPARATUS OF RAT LIVER 80 S RIBOSOMES AND 40 S RIBOSOMAL SUBUNITS YOSHIAKI TAKAHASHI and KIKUO OGATA
Department of Biochemistry, Niigata University School of Medicine, Niigata 951 (Japan) (Received November 13th, 1981)
Key words: Ethionine; 40 S ribosomal subuniC Protein synthesis; Initiation factor," Globin mRNA; (Rat liver)
The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete ceil-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsC! isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [t4C]- or [3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that elF-3 subunits and two unidentified protein fractions of molecular weight of 2.3.10 4 and 2.1.10 4 were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[3H]ethylmaleimide or N-[ 14C]ethyimaleimide, and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially elF-3, as the results of conformational changes of 40 S subunits.
Introduction It was shown that administration of DLethionine to female rats resulted in a rapid depletion of hepatic ATP [1-3]. Protein synthesis was then decreased with a concomitant breakdown of polysomes into monosomes [4,5]. These effects of ethionine were reversed by treatment with adenine and/or methionine which reaggregated liver polysomes and increased protein synthesis in the liver [6,7]. However, the sites of the protein-synthesizing 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
apparatus which were impaired by ethionine administration, have not yet completely been clarified. From in vivo labeling kinetics, it was reported that ethionine treatment caused the inhibition of protein synthesis by reducing the rate of the initiation [8]. Murty et al. [9] using poly(U) as a template, indicated that administration of ethionine led to a significant reduction in the activities of rat liver for peptide bond initiation by affecting both initiaton factors and ribosomes. Recently, Kisilevsky et al. [10] demonstrated that administration of ethionine caused not only con-
102
formational changes of rat liver ribosomes but also decreased the levels of phosphorylation of ribosomal protein $6 * of rat liver [12]. They also reported that the extent of phosphorylation of rat liver $6 protein during ethionine intoxication and recovery was closely related to the proteinsynthesizing activity of the liver [13]. Another effect of ethionine on protein synthesis was that methyl-deficient or ethylated RNAs were accumulated in rat liver by ethionine administration [14]. Goswami and Sharma [15] reported that methyl deficiency of the 5' cap structure of mRNA was observed when rats were fed with ethionine in their diet for 7 days, and the translational activity of methyl-deficient m R N A was decreased when added to a wheat germ cell-free system [15]. When ethionine was administered to rats, ethylated tRNAs were observed in rat liver and especially lysine t R N A 2 [16] was highly ethylated. Methyl-deficient tRNAs were accumulated in the liver of ethionine-administrated rats. When assayed in injected oocytes, treatment of rats with ethionine caused severe impairments in the amino acylation capacity of some kinds of tRNA. Furthermore, the participation of aminoacyl tRNA from ethionine-treated rat liver in the translation of various mRNAs was severely impaired in both injected oocyte and a cell-free system from wheat germ [17]. Thus, ethionine appears to have pleiotropic effects on liver protein-synthesizing apparatus. The present study was undertaken to elucidate whether rat liver ribosomes and especially, their 40 S subunits were altered by ethionine treatment for short time periods of 4 h in vivo. The effects of ethionine treatment on activities of 40 S subunits for the translation of globin mRNA and for the interaction with initiation factors were investigated. Conformational changes of ethioninetreated rat liver 40 S subunits were examined. This report describes the results of these experiments. Materials and Methods
Chemicals. L-[4,5-3H]Leucine (54 Ci/mmol), [614C]orotic acid monohydrate (61 m C i / m m o l ) and * Individual ribosomal proteins were designated according to the proposed u n i f o r m n o m e n c l a t u r e [ I I].
[5-3H]orotic acid (21 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, U.K. N-[ethyl-lJ4C]Ethylmaleimide (23.7 mCi/mmol) and N-[ethyl-2-3H]ethylmaleimide (692.0 m C i / m mol) were purchased from New England Nuclear, Boston. All other chemicals were of reagent grade.
Ethionine treatment and preparation of ribosomes and ribosomal subunits. Female Wistar strain rats weighing 150-200g were fasted overnight. DLEthionine was administered intraperitoneally at a dose of 1 g / k g body weight. Control rats received an equivalent volume of 0.9% saline [12]. Rats were killed 4 h later. The livers were rapidly excised and homogenized in 3 vol. ice-cold Medium A containing 0.25 M sucrose/25 mM KCI/5 mM MgC12/50 mM Tris-HCl, pH 7.6. All subsequent procedures were carried out at 0-4°C. Postmitochondrial supernatant was prepared by centrifugation of the homogenate at 10000 × g for 10 min. After sodium deoxycholate treatment [12], 18 ml supernatant were layered over 20 ml of 0 . 5 M sucrose, containing mediumA, and ribosomes were pelleted in a Beckman type 60 Ti rotor by centrifugation at 105000 X g for 1 h. The ribosomes were then run off by incubation with the rat liver pH 5 fraction and precipitated by centrifugation at 130000 × g for 15 h [18], these were designated as 80 S ribosomes. Rat liver 40 S subunits were then prepared by centrifugation of run-off 80S ribosomes at 2°C through a 15-30% linear sucrose density-gradient containing 0.3 M KC1/ 3 mM MgC12/10 mM 2-mercaptoethanol/20 mM Tris-HC1, p H 7.6 and were pelleted in a Beckman type 65 rotor by centrifugation at 130000 X g for 15 h [18]. Mouse liver 60 S subunits were prepared by the same methods as described above, except that two cycles of sucrose-gradient centrifugation were carried out.
Preparation of partially purified rabbit initiation factors and 9 S globin mRNA. Reticulocyte lysate and polysomes were prepared from phenylhydragine-treated rabbits according to the method of Schreier and Staehelin [18]. Partially purified initiation factors were prepared by DEAE-cellulose column chromatography of 0.5 M KC1 wash of rabbit reticulocyte polysomes, followed by 0-40% and 40-70% ammonium sulfate precipitation. The factors thus obtained were designated as elF 0 -
103
40% (NH4)2SO 4 and elF 40-70% (NH4)2SO4, respectively [18]. Globin mRNA was prepared from total RNA from 0.5 M KC1 wash of rabbit reticulocyte polysomes by two cycles of oligo-dT celluose chromatography by the method of Aviv and Leder [ 19]. Purification of elF-3, eIF-3 was purified from rat liver total native subunits [20], according to the methods of Schreier et al. [21], followed by the method of Jones et al. [22]. In brief, rat liver total native subunits were incubated in the presence of 0.5 M KC1 at 0°C for 15 min. Subsequently, the total subunits were sedimented at 113000 × g for 15h. Crystalline (NH4)2SO4 was added to the supernatant to a saturation of 40%. The precipitate was collected by centrifugation at 10000 X g for 10 min and dissolved in Medium B (20 mM Tris-HCl, pH 7.6/0.1 mM EDTA/1 mM dithiothreitol/10% glycerol) containing 0.1 M KCI and dialysed for 12h against the same buffer. This fraction was applied onto the DEAE-cellulose column equilibrated with Medium B. After passage of non-absorbed proteins, retained protein fractions were eluted with 0.3 M KCI in Medium B. The eluent was dialysed for 12h against MediumC (30 mM potassium phosphate, pH 7.0/0.1 mM E D T A / l mM dithiothreitol/10% glycerol) containing 0.1 M KC1 and then applied onto the phosphocellulose column equilibrated with Medium C. The protein fractions, retained to 0.1 M KC1, were eluted with 0.4 M KC1 in Medium C. The eluent was further purified by 10-30% linear glycerol gradientcentrifugation following the methods of Jones et al. [22]. The fractions having subunit anti-association activity, were pooled as eIF-3 and concentrated by adding crystalline (NH4)2SO4 t o a saturation of 50%. The purity of eIF-3 was checked by SDS-polyacrylamide gel electrophoresis and by 3.6% polyacrylamide gel electrophoresis under non-dissociating conditions [21]. After staining with Coomassie brillant blue R250, the density at 600 nm was traced with Joyce Loebl Chromoscan 200. The results are described in the later section.
Preparation of native 40 S ribosomes uniformly labeled with [~H]- or [HC]orotic acid. 5/~Ci [14C]orotic acid or 70 #Ci [aH]orotic acid were intraperitoneally injected into rats three times at an interval of 12 h. Ethionine or 0.9% saline was injected 20h after the final isotope injection and
the rats were killed 4 h later. Native 40 S subunits were pelleted from postmicrosomal supernatant of rat liver by centrifugation at 130000 X g for 15 h and further purified by centrifugation through a 10-45% hyperboric sucrose-density gradient according to a modification of the method of Sundkvist and Staehelin [23]. Translation of globin mRNA. The translation of globin mRNA was carried out according to the method of Schreier and Staehelin [18]. The complete reaction mixture of 0.1 ml contained the following components: 0.14 A260 U of rat liver 40 S subunits and 0.36 A260 U of mouse liver 60 S subunits, or 0.5 A26o U of rat liver 80 S ribosomes, 60 #g protein of elF 0-40% (NH4)2SO4, 60 #g protein of elF 40-70% (NH4)2SO4, 125 #g protein of the pH 5 fraction from rat liver [24], 0.05 A260 U of globin mRNA, 1 mM ATP, 0.4 mM GTP, 20 mM creatine phosphate, 0.38 U creatine kinase, 1 mM dithiothreitol, an amino acid mixture containing 30 /tM of each unlabeled amino acid except for leucine, 4mM MgC12, 70 mM KC1, 20 mM Tris-HCl, pH 7.6 and 1 /tCi [3H]leucine. Incubation was carried out at 30°C for 2 h. The reaction was stopped by the addition of 0.1 ml 10% trichloroacetic acid. After 2ml 5% trichloroacetic acid were added, the mixture was heated at 90°C for 10 min. The resulting precipitate was collected on a Whatman G F / C glass fiber filter, washed three times with 5% trichloroacetic acid and its radioactivity was measured in a model LS-3155T Beckman liquid scintillation counter. CsCI isopycnic centrifugation. CsC1 isopycnic centrifugation was carried out according to the method of Brunk and Leick [25]. In brief, native 40 S subunits from ethionine-treated rats and those from control saline-treated rats were labeled with [3H]- and [14C]orotic acid, respectively, as described above. The labeled native subunits (0.7 4260 U) were suspended in a medium containing 30 mM KC1/5 mM MgC12/50 mM triethanolamine-HCl buffer, pH 7.6. They were fixed with 6% formaldehyde for 20 h separately and then combined. An excess of formaldehyde was removed by dialysing against 50 mM triethanolamine-HCl buffer, pH 7.6 for 24 h. The sample was layered over 45-90% (w/v) of a discontinuous CsC1 density-gradient and centrifuged at 149000 × g for
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20h in a Beckman SW 50.1 rotor. The gradient was fractionated into 20 fractions, p values were determined by the reflective index at 25°C according to the method of Vinograd and Hearst [26]. Trichloroacetic acid-insoluble 3H and 14C radioactivities of each sample were measured as described above.
Labeling of 40 S subunits with N-[14C]- and N[3H]ethylmaleimide and analyses of labeled 40S proteins by two-dimensional polyacrylamide gel electrophoresis. Proteins of control and ethioninetreated 40 S subunits were labeled as described by Terao and Ogata [27]. In brief, 2 mg 40 S subunits were incubated with 5 #Ci N-[14C]ethylmaleimide or 50 #Ci N-[3H]ethylmaleimide in 2.0 ml of MediumA at 37°C for 2h. Ribosomal proteins were then extracted with 4 M u r e a / 2 M LiC1. The specific activities of the 14C-labeled proteins ranged from 1.0 to 1.5.105 c p m / m g and those of the 3H-labeled proteins from 0.4 to 0.6.105 c p m / m g . 40 S ribosomal proteins from control rats labeled with ~4C and those from ethionine-treated animals labeled with 3H, or vice versa, were combined and subjected to two-dimensional polyacrylamide gel electrophoresis according to a slight modification [27] of the method of Kaltschmidt and Wittmann [28]. After electrophoresis, the gel was stained with Amido black 10B. Protein spots were cut out and solubilized with NCS solubilizer, and then their radioactivity was measured with a Beckman liquid scintillation counter with the counting efficiencies of approx. 20% for 3H and 60% for 14C. Individual ribosomal proteins were designated according to the proposed uniform nomenclature [ 11]. Determination of protein concentrations. Protein concentrations were determined by the method of Lowry et al. [29] using bovine serum albumin as standard.
Results
Inhibitory effects of ethionine administration on the translating activity of rat liver 80 S ribosomes and 40 S subunits To examine the effects of ethionine administration to female rats on the translational activity of liver ribosomes, the activity of run-off 80 S ribosomes from ethionine-treated rats and that of control saline-treated rats for translation of rabbit globin mRNA were compared with each other in the complete system containing [3H]leucine, saturated amounts of partially purified initiation factors [18] and the rat liver pH 5 fraction [24]. As shown in Table I, the incorporation of [3 H]leucine into total protein by 80 S ribosomes was markedly decreased 4 h after ethionine administration, indicating that ethionine administration injures the translating activity of 80 S ribosomes. As the next step, we attempted to elucidate which subunits were injured. We could not, however, prepare rat liver 60 S subunits free from the contamination with 40 S subunits even with three
TABLE I E F F E C T S OF E T H I O N I N E A D M I N I S T R A T I O N ON T H E T R A N S L A T I O N OF RABBIT GLOBIN m R N A BY R A T LIVER 80 S RIBOSOMES A N D 40 S S U B U N I T S The reaction mixture is described in Materials and Methods. (a) 0.5 A260 U of 80 S liver ribosomes from control or ethionine-treated rats was used. (b) 0.14 ,4260 U of liver 4 0 S subunits from saline or ethionine-treated rats and 0.36 ,4260 U of mouse liver 60 S subunits were used. Incubation was carried out at 30°C for 2 h. The radioactivity of the hot trichloroacetic acid-insoluble fraction was measured. Six independent experiments are shown in this table. Percent of the control radioactivity is shown in parentheses. [ 3H]Leucineincorporation (cpm)
SDS-polyacrylamide gel electrophoresis of native 40S subunits. 0.5 A260 U of derived 40S subunits or 1 A260 U of native 40 S subunits was dissolved in 8 M u r e a / 2 0 mM, dithiothreitol/l% S D S / 1 0 mM Tris-HCl, pH 7.5. After heating for 1 min at 90°C, the solution was subjected to SDS-polyacrylamide gel electrophoresis according to the procedure of Laemmli [30], except that the concentration of polyacrylamide was 15%. The gel was stained with Coomassie brillant blue [30].
control
ethionine treatment
(a) 80 S ribosomes
23445 34395 6581
7723 (33%) 5297 (15%/ 2 170 (33%)
(b) 40 S ribosomal subunits
21894 20938 5807
14297 (65%) 5 985 (29%) 3218 (55%)
105 cycles of sucrose density-gradient centrifugation of rat liver 60 S subunits. Therefore, we used mouse liver 60 S subunits, which were shown to be sufficiently pure after two cycles of sucrose densitygradient centrifugation of run-off ribosomes, as judged by the fact that 60 S subunits alone had protein synthesizing activity less than 10% of that in the presence of rat liver 40 S subunits in the c o m p l e t e s y s t e m . W h e n the activity of ethionine-treated rat liver 40 S subunits, for the translation of globin m R N A , was compared with that of control 40 S subunits in the presence of mouse liver 60S subunits, it was found that ethionine administration markedly inhibited the activity of 40 S subunits for the translation of globin m R N A (Table I). Thus, ethionine intoxication inactivated 40 S ribosomal subunits. Therefore, at least part of the decrease in the activity of 80 S ribosomes from ethionine-treated rats was due to the inactivation of 40 S subunits.
Polysome formation of 80 S ribosomes and of 40 S subunits with rabbit globin mRNA To investigate whether the decrease in the translating activity of ribosomes from ethionine-treated rat liver was due to their defects in the interaction with initiation factors, 80 S ribosomes from ethionine-treated or from control rat livers were incubated with globin m R N A in the presence of the rat liver p H 5 fraction and initiation factors partially purified from rabbit reticulocyte [18]. After incubation at 30°C for 10 min, the reaction mixture was analyzed by sucrose density-gradient centrifugation. As shown in Fig. 1, while polysomes up to the pentamer were formed in the case of 80 S ribosomes from control rat liver, the polysome formation was highly impaired in the case of 80 S ribosomes from ethionine-treated rat liver, and only monomers and dimers were observed. In addition, 40 S subunits were not detected in the case of 80 S ribosomes from ethionine-treated rat liver, whereas in the case of 80 S ribosomes from control rat liver a small but distinct peak of 40 S subunits was observed. Furthermore, the 80 S peak was markedly smaller, while the 60 S peak was larger in the case of ethionine-treated 80 S ribosomes than the corresponding peak in the case of the control 80 S ribosomes. The results may be explained by the low activ-
0.4
i~ 80s
~4~s 6~s 0.2
Fig. 1. The polysome formation of 80 S ribosomes with rabbit globin mRNA. The polysome formation was carried out in the same system as described in Table I. 0.5 ml reaction mixture contained: 5 A26o U of liver 80S ribosomes from saline or ethionine-administered rats, 300 v,g elF 0-40% (NH4)2SO4, 300 #g elF 40-70% (NH,,)2SO4, 625 #g protein of the rat liver
pH 5 fraction. All other reagents were the same as described in Table I except that unlabeled leucine was used instead of [3H]leucine. The mixture was incubated at 30°C for 10 min. After incubation, the mixture was chilled in ice and diluted to 2 ml with ice-cold buffer containing 70 mM KCI/5 mM MgC12/20 mM Tris-HCl, pH 7.5, and then centrifuged at 96300×g for 220 rain through 38 ml of a hyperbolic gradient of 10-46%"sucrose containing the same buffer as described above. The absorbance at 254 nm was recorded continuously through the ultraviolet flow cell of an ISCO spectrophotometer. - - , control; . . . . . . , ethionine.
ity of ethionine-treated 80S ribosomes for the initiation complex formation by initiation factors. When ethionine-treated and control rat liverderived 40S subunits, uniformly labeled with [14C]orotic acid in vivo, were incubated in reticulocyte lysate [31] similar results were obtained. That is, the negligible formation of polysomes as well as 80 S particles probably containing the 80 S initiation complex, the marked accumulation of labeled 60 S particles and the disappearance of labeled 40 S subunits were observed in the case of ethionine-treated 40 S subunits, whereas the low but definite formation of polysomes as well as 80 S particles, the low accumulation of labeled 60 S particles and the presence of labeled 40 S subunits were observed in the case of control 40 S subunits (data not shown). These findings may indicate that ethionine-treated 40 S subunits have a very low activity for the initiation complex formation. The disappearance of labeled 40 S subunits with the concomitant accumulation of 60 S particles in the
106
case of ethionine-treated 40 S subunits is thought to be due to the dimer formation of 40 S subunits which do not participate in the initiation [20,32], since the dimer was sedimented at the position of 60 S subunits [33]. A similar situation was observed in the case of ethionine-treated 80 S subunits as described above.
CsCl-buoyant density-gradient centrifugation of native 40 S subunits It is well known that native 40 S subunits of rat liver contain initiation factors [20,23,34]. When ethionine administration inhibits the activities of 80 S ribosomes and of 40 S subunits for the peptide bond initiation as described above, the interaction of ethionine-treated 40S subunits with initiation factors may be impaired. As a result, it is possible that the buoyant density of native 40S subunits from ethionine-treated rat liver may be higher than that of control native 40 S subunits. To test such a hypothesis, native 40S subunits from ethionine-treated and those from control rat livers which had been labeled uniformly with [14C]orotic acid and [3H]orotic acid, respectively, were separated from post-ribosomal supernatant and purified by 10-45% sucrose density-gradient centrifu-
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Fig. 2. The pattern of CsC1 isopycnic centrifugation of native 40 S subunits from saline- and ethionine-treated rats. Formaldehyde-fixed native 40 S subunits uniformly labeled with [14C1- (0) or [3H]orotic acid (A) were analysed in 5 ml CsCI gradient centrifugation as described in Materials and Methods. ..... ; absorbance at 260 nrn ( . ) of derived 40 S subunits as the marker, - - , 14C radioactivity of saline-treated native 40 S subunits, - . . . . . ; 3H radioactivity of ethioninetreated native 40 S subunits.
gation. After fixing two kinds of subunits with 6% formaldehyde independent/y, they were combined and the mixture was subjected to CsC1 densitygradient centrifugation. The pattern is shown in Fig. 2. Two distinct components were observed with buoyant densities of 1.470 and 1.523 in the case of control native 40 S subunits, in agreement with the results of other investigators [35,36]. Since the p value of derived 40 S subunits is 1.548, the major component with the p value of 1.470 was the 40 S subunits containing additional non-ribosomal proteins including initiation factors. The minor component with a density of 1.523 was the 40 S subunit containing fewer extra proteins than the major component. On the other hand, native 40 S subunits from ethionine-treated rat liver showed a broad radioactive peak with the p value of 1.523 and a shoulder with the p value of around 1.624. Thus, native 40 S subunits from ethionine-treated rat liver contained less amounts of nonribosomal proteins. The results may be explained by assuming that the interaction of rat liver 40 S subunits with initiation factors is injured by ethionine intoxication.
Analyses of the protein moiety of native 40S subunits To examine which initiation factors were inhibited in the interaction with 40 S subunits by ethionine treatment, we compared the protein moiety of native 40 S subunits of ethionine-treated rat liver with that of native 40 S subunits of the control rat liver by SDS-polyacrylamide slab gel electrophoresis. The results are shown in Fig. 3. The protein pattern of native 40 S subunits showed that, in addition to 40 S ribosomal structural proteins, a number of non-ribosomal proteins which might contain many initiation factors, were seen in high molecular weight areas [20,23,34]. It was obvious that the staining intensities of many bands of non-ribosomal proteins of ethionine-treated native 40 S subunits were fainter as compared with those of corresponding bands of the control native 40S subunits (Fig. 3, slots c and d), while the staining intensities of structural proteins of both derived and native subunits were similar between two kinds of rat liver (Fig. 3, slots a and b). These results showed that native 40 S subunits from ethionine-treated rat liver contained smaller
107
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6.5k
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Fig. 3. SDS-polyacrylamide gel electrophoresis of the proteins of the native and derived 40 S ribosomal subunits from control and ethionine-treated rats. The SDS-polyacrylamide gel electrophoresis was carried out as described in Materials and Methods. Slot a, derived 40 S subunits from saline-treated rats; slot b, derived 40 S subunits from ethionine-treated rats; slot c, native 40 S subunits from control rats; slot d, native 40 S subunits from ethionine-treated rats; slot e, standard proteins as molecular weight markers: phosphorylase b (M r 96 000), rat albumin (65000), ovalbumin (45000), carbonic anhydrase (29000) and soybean trypsin inhibitor (20000).
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a m o u n t s of n o n - r i b o s o m a l proteins, which might i n c l u d e initiation factors, than those f r o m n o r m a l rat liver. These findings m a y explain the higher b u o y a n t d e n s i t y of native 40 S s u b u n i t s , of e t h i o n i n e - t r e a t e d rats t h a n that o f control native subunits, as d e s c r i b e d in the f o r m e r section. Since these n o n - r i b o s o m a l p r o t e i n s c o n t a i n e d c o m p o n e n t s o f large m o l e c u l a r weight characteristics of the subunits of eIF-3, we tried to identify these p r o t e i n s with e I F - 3 subunits. F o r this p u r p o s e we p u r i f i e d e I F - 3 from total n a t i v e subunits a n d used this as the reference for the identification. In such e x p e r i m e h t s the p u r i t y o f e I F - 3 was t h o u g h t to b e critical. I n a c r y l a m i d e gel electrophoresis of e I F - 3 u n d e r n o n - d i s s o c i a t i n g c o n d i t i o n s [21], there was o n e m a i n c o m p o n e n t o c c u p y i n g m o r e t h a n 60% of the p r o t e i n s in this
2. Ok
a
b
c
•Fig. 4. SDS-polyacrylamide gel electrophoretograms of the high KC1 wash of rat liver native 40 S subunits and eIF-3. Slot a, high KCI wash. l-11 are protein bands corresponding to protein I-ll of eIF-3 as shown in Fig. 5. Slotb, eIF-3 subunits prepared as described in Materials and Methods. Slot c, standard proteins as molecular weight markers. Insert; the densitomatic pattern of slot b.
gel, a n d showing a similar S value to that of e l F - 3 [21], in a d d i t i o n to four m i n o r b a n d s in the heavier region t h a n the m a i n c o m p o n e n t s . Since there were no i n i t i a t i o n factors greater t h a n eIF-3, we
108
suspected that these minor proteins could be aggregate forms of elF-3. Therefore, each minor component was cut off from the gel and labeled with NalZ5I [37]. After the extraction of the labeled components with phosphate buffer containing 1% SDS, the extracts were analyzed by SDS-polyacrylamide slab gel electrophoresis, followed by radioautography. The results showed that all minor components consisted of the same subunits as those of the major components, indicating that they were aggregated forms of elF-3 (data not shown). The electrophoretograms of SDS-polyacrylamide slab gel electrophoresis of elF-3 showed 11 bands, in agreement with the result of reticulocyte elF-3 [21,38] (Fig. 4, slotB). Densitometric tracing of the bands in the SDS-polyacrylamide slab gel electrophoresis indicated that the purity of our elF-3 preparation was more than 80% and high enough to use as the reference (Fig. 4, insert). The pattern of SDS-polyacrylamide slab gel electrophoresis of elF-3 is shown in Fig. 4. 11 bands of molecular weight ranging from 148000 to 37000 were detected, which were, on the whole, similar to the band pattern of rabbit reticulocyte elF-3 [21,38]. The protein bands were numbered according to their molecular weights from 1 to 11 (Fig. 4, slot b). To identify protein bands in SDS-polyacrylamide gel electrophoresis of native subunits the following methods were employed. Comparing the band patterns of 0.5 M KC1 extract of 40 S subunits with elF-3, we could identify 1-11 protein bands to 1-11 proteins of elF-3 (Fig. 4, slots a and b). Then we compared the band pattern on SDSpolyacrylamide gel electrophoresis of control or ethionine-treated native 40 S subunits with that of 0.5 M KCI extract, and identified 1-11 proteins as 1-11 proteins of elF-3, although the bands of proteins4 and 5 of control and ethionine-treated native 40 S subunits were very faint. In addition, several unidentified bands of non-ribosomal proteins and many ribosomal protein bands which were located in the low molecular region, are observed in the case of both kinds of native subunits. The results of densitometric tracing of SDSpolyacrylamide gel electrophoresis of the control and ethionine-treated 40 S subunits are shown in Fig. 5a and b, respectively. It must be emphasized
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1| ::: 1| Fig. 5. Densitometry of the electrophoretogramof the proteins of liver native 40 S subunits from control or ethionine-treated rats. The gels shown in Fig. 3c and d were scanned spectrophotometrically at 590 nm by using a Joyce Loeble microdensitometer. a, the gel pattern of native 40 S subunits from control rats; b, that of native 40 S subunits from ethionine-treated rats. I- 11 represent the protein bands corresponding to proteins I-I 1 of elF-3 shown in Fig. 4. The shaded area represents the staining pattern of 40 S ribosomal structural proteins.
that the amounts of protein corresponding to the subunits of elF-3 were definitely smaller in ethionine-treated 40 S subunits than in control 40 S subunits except for proteins 4 and 5 which were in very small a m o u n t s in both control and
109
ethionine-treated ribosomes, although they were present as definite bands in 0.5 M KC1 extract of native 40 S subunits (Fig. 4, slot a). On the other hand, the amount of ribosomal protein was similar between two kinds of native subunits, shown as shaded areas in Fig. 5a and b. It is noticeable that the amounts of proteins a and b of molecular weights of 2.3- 104 and 2.1.10 4, respectively, were smaller in ethionine-treated native 40 S subunits than in control native subunits. Although these peaks contain ribosomal proteins, the amounts of ribosomal proteins are similar between control and ethionine-treated rats. Therefore, the difference in the amounts of peaks a and b was due to non-ribosomal proteins. These proteins were present in the 0.5 M KC1 wash of 40 S subunits as shown in slot a o f Fig. 4. Since these two proteins were major components and markedly decreased by ethionine treatment, the decrease in the amount of proteins a and b in the case of ethionine-treated 40 S native subunits contributed markedly to the differences of the # values between the two kinds of native 40 S subunits as described above. Further studies should be done to identify these proteins. It must be added that several non-ribosomal protein peaks other than those of elF-3, were similar between these two kinds of polysome (Fig. 5a and b). The above results may indicate that native 40 S subunits from ethionine-treated rat liver contain smaller amounts of elF-3 than those from normal rat liver, whereas the amounts of ribosomal structural proteins are similar between two kinds of rat liver. Conformational changes of rat liver 40 S subunits by ethionine treatment The low activity of 40 S subunits for interaction with initiation factors may be induced by conformational changes of rat liver 40 S subunits by ethionine treatment. To test this possibility, we used labeled N-ethylmaleimide, because this SH reagent was widely used to investigate conformational changes of E. coli ribosomes and reported to be very useful in discerning differential forms of E. coli ribosomes and ribosomal subunits which correlated with differences in their functional states [39-41]. 40 S subunits prepared from control and
ethionine-treated rat livers were labeled with N[t4c]- and N-[3H]ethylmaleimide, respectively, or vice versa, and then combined. The 40 S proteins were prepared from the combined subunits and subjected to two-dimensional polyacrylamide gel electrophoresis. In the preliminary experiments, 14 kinds of 40 S proteins were shown to be definitely labeled with N-[14C]ethylmaleimide on two-dimensional polyacrylamide gel electrophoresis. Therefore, the ratios of ~4C to 3H radioactivity of these 14 proteins were calculated. The ratios of individual proteins were normalized by taking the ratio of S11 protein to be one, because this protein was labeled strongly with N-ethylmaleimide in both cases of control and ethionine-treated rat livers. As shown in Table II, the reaction rates of $6, $9, $23, $25 and $26 proteins with N-
TABLE II Reactions of 40 S proteins of ethionine-treated and control rat livers with labeled N-ethylmaleimide. In Expt. 1, ethioninetreated rat liver 40 S ribosomal proteins labeled with N[laC]ethylmaleimide (14350 cpm) and control 40 S ribosomal proteins labeled with N-[ 3H]ethylmaleimide (13 400 cpm) were combined. In Expt. 2, ethionine-treated 3H-labeled proteins (14600 cpm) and control 14C-labeled proteins (19500 cpm) were combined. The combined proteins were subjected to twodimensional polyacrylamide gel electrophoresis. The radioactivities of individual 40 S proteins were counted and expressed as dpm. The 14C to 3H ratio (Expt. 1) or 3H to 14C ratio (Expt. 2) of a given ribosomal protein was normalized by taking the ratio of S11 protein to be 1.
$2 $3/$3a S3b $4 $6 S8 $9 Sll S17 S20 $23 $25 $26 $27
Expt. 1
Expt. 2
14C dpm (Ethionine)
3H dpm (Ethionine)
3H dpm (Control)
14C dpm (Control)
1.19 1.01 1.17 0.86 2.51 1.16 2.87 1.00 0.62 1.10 1.89 1.49 1.55 0.47
0.97 1.18 1.00 0.72 3.32 1.38 3.30 1.00 0.43 0.92 2.13 1.86 1.74 0.59
110
ethylmaleimide became higher by ethioninetreatment, whereas the reaction rates of S17 and $27 proteins became lower. These observations may indicate that treatment with ethionine induces conformational changes of rat liver 40 S subunits. By using reductive methylation, Kisilevsky et al. [10] showed that ethioninetreatment induced conformational changes of both ribosomal subunits of rat liver, in agreement with the results described above. Discussion Translation of rabbit reticulocyte globin mRNA by rat liver 80 S ribosomes or 40 S subunits was markedly decreased by ethionine treatment of female rats. Taking these findings together with the results reported previously that rat liver polysomes were degraded by ethionine administration [4,5], it is indicated that the activity of 40 S subunits for peptide bond initiation is markedly inhibited by ethionine treatment. We could not examine the changes in the activity of rat liver 60 S subunits by ethionine treatment owing to the contamination with 40 S subunits. Therefore, in the present experiments our interest was centered on the changes of 80 S ribosomes and of 40 S subunits of rat liver by ethionine administration. In this respect, Treloar et al. reported that $6 proteins of 40 S subunits of rat liver were dephosphorylated by ethionine administration to the female rat while four or five phosphorylated derivatives of $6 protein were observed in 40 S subunits of control saline-treated rats and of recovered rats which received adenine after ethionine treatment [12]. From these results they indicated that the different extents of phosphorylation of the $6 protein related to protein synthesizing activities of three kinds of rat liver 40 S subunits as described above. These findings attracted our attention, since we showed that [42,43] poly(U) was bound to rat liver $6 proteins by ultraviolet-irradiation of 40S subunits and suggested the possibility that phosphorylation of $6 proteins changes the interaction of mRNA to 40 S ribosomes and, as a result the protein synthesizing activity of ribosomes is affected [13]. We could not, however, reproduce the result that the $6 protein of control saline-treated rat liver was more
highly phosphorylated than that of ethioninetreated rat liver, although the $6 protein of 40 S subunits from recovered rat liver showed a higher translating activity than ethionine-treated rat liver and was highly phosphorylated (data not shown). It was reported that phosphorylation of $6 protein is not always correlated to the protein-synthesizing activity of liver ribosomes (see review from Refs. 44, 45). To obtain direct evidence for the impairment of rat liver 80 S ribosomes in the initiation of protein biosynthesis by ethionine-treatment, we used a heterogeneous cell-free system containing partially purified initiation factors from reticulocytes, the rat liver pH 5 fraction and globin mRNA. The polysomal formation of 80 S ribosomes was markedly inhibited by ethionine treatment. Similar results were obtained by adding control and ethionine-treated 40 S subunits to rabbit reticulocyte lysate (data not shown). These findings may indicate the impairment of the activity of rat liver 40 S subunits for the peptide bond initiation by ethionine treatment. It was observed that 40S dimers increased and 40 S subunits markedly decreased in the case of ethionine treatment. These findings may be explained by the impairment of the interaction of ethionine-treated 40 S subunits with eIF-3, since eIF-3 has the activity to dissociate 40 S dimers [20,22]. To obtain in vivo evidence for this indication, we isolated native 40 S subunits from rat liver. Their p values measured by CsCI isopycnic centrifugation actually showed that the attachment of non-ribosomal proteins with 40 S subunits was markedly impaired in the case of ethionine-treated rat liver, since the contents of ribosomal structural protein were similar between control and ethionine-treated rat liver 40 S subunits (Figs. 3 and 5). To investigate the factors with which the interaction of 40 S subunits were impaired, the patterns of SDS-polyaerylamide gel electrophoresis were compared, between liver native 40 S subunits from ethionine-treated rats and those from control rats, by using elF-3 purified from rat liver native subunits as the reference. The results indicate that at least the interaction of 40 S subunits with elF-3 may be inhibited by ethionine treatment, which is known to play an important role in the dissociation of 80 S ribosomes and the attachment of 40 S
I11
subunits to mRNA [46]. In addition, proteins a and b were decreased in the native 40 S subunits by ethionine treatment. The natures of these two major proteins must be clarified. Concerning the protein moiety of native 40 S proteins, the presence of free messenger ribonucleoprotein particles must be considered. However, from the methods of purification and the p values, our native 40 S subunits are thought to correspond to the native 40 S particles of Henshaw and Loebenstein [36]. Since RNA purified from the particles was reported to be almost entirely 18 S rRNA, the contamination of native 40 S subunits with messenger ribonucleoprotein particles may be small, if any [47]. From these results it was that the conformation of 40 S subunits was changed by ethionine treatment. Therefore, interactions of individual 40 S ribosomal proteins with N-ethylmaleimide in 40 S ribosomes were examined. The results may indicate that the conformation of ethionine-treated 40 S subunits actually changes. Summing up the results described above, it may be concluded that ethionine administration changes the conformation of the 40 S subunits and, as a result, impairs the interaction of the 40 S subunit with initiation factors, especially with eIF-3. These inhibitions may be one of the important factors causing the decrease in protein synthesis and degradation of polysomes in rat liver treated with ethionine. It is important to know the mechanism by which ethionine treatment causes the conformational changes of rat liver 40 S subunits. Some changes secondary to the depletion of ATP must be considered, although low phosphorylation of the $6 protein may not be the factor causing such changes as described above. Another factor may be the impairment of the methylation of ribosomal proteins, because we observed that the incorporation of [3H]methyl into ribosomal protein, 30 min after the injection of [3H]methyl-labeled methionine into the tail vain, was markedly decreased by ethionine treatment for 3.5 h (one-tenth of the control in the case of 60 S proteins and one-seventh in that of 40 S subunits). Further studies should be made to elucidate these points.
Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research (No. 448401 and No. 577140) from the Ministry of Education, Science and Culture of Japan.
References 1 Shull, K.H. (1962) J. Biol. Chem. 237, 1734-1735 2 Shull, K.H., McConomy, J.M., Vogt, M., Castillo, A. and Farber, E. ([966) J. Biol. Chem. 241, 5060-5070 3 Kotite, L. and Tarver, H. (1969) Proc. Soc. Exp. Biol. Med. 131,403-408 4 Villa-Trevino, S., Farber, E., Staehelin, T., Wettstein, F.O. and Noll, H. (1964) J. Biol. Chem. 239, 3826-3833 5 Baglio, L.M. and Farber, E. (1965) J. Mol. Biol. 12,466-467 6 Stewart, G.A. and Farber, E. (1967) Science 157, 67-69 7 Farber, J.L., Shinozuka, H., Serroni, A. and Farber, E. (1974) Lab. Invest. 31,465-472 8 Kisilevsky, R. (1972) Biochim. Biophys. Acta 272, 463-472 9 Murty, C.N., Verney, E. and Sidransky, H. (1977) Exp. Mol. Pathol. 27, 392-402 l0 Kisilevsky, R, Weiler, L. and Treloar, M. (1978) J. Biol. Chem. 253, 7101-7108 11 McConkey, E.H., Bielka, H., Gordon, J., Lastic, S.M., Lin, A., Ogata, K., Rebou, J.-P., Traugh, J.A., Traut, R.R., Warner, J.R., Welfle, H. and Wool, I.G. (1979) Mol. Gen. Genet. 169, 1-6 12 Treloar, M.A., Treloar, M.E. and Kisilevsky, R. (1977) J. Biol. Chem. 252, 6217-6221 13 Treloar, M.A. and Kisilevsky, R. (1978) Can. J. Biochem. 57, 209-215 14 Rajalakshmi, S. (1973) Proc. Am. Assoc. Cancer Res. 14, 39 (Abstr.) 15 Goswami, B.B. and Sharma, O.K. (1980) Biochemistry 19, 2101-2108 16 Kuchino, Y., Sharma, O.K. and Borek, E. (1978) Biochemistry 17, 144-147 17 Ginzburg, I., Cornelis, P., Giveon, D. and Littauer, U.Z. (1979) Nucl. Acid Res. 6, 657-672 18 Schreier, M.H. and Staehelin, T. (1973) J. Mol. Biol. 73, 329-349 19 Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 1408-1412 20 Thompson, H.A., Sadnik, I., Scheinbuks, J. and Moldave, K. (1977) Biochemistry 16, 2221-2230 21 Schreier, M.H., Erni, B. and Staehelin, T. (1977) J. Mol. Biol. 116, 727-753 22 Jones, R.L., Sadnik, I., Thompson, H.A. and Moldave, K. (1980) Arch. Biochem. Biophys. 199, 277-285 23 Sundkvist, I.C. and Staehelin, T. (1975) J. Mol. Biol. 99, 401-418 24 Ogata, K. and Nohara, H. (1959) Biochim. Biophys. Acta 31, 149-157
112 25 Brunk, C.F. and Leick, V. (1969) Biochim. Biophys. Acta 179, 136-144 26 Vinograd, J. and Hearst, J.E. (1962) Fortsch, Chem. Org. Nature 20, 372-422 27 Terao, K. and Ogata, K. (1978) J. Biochem. 84, 1119-1123 28 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 36, 401-412 29 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (195t) J. Biol. Chem. 193, 265-275 30 Laemmli, U.K. (1970) Nature 227, 680-685 31 Schimke, R.T., Rhoads, R.E. and McKnight, G.S. (1974) Methods Enzymol. 30, 694-701 32 Goumans, H., Thomas, A., Verhoeven, A., Voorma, H.O. and Benne, R. (1980) Biochim. Biophys. Acta 608, 39-46 33 Wettenhall, R.E.H., Wool, I.G. and Sherton, C.C. (1973) Biochemistry 12, 2403-2411 34 Herrera, F., Sadnik, I., Gough, G. and Moldave, K. (1977) Biochemistry 16, 4664-4672 35 Sugano, H., Suda, D., Kawada, T. and Sugano, I. (1971) Biochim. Biophys. Acta 238, 139-149 36 Henshaw, F.C. and Loebenstein, J. (1970) Biochim. Biophys. Acta 199, 405-420
37 Terao, K., Uchiumi, T., Kobayashi, Y. and Ogata, K. (1980) Biochim. Biophys. Acta 621.72-82 38 Benne, R. and Hershey, W.B.J. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3005-3009 39 Ginzburg, I., Miskin, R. and Zamir, A. (1973) J. Mol. Biol. 79, 481-49 40 Ginzburg, I. and Zamir, A. (1975) J. Mol. Biol. 93,465-476 41 Ghosh, N. and Moore, P.B. (1979) Eur. J. Biochem. 93, 147-156 42 Terao, K. and Ogata, K. (1979) J. Biochem. 86, 597-603 43 Terao, K. and Ogata, K. (1979) J. Biochem. 86, 605-617 44 Wool, I.G. (1972) Ann. Rev. Biochem 48, 719-754 45 Leder, D.P. (1980) Newly discovered systems of enzyme regulation by reversible phosphorylation (Cohen, P., ed.), pp. 204-233, Elsevier-North Holland Biomedical Press, Amsterdam 46 Trachsel, H., Erni, B., Schreier, M.H. and Staehelin, T. (1977) J. Mol. Biol. 116, 755-767 47 Henshaw, F.C., Revel, M. and Hiatt, H.H. (1965) J. Mol. Biol. 14, 241-256
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Elsevier Biomedical Press BBA 91051
EFFECTS OF ETHIONINE TREATMENT ON PROTEIN-SYNTHESIZING APPARATUS OF RAT LIVER 80 S RIBOSOMES AND 40 S RIBOSOMAL SUBUNITS YOSHIAKI TAKAHASHI and KIKUO OGATA
Department of Biochemistry, Niigata University School of Medicine, Niigata 951 (Japan) (Received November 13th, 1981)
Key words: Ethionine; 40 S ribosomal subuniC Protein synthesis; Initiation factor," Globin mRNA; (Rat liver)
The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete ceil-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsC! isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [t4C]- or [3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that elF-3 subunits and two unidentified protein fractions of molecular weight of 2.3.10 4 and 2.1.10 4 were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[3H]ethylmaleimide or N-[ 14C]ethyimaleimide, and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially elF-3, as the results of conformational changes of 40 S subunits.
Introduction It was shown that administration of DLethionine to female rats resulted in a rapid depletion of hepatic ATP [1-3]. Protein synthesis was then decreased with a concomitant breakdown of polysomes into monosomes [4,5]. These effects of ethionine were reversed by treatment with adenine and/or methionine which reaggregated liver polysomes and increased protein synthesis in the liver [6,7]. However, the sites of the protein-synthesizing 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
apparatus which were impaired by ethionine administration, have not yet completely been clarified. From in vivo labeling kinetics, it was reported that ethionine treatment caused the inhibition of protein synthesis by reducing the rate of the initiation [8]. Murty et al. [9] using poly(U) as a template, indicated that administration of ethionine led to a significant reduction in the activities of rat liver for peptide bond initiation by affecting both initiaton factors and ribosomes. Recently, Kisilevsky et al. [10] demonstrated that administration of ethionine caused not only con-
102
formational changes of rat liver ribosomes but also decreased the levels of phosphorylation of ribosomal protein $6 * of rat liver [12]. They also reported that the extent of phosphorylation of rat liver $6 protein during ethionine intoxication and recovery was closely related to the proteinsynthesizing activity of the liver [13]. Another effect of ethionine on protein synthesis was that methyl-deficient or ethylated RNAs were accumulated in rat liver by ethionine administration [14]. Goswami and Sharma [15] reported that methyl deficiency of the 5' cap structure of mRNA was observed when rats were fed with ethionine in their diet for 7 days, and the translational activity of methyl-deficient m R N A was decreased when added to a wheat germ cell-free system [15]. When ethionine was administered to rats, ethylated tRNAs were observed in rat liver and especially lysine t R N A 2 [16] was highly ethylated. Methyl-deficient tRNAs were accumulated in the liver of ethionine-administrated rats. When assayed in injected oocytes, treatment of rats with ethionine caused severe impairments in the amino acylation capacity of some kinds of tRNA. Furthermore, the participation of aminoacyl tRNA from ethionine-treated rat liver in the translation of various mRNAs was severely impaired in both injected oocyte and a cell-free system from wheat germ [17]. Thus, ethionine appears to have pleiotropic effects on liver protein-synthesizing apparatus. The present study was undertaken to elucidate whether rat liver ribosomes and especially, their 40 S subunits were altered by ethionine treatment for short time periods of 4 h in vivo. The effects of ethionine treatment on activities of 40 S subunits for the translation of globin mRNA and for the interaction with initiation factors were investigated. Conformational changes of ethioninetreated rat liver 40 S subunits were examined. This report describes the results of these experiments. Materials and Methods
Chemicals. L-[4,5-3H]Leucine (54 Ci/mmol), [614C]orotic acid monohydrate (61 m C i / m m o l ) and * Individual ribosomal proteins were designated according to the proposed u n i f o r m n o m e n c l a t u r e [ I I].
[5-3H]orotic acid (21 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, U.K. N-[ethyl-lJ4C]Ethylmaleimide (23.7 mCi/mmol) and N-[ethyl-2-3H]ethylmaleimide (692.0 m C i / m mol) were purchased from New England Nuclear, Boston. All other chemicals were of reagent grade.
Ethionine treatment and preparation of ribosomes and ribosomal subunits. Female Wistar strain rats weighing 150-200g were fasted overnight. DLEthionine was administered intraperitoneally at a dose of 1 g / k g body weight. Control rats received an equivalent volume of 0.9% saline [12]. Rats were killed 4 h later. The livers were rapidly excised and homogenized in 3 vol. ice-cold Medium A containing 0.25 M sucrose/25 mM KCI/5 mM MgC12/50 mM Tris-HCl, pH 7.6. All subsequent procedures were carried out at 0-4°C. Postmitochondrial supernatant was prepared by centrifugation of the homogenate at 10000 × g for 10 min. After sodium deoxycholate treatment [12], 18 ml supernatant were layered over 20 ml of 0 . 5 M sucrose, containing mediumA, and ribosomes were pelleted in a Beckman type 60 Ti rotor by centrifugation at 105000 X g for 1 h. The ribosomes were then run off by incubation with the rat liver pH 5 fraction and precipitated by centrifugation at 130000 × g for 15 h [18], these were designated as 80 S ribosomes. Rat liver 40 S subunits were then prepared by centrifugation of run-off 80S ribosomes at 2°C through a 15-30% linear sucrose density-gradient containing 0.3 M KC1/ 3 mM MgC12/10 mM 2-mercaptoethanol/20 mM Tris-HC1, p H 7.6 and were pelleted in a Beckman type 65 rotor by centrifugation at 130000 X g for 15 h [18]. Mouse liver 60 S subunits were prepared by the same methods as described above, except that two cycles of sucrose-gradient centrifugation were carried out.
Preparation of partially purified rabbit initiation factors and 9 S globin mRNA. Reticulocyte lysate and polysomes were prepared from phenylhydragine-treated rabbits according to the method of Schreier and Staehelin [18]. Partially purified initiation factors were prepared by DEAE-cellulose column chromatography of 0.5 M KC1 wash of rabbit reticulocyte polysomes, followed by 0-40% and 40-70% ammonium sulfate precipitation. The factors thus obtained were designated as elF 0 -
103
40% (NH4)2SO 4 and elF 40-70% (NH4)2SO4, respectively [18]. Globin mRNA was prepared from total RNA from 0.5 M KC1 wash of rabbit reticulocyte polysomes by two cycles of oligo-dT celluose chromatography by the method of Aviv and Leder [ 19]. Purification of elF-3, eIF-3 was purified from rat liver total native subunits [20], according to the methods of Schreier et al. [21], followed by the method of Jones et al. [22]. In brief, rat liver total native subunits were incubated in the presence of 0.5 M KC1 at 0°C for 15 min. Subsequently, the total subunits were sedimented at 113000 × g for 15h. Crystalline (NH4)2SO4 was added to the supernatant to a saturation of 40%. The precipitate was collected by centrifugation at 10000 X g for 10 min and dissolved in Medium B (20 mM Tris-HCl, pH 7.6/0.1 mM EDTA/1 mM dithiothreitol/10% glycerol) containing 0.1 M KCI and dialysed for 12h against the same buffer. This fraction was applied onto the DEAE-cellulose column equilibrated with Medium B. After passage of non-absorbed proteins, retained protein fractions were eluted with 0.3 M KCI in Medium B. The eluent was dialysed for 12h against MediumC (30 mM potassium phosphate, pH 7.0/0.1 mM E D T A / l mM dithiothreitol/10% glycerol) containing 0.1 M KC1 and then applied onto the phosphocellulose column equilibrated with Medium C. The protein fractions, retained to 0.1 M KC1, were eluted with 0.4 M KC1 in Medium C. The eluent was further purified by 10-30% linear glycerol gradientcentrifugation following the methods of Jones et al. [22]. The fractions having subunit anti-association activity, were pooled as eIF-3 and concentrated by adding crystalline (NH4)2SO4 t o a saturation of 50%. The purity of eIF-3 was checked by SDS-polyacrylamide gel electrophoresis and by 3.6% polyacrylamide gel electrophoresis under non-dissociating conditions [21]. After staining with Coomassie brillant blue R250, the density at 600 nm was traced with Joyce Loebl Chromoscan 200. The results are described in the later section.
Preparation of native 40 S ribosomes uniformly labeled with [~H]- or [HC]orotic acid. 5/~Ci [14C]orotic acid or 70 #Ci [aH]orotic acid were intraperitoneally injected into rats three times at an interval of 12 h. Ethionine or 0.9% saline was injected 20h after the final isotope injection and
the rats were killed 4 h later. Native 40 S subunits were pelleted from postmicrosomal supernatant of rat liver by centrifugation at 130000 X g for 15 h and further purified by centrifugation through a 10-45% hyperboric sucrose-density gradient according to a modification of the method of Sundkvist and Staehelin [23]. Translation of globin mRNA. The translation of globin mRNA was carried out according to the method of Schreier and Staehelin [18]. The complete reaction mixture of 0.1 ml contained the following components: 0.14 A260 U of rat liver 40 S subunits and 0.36 A260 U of mouse liver 60 S subunits, or 0.5 A26o U of rat liver 80 S ribosomes, 60 #g protein of elF 0-40% (NH4)2SO4, 60 #g protein of elF 40-70% (NH4)2SO4, 125 #g protein of the pH 5 fraction from rat liver [24], 0.05 A260 U of globin mRNA, 1 mM ATP, 0.4 mM GTP, 20 mM creatine phosphate, 0.38 U creatine kinase, 1 mM dithiothreitol, an amino acid mixture containing 30 /tM of each unlabeled amino acid except for leucine, 4mM MgC12, 70 mM KC1, 20 mM Tris-HCl, pH 7.6 and 1 /tCi [3H]leucine. Incubation was carried out at 30°C for 2 h. The reaction was stopped by the addition of 0.1 ml 10% trichloroacetic acid. After 2ml 5% trichloroacetic acid were added, the mixture was heated at 90°C for 10 min. The resulting precipitate was collected on a Whatman G F / C glass fiber filter, washed three times with 5% trichloroacetic acid and its radioactivity was measured in a model LS-3155T Beckman liquid scintillation counter. CsCI isopycnic centrifugation. CsC1 isopycnic centrifugation was carried out according to the method of Brunk and Leick [25]. In brief, native 40 S subunits from ethionine-treated rats and those from control saline-treated rats were labeled with [3H]- and [14C]orotic acid, respectively, as described above. The labeled native subunits (0.7 4260 U) were suspended in a medium containing 30 mM KC1/5 mM MgC12/50 mM triethanolamine-HCl buffer, pH 7.6. They were fixed with 6% formaldehyde for 20 h separately and then combined. An excess of formaldehyde was removed by dialysing against 50 mM triethanolamine-HCl buffer, pH 7.6 for 24 h. The sample was layered over 45-90% (w/v) of a discontinuous CsC1 density-gradient and centrifuged at 149000 × g for
104
20h in a Beckman SW 50.1 rotor. The gradient was fractionated into 20 fractions, p values were determined by the reflective index at 25°C according to the method of Vinograd and Hearst [26]. Trichloroacetic acid-insoluble 3H and 14C radioactivities of each sample were measured as described above.
Labeling of 40 S subunits with N-[14C]- and N[3H]ethylmaleimide and analyses of labeled 40S proteins by two-dimensional polyacrylamide gel electrophoresis. Proteins of control and ethioninetreated 40 S subunits were labeled as described by Terao and Ogata [27]. In brief, 2 mg 40 S subunits were incubated with 5 #Ci N-[14C]ethylmaleimide or 50 #Ci N-[3H]ethylmaleimide in 2.0 ml of MediumA at 37°C for 2h. Ribosomal proteins were then extracted with 4 M u r e a / 2 M LiC1. The specific activities of the 14C-labeled proteins ranged from 1.0 to 1.5.105 c p m / m g and those of the 3H-labeled proteins from 0.4 to 0.6.105 c p m / m g . 40 S ribosomal proteins from control rats labeled with ~4C and those from ethionine-treated animals labeled with 3H, or vice versa, were combined and subjected to two-dimensional polyacrylamide gel electrophoresis according to a slight modification [27] of the method of Kaltschmidt and Wittmann [28]. After electrophoresis, the gel was stained with Amido black 10B. Protein spots were cut out and solubilized with NCS solubilizer, and then their radioactivity was measured with a Beckman liquid scintillation counter with the counting efficiencies of approx. 20% for 3H and 60% for 14C. Individual ribosomal proteins were designated according to the proposed uniform nomenclature [ 11]. Determination of protein concentrations. Protein concentrations were determined by the method of Lowry et al. [29] using bovine serum albumin as standard.
Results
Inhibitory effects of ethionine administration on the translating activity of rat liver 80 S ribosomes and 40 S subunits To examine the effects of ethionine administration to female rats on the translational activity of liver ribosomes, the activity of run-off 80 S ribosomes from ethionine-treated rats and that of control saline-treated rats for translation of rabbit globin mRNA were compared with each other in the complete system containing [3H]leucine, saturated amounts of partially purified initiation factors [18] and the rat liver pH 5 fraction [24]. As shown in Table I, the incorporation of [3 H]leucine into total protein by 80 S ribosomes was markedly decreased 4 h after ethionine administration, indicating that ethionine administration injures the translating activity of 80 S ribosomes. As the next step, we attempted to elucidate which subunits were injured. We could not, however, prepare rat liver 60 S subunits free from the contamination with 40 S subunits even with three
TABLE I E F F E C T S OF E T H I O N I N E A D M I N I S T R A T I O N ON T H E T R A N S L A T I O N OF RABBIT GLOBIN m R N A BY R A T LIVER 80 S RIBOSOMES A N D 40 S S U B U N I T S The reaction mixture is described in Materials and Methods. (a) 0.5 A260 U of 80 S liver ribosomes from control or ethionine-treated rats was used. (b) 0.14 ,4260 U of liver 4 0 S subunits from saline or ethionine-treated rats and 0.36 ,4260 U of mouse liver 60 S subunits were used. Incubation was carried out at 30°C for 2 h. The radioactivity of the hot trichloroacetic acid-insoluble fraction was measured. Six independent experiments are shown in this table. Percent of the control radioactivity is shown in parentheses. [ 3H]Leucineincorporation (cpm)
SDS-polyacrylamide gel electrophoresis of native 40S subunits. 0.5 A260 U of derived 40S subunits or 1 A260 U of native 40 S subunits was dissolved in 8 M u r e a / 2 0 mM, dithiothreitol/l% S D S / 1 0 mM Tris-HCl, pH 7.5. After heating for 1 min at 90°C, the solution was subjected to SDS-polyacrylamide gel electrophoresis according to the procedure of Laemmli [30], except that the concentration of polyacrylamide was 15%. The gel was stained with Coomassie brillant blue [30].
control
ethionine treatment
(a) 80 S ribosomes
23445 34395 6581
7723 (33%) 5297 (15%/ 2 170 (33%)
(b) 40 S ribosomal subunits
21894 20938 5807
14297 (65%) 5 985 (29%) 3218 (55%)
105 cycles of sucrose density-gradient centrifugation of rat liver 60 S subunits. Therefore, we used mouse liver 60 S subunits, which were shown to be sufficiently pure after two cycles of sucrose densitygradient centrifugation of run-off ribosomes, as judged by the fact that 60 S subunits alone had protein synthesizing activity less than 10% of that in the presence of rat liver 40 S subunits in the c o m p l e t e s y s t e m . W h e n the activity of ethionine-treated rat liver 40 S subunits, for the translation of globin m R N A , was compared with that of control 40 S subunits in the presence of mouse liver 60S subunits, it was found that ethionine administration markedly inhibited the activity of 40 S subunits for the translation of globin m R N A (Table I). Thus, ethionine intoxication inactivated 40 S ribosomal subunits. Therefore, at least part of the decrease in the activity of 80 S ribosomes from ethionine-treated rats was due to the inactivation of 40 S subunits.
Polysome formation of 80 S ribosomes and of 40 S subunits with rabbit globin mRNA To investigate whether the decrease in the translating activity of ribosomes from ethionine-treated rat liver was due to their defects in the interaction with initiation factors, 80 S ribosomes from ethionine-treated or from control rat livers were incubated with globin m R N A in the presence of the rat liver p H 5 fraction and initiation factors partially purified from rabbit reticulocyte [18]. After incubation at 30°C for 10 min, the reaction mixture was analyzed by sucrose density-gradient centrifugation. As shown in Fig. 1, while polysomes up to the pentamer were formed in the case of 80 S ribosomes from control rat liver, the polysome formation was highly impaired in the case of 80 S ribosomes from ethionine-treated rat liver, and only monomers and dimers were observed. In addition, 40 S subunits were not detected in the case of 80 S ribosomes from ethionine-treated rat liver, whereas in the case of 80 S ribosomes from control rat liver a small but distinct peak of 40 S subunits was observed. Furthermore, the 80 S peak was markedly smaller, while the 60 S peak was larger in the case of ethionine-treated 80 S ribosomes than the corresponding peak in the case of the control 80 S ribosomes. The results may be explained by the low activ-
0.4
i~ 80s
~4~s 6~s 0.2
Fig. 1. The polysome formation of 80 S ribosomes with rabbit globin mRNA. The polysome formation was carried out in the same system as described in Table I. 0.5 ml reaction mixture contained: 5 A26o U of liver 80S ribosomes from saline or ethionine-administered rats, 300 v,g elF 0-40% (NH4)2SO4, 300 #g elF 40-70% (NH,,)2SO4, 625 #g protein of the rat liver
pH 5 fraction. All other reagents were the same as described in Table I except that unlabeled leucine was used instead of [3H]leucine. The mixture was incubated at 30°C for 10 min. After incubation, the mixture was chilled in ice and diluted to 2 ml with ice-cold buffer containing 70 mM KCI/5 mM MgC12/20 mM Tris-HCl, pH 7.5, and then centrifuged at 96300×g for 220 rain through 38 ml of a hyperbolic gradient of 10-46%"sucrose containing the same buffer as described above. The absorbance at 254 nm was recorded continuously through the ultraviolet flow cell of an ISCO spectrophotometer. - - , control; . . . . . . , ethionine.
ity of ethionine-treated 80S ribosomes for the initiation complex formation by initiation factors. When ethionine-treated and control rat liverderived 40S subunits, uniformly labeled with [14C]orotic acid in vivo, were incubated in reticulocyte lysate [31] similar results were obtained. That is, the negligible formation of polysomes as well as 80 S particles probably containing the 80 S initiation complex, the marked accumulation of labeled 60 S particles and the disappearance of labeled 40 S subunits were observed in the case of ethionine-treated 40 S subunits, whereas the low but definite formation of polysomes as well as 80 S particles, the low accumulation of labeled 60 S particles and the presence of labeled 40 S subunits were observed in the case of control 40 S subunits (data not shown). These findings may indicate that ethionine-treated 40 S subunits have a very low activity for the initiation complex formation. The disappearance of labeled 40 S subunits with the concomitant accumulation of 60 S particles in the
106
case of ethionine-treated 40 S subunits is thought to be due to the dimer formation of 40 S subunits which do not participate in the initiation [20,32], since the dimer was sedimented at the position of 60 S subunits [33]. A similar situation was observed in the case of ethionine-treated 80 S subunits as described above.
CsCl-buoyant density-gradient centrifugation of native 40 S subunits It is well known that native 40 S subunits of rat liver contain initiation factors [20,23,34]. When ethionine administration inhibits the activities of 80 S ribosomes and of 40 S subunits for the peptide bond initiation as described above, the interaction of ethionine-treated 40S subunits with initiation factors may be impaired. As a result, it is possible that the buoyant density of native 40S subunits from ethionine-treated rat liver may be higher than that of control native 40 S subunits. To test such a hypothesis, native 40S subunits from ethionine-treated and those from control rat livers which had been labeled uniformly with [14C]orotic acid and [3H]orotic acid, respectively, were separated from post-ribosomal supernatant and purified by 10-45% sucrose density-gradient centrifu-
75
300
~o "~ 5 0
,; .
-
,,"
.
.
~_oo
.
;
~_
075
u
_u o
0.25
=
m'l ~ m ~
1.7
I 16 ~) density
"', I " .
.
.
15
"[I "m-'r"-~"" -'ll~-Wl .
.
14
.
.
13
"~
~. 0
0
( g / c r n 3)
Fig. 2. The pattern of CsC1 isopycnic centrifugation of native 40 S subunits from saline- and ethionine-treated rats. Formaldehyde-fixed native 40 S subunits uniformly labeled with [14C1- (0) or [3H]orotic acid (A) were analysed in 5 ml CsCI gradient centrifugation as described in Materials and Methods. ..... ; absorbance at 260 nrn ( . ) of derived 40 S subunits as the marker, - - , 14C radioactivity of saline-treated native 40 S subunits, - . . . . . ; 3H radioactivity of ethioninetreated native 40 S subunits.
gation. After fixing two kinds of subunits with 6% formaldehyde independent/y, they were combined and the mixture was subjected to CsC1 densitygradient centrifugation. The pattern is shown in Fig. 2. Two distinct components were observed with buoyant densities of 1.470 and 1.523 in the case of control native 40 S subunits, in agreement with the results of other investigators [35,36]. Since the p value of derived 40 S subunits is 1.548, the major component with the p value of 1.470 was the 40 S subunits containing additional non-ribosomal proteins including initiation factors. The minor component with a density of 1.523 was the 40 S subunit containing fewer extra proteins than the major component. On the other hand, native 40 S subunits from ethionine-treated rat liver showed a broad radioactive peak with the p value of 1.523 and a shoulder with the p value of around 1.624. Thus, native 40 S subunits from ethionine-treated rat liver contained less amounts of nonribosomal proteins. The results may be explained by assuming that the interaction of rat liver 40 S subunits with initiation factors is injured by ethionine intoxication.
Analyses of the protein moiety of native 40S subunits To examine which initiation factors were inhibited in the interaction with 40 S subunits by ethionine treatment, we compared the protein moiety of native 40 S subunits of ethionine-treated rat liver with that of native 40 S subunits of the control rat liver by SDS-polyacrylamide slab gel electrophoresis. The results are shown in Fig. 3. The protein pattern of native 40 S subunits showed that, in addition to 40 S ribosomal structural proteins, a number of non-ribosomal proteins which might contain many initiation factors, were seen in high molecular weight areas [20,23,34]. It was obvious that the staining intensities of many bands of non-ribosomal proteins of ethionine-treated native 40 S subunits were fainter as compared with those of corresponding bands of the control native 40S subunits (Fig. 3, slots c and d), while the staining intensities of structural proteins of both derived and native subunits were similar between two kinds of rat liver (Fig. 3, slots a and b). These results showed that native 40 S subunits from ethionine-treated rat liver contained smaller
107
:
i
23
719 8
x
h..J ~ ...... 2 , 0
----
x 9.6k
6.5k
4.5k
ii ¸
1 9.6k
7 a
b
c
d
~;~: . ~ ! ~
67 ~
6.5k
e 8
Fig. 3. SDS-polyacrylamide gel electrophoresis of the proteins of the native and derived 40 S ribosomal subunits from control and ethionine-treated rats. The SDS-polyacrylamide gel electrophoresis was carried out as described in Materials and Methods. Slot a, derived 40 S subunits from saline-treated rats; slot b, derived 40 S subunits from ethionine-treated rats; slot c, native 40 S subunits from control rats; slot d, native 40 S subunits from ethionine-treated rats; slot e, standard proteins as molecular weight markers: phosphorylase b (M r 96 000), rat albumin (65000), ovalbumin (45000), carbonic anhydrase (29000) and soybean trypsin inhibitor (20000).
"
~. . . . . . . . . . . .
9 i0
~
4.5k
~
2.9k
ii
.a
b
a m o u n t s of n o n - r i b o s o m a l proteins, which might i n c l u d e initiation factors, than those f r o m n o r m a l rat liver. These findings m a y explain the higher b u o y a n t d e n s i t y of native 40 S s u b u n i t s , of e t h i o n i n e - t r e a t e d rats t h a n that o f control native subunits, as d e s c r i b e d in the f o r m e r section. Since these n o n - r i b o s o m a l p r o t e i n s c o n t a i n e d c o m p o n e n t s o f large m o l e c u l a r weight characteristics of the subunits of eIF-3, we tried to identify these p r o t e i n s with e I F - 3 subunits. F o r this p u r p o s e we p u r i f i e d e I F - 3 from total n a t i v e subunits a n d used this as the reference for the identification. In such e x p e r i m e h t s the p u r i t y o f e I F - 3 was t h o u g h t to b e critical. I n a c r y l a m i d e gel electrophoresis of e I F - 3 u n d e r n o n - d i s s o c i a t i n g c o n d i t i o n s [21], there was o n e m a i n c o m p o n e n t o c c u p y i n g m o r e t h a n 60% of the p r o t e i n s in this
2. Ok
a
b
c
•Fig. 4. SDS-polyacrylamide gel electrophoretograms of the high KC1 wash of rat liver native 40 S subunits and eIF-3. Slot a, high KCI wash. l-11 are protein bands corresponding to protein I-ll of eIF-3 as shown in Fig. 5. Slotb, eIF-3 subunits prepared as described in Materials and Methods. Slot c, standard proteins as molecular weight markers. Insert; the densitomatic pattern of slot b.
gel, a n d showing a similar S value to that of e l F - 3 [21], in a d d i t i o n to four m i n o r b a n d s in the heavier region t h a n the m a i n c o m p o n e n t s . Since there were no i n i t i a t i o n factors greater t h a n eIF-3, we
108
suspected that these minor proteins could be aggregate forms of elF-3. Therefore, each minor component was cut off from the gel and labeled with NalZ5I [37]. After the extraction of the labeled components with phosphate buffer containing 1% SDS, the extracts were analyzed by SDS-polyacrylamide slab gel electrophoresis, followed by radioautography. The results showed that all minor components consisted of the same subunits as those of the major components, indicating that they were aggregated forms of elF-3 (data not shown). The electrophoretograms of SDS-polyacrylamide slab gel electrophoresis of elF-3 showed 11 bands, in agreement with the result of reticulocyte elF-3 [21,38] (Fig. 4, slotB). Densitometric tracing of the bands in the SDS-polyacrylamide slab gel electrophoresis indicated that the purity of our elF-3 preparation was more than 80% and high enough to use as the reference (Fig. 4, insert). The pattern of SDS-polyacrylamide slab gel electrophoresis of elF-3 is shown in Fig. 4. 11 bands of molecular weight ranging from 148000 to 37000 were detected, which were, on the whole, similar to the band pattern of rabbit reticulocyte elF-3 [21,38]. The protein bands were numbered according to their molecular weights from 1 to 11 (Fig. 4, slot b). To identify protein bands in SDS-polyacrylamide gel electrophoresis of native subunits the following methods were employed. Comparing the band patterns of 0.5 M KC1 extract of 40 S subunits with elF-3, we could identify 1-11 protein bands to 1-11 proteins of elF-3 (Fig. 4, slots a and b). Then we compared the band pattern on SDSpolyacrylamide gel electrophoresis of control or ethionine-treated native 40 S subunits with that of 0.5 M KCI extract, and identified 1-11 proteins as 1-11 proteins of elF-3, although the bands of proteins4 and 5 of control and ethionine-treated native 40 S subunits were very faint. In addition, several unidentified bands of non-ribosomal proteins and many ribosomal protein bands which were located in the low molecular region, are observed in the case of both kinds of native subunits. The results of densitometric tracing of SDSpolyacrylamide gel electrophoresis of the control and ethionine-treated 40 S subunits are shown in Fig. 5a and b, respectively. It must be emphasized
\L
•
,' s
it,
1| ::: 1| Fig. 5. Densitometry of the electrophoretogramof the proteins of liver native 40 S subunits from control or ethionine-treated rats. The gels shown in Fig. 3c and d were scanned spectrophotometrically at 590 nm by using a Joyce Loeble microdensitometer. a, the gel pattern of native 40 S subunits from control rats; b, that of native 40 S subunits from ethionine-treated rats. I- 11 represent the protein bands corresponding to proteins I-I 1 of elF-3 shown in Fig. 4. The shaded area represents the staining pattern of 40 S ribosomal structural proteins.
that the amounts of protein corresponding to the subunits of elF-3 were definitely smaller in ethionine-treated 40 S subunits than in control 40 S subunits except for proteins 4 and 5 which were in very small a m o u n t s in both control and
109
ethionine-treated ribosomes, although they were present as definite bands in 0.5 M KC1 extract of native 40 S subunits (Fig. 4, slot a). On the other hand, the amount of ribosomal protein was similar between two kinds of native subunits, shown as shaded areas in Fig. 5a and b. It is noticeable that the amounts of proteins a and b of molecular weights of 2.3- 104 and 2.1.10 4, respectively, were smaller in ethionine-treated native 40 S subunits than in control native subunits. Although these peaks contain ribosomal proteins, the amounts of ribosomal proteins are similar between control and ethionine-treated rats. Therefore, the difference in the amounts of peaks a and b was due to non-ribosomal proteins. These proteins were present in the 0.5 M KC1 wash of 40 S subunits as shown in slot a o f Fig. 4. Since these two proteins were major components and markedly decreased by ethionine treatment, the decrease in the amount of proteins a and b in the case of ethionine-treated 40 S native subunits contributed markedly to the differences of the # values between the two kinds of native 40 S subunits as described above. Further studies should be done to identify these proteins. It must be added that several non-ribosomal protein peaks other than those of elF-3, were similar between these two kinds of polysome (Fig. 5a and b). The above results may indicate that native 40 S subunits from ethionine-treated rat liver contain smaller amounts of elF-3 than those from normal rat liver, whereas the amounts of ribosomal structural proteins are similar between two kinds of rat liver. Conformational changes of rat liver 40 S subunits by ethionine treatment The low activity of 40 S subunits for interaction with initiation factors may be induced by conformational changes of rat liver 40 S subunits by ethionine treatment. To test this possibility, we used labeled N-ethylmaleimide, because this SH reagent was widely used to investigate conformational changes of E. coli ribosomes and reported to be very useful in discerning differential forms of E. coli ribosomes and ribosomal subunits which correlated with differences in their functional states [39-41]. 40 S subunits prepared from control and
ethionine-treated rat livers were labeled with N[t4c]- and N-[3H]ethylmaleimide, respectively, or vice versa, and then combined. The 40 S proteins were prepared from the combined subunits and subjected to two-dimensional polyacrylamide gel electrophoresis. In the preliminary experiments, 14 kinds of 40 S proteins were shown to be definitely labeled with N-[14C]ethylmaleimide on two-dimensional polyacrylamide gel electrophoresis. Therefore, the ratios of ~4C to 3H radioactivity of these 14 proteins were calculated. The ratios of individual proteins were normalized by taking the ratio of S11 protein to be one, because this protein was labeled strongly with N-ethylmaleimide in both cases of control and ethionine-treated rat livers. As shown in Table II, the reaction rates of $6, $9, $23, $25 and $26 proteins with N-
TABLE II Reactions of 40 S proteins of ethionine-treated and control rat livers with labeled N-ethylmaleimide. In Expt. 1, ethioninetreated rat liver 40 S ribosomal proteins labeled with N[laC]ethylmaleimide (14350 cpm) and control 40 S ribosomal proteins labeled with N-[ 3H]ethylmaleimide (13 400 cpm) were combined. In Expt. 2, ethionine-treated 3H-labeled proteins (14600 cpm) and control 14C-labeled proteins (19500 cpm) were combined. The combined proteins were subjected to twodimensional polyacrylamide gel electrophoresis. The radioactivities of individual 40 S proteins were counted and expressed as dpm. The 14C to 3H ratio (Expt. 1) or 3H to 14C ratio (Expt. 2) of a given ribosomal protein was normalized by taking the ratio of S11 protein to be 1.
$2 $3/$3a S3b $4 $6 S8 $9 Sll S17 S20 $23 $25 $26 $27
Expt. 1
Expt. 2
14C dpm (Ethionine)
3H dpm (Ethionine)
3H dpm (Control)
14C dpm (Control)
1.19 1.01 1.17 0.86 2.51 1.16 2.87 1.00 0.62 1.10 1.89 1.49 1.55 0.47
0.97 1.18 1.00 0.72 3.32 1.38 3.30 1.00 0.43 0.92 2.13 1.86 1.74 0.59
110
ethylmaleimide became higher by ethioninetreatment, whereas the reaction rates of S17 and $27 proteins became lower. These observations may indicate that treatment with ethionine induces conformational changes of rat liver 40 S subunits. By using reductive methylation, Kisilevsky et al. [10] showed that ethioninetreatment induced conformational changes of both ribosomal subunits of rat liver, in agreement with the results described above. Discussion Translation of rabbit reticulocyte globin mRNA by rat liver 80 S ribosomes or 40 S subunits was markedly decreased by ethionine treatment of female rats. Taking these findings together with the results reported previously that rat liver polysomes were degraded by ethionine administration [4,5], it is indicated that the activity of 40 S subunits for peptide bond initiation is markedly inhibited by ethionine treatment. We could not examine the changes in the activity of rat liver 60 S subunits by ethionine treatment owing to the contamination with 40 S subunits. Therefore, in the present experiments our interest was centered on the changes of 80 S ribosomes and of 40 S subunits of rat liver by ethionine administration. In this respect, Treloar et al. reported that $6 proteins of 40 S subunits of rat liver were dephosphorylated by ethionine administration to the female rat while four or five phosphorylated derivatives of $6 protein were observed in 40 S subunits of control saline-treated rats and of recovered rats which received adenine after ethionine treatment [12]. From these results they indicated that the different extents of phosphorylation of the $6 protein related to protein synthesizing activities of three kinds of rat liver 40 S subunits as described above. These findings attracted our attention, since we showed that [42,43] poly(U) was bound to rat liver $6 proteins by ultraviolet-irradiation of 40S subunits and suggested the possibility that phosphorylation of $6 proteins changes the interaction of mRNA to 40 S ribosomes and, as a result the protein synthesizing activity of ribosomes is affected [13]. We could not, however, reproduce the result that the $6 protein of control saline-treated rat liver was more
highly phosphorylated than that of ethioninetreated rat liver, although the $6 protein of 40 S subunits from recovered rat liver showed a higher translating activity than ethionine-treated rat liver and was highly phosphorylated (data not shown). It was reported that phosphorylation of $6 protein is not always correlated to the protein-synthesizing activity of liver ribosomes (see review from Refs. 44, 45). To obtain direct evidence for the impairment of rat liver 80 S ribosomes in the initiation of protein biosynthesis by ethionine-treatment, we used a heterogeneous cell-free system containing partially purified initiation factors from reticulocytes, the rat liver pH 5 fraction and globin mRNA. The polysomal formation of 80 S ribosomes was markedly inhibited by ethionine treatment. Similar results were obtained by adding control and ethionine-treated 40 S subunits to rabbit reticulocyte lysate (data not shown). These findings may indicate the impairment of the activity of rat liver 40 S subunits for the peptide bond initiation by ethionine treatment. It was observed that 40S dimers increased and 40 S subunits markedly decreased in the case of ethionine treatment. These findings may be explained by the impairment of the interaction of ethionine-treated 40 S subunits with eIF-3, since eIF-3 has the activity to dissociate 40 S dimers [20,22]. To obtain in vivo evidence for this indication, we isolated native 40 S subunits from rat liver. Their p values measured by CsCI isopycnic centrifugation actually showed that the attachment of non-ribosomal proteins with 40 S subunits was markedly impaired in the case of ethionine-treated rat liver, since the contents of ribosomal structural protein were similar between control and ethionine-treated rat liver 40 S subunits (Figs. 3 and 5). To investigate the factors with which the interaction of 40 S subunits were impaired, the patterns of SDS-polyaerylamide gel electrophoresis were compared, between liver native 40 S subunits from ethionine-treated rats and those from control rats, by using elF-3 purified from rat liver native subunits as the reference. The results indicate that at least the interaction of 40 S subunits with elF-3 may be inhibited by ethionine treatment, which is known to play an important role in the dissociation of 80 S ribosomes and the attachment of 40 S
I11
subunits to mRNA [46]. In addition, proteins a and b were decreased in the native 40 S subunits by ethionine treatment. The natures of these two major proteins must be clarified. Concerning the protein moiety of native 40 S proteins, the presence of free messenger ribonucleoprotein particles must be considered. However, from the methods of purification and the p values, our native 40 S subunits are thought to correspond to the native 40 S particles of Henshaw and Loebenstein [36]. Since RNA purified from the particles was reported to be almost entirely 18 S rRNA, the contamination of native 40 S subunits with messenger ribonucleoprotein particles may be small, if any [47]. From these results it was that the conformation of 40 S subunits was changed by ethionine treatment. Therefore, interactions of individual 40 S ribosomal proteins with N-ethylmaleimide in 40 S ribosomes were examined. The results may indicate that the conformation of ethionine-treated 40 S subunits actually changes. Summing up the results described above, it may be concluded that ethionine administration changes the conformation of the 40 S subunits and, as a result, impairs the interaction of the 40 S subunit with initiation factors, especially with eIF-3. These inhibitions may be one of the important factors causing the decrease in protein synthesis and degradation of polysomes in rat liver treated with ethionine. It is important to know the mechanism by which ethionine treatment causes the conformational changes of rat liver 40 S subunits. Some changes secondary to the depletion of ATP must be considered, although low phosphorylation of the $6 protein may not be the factor causing such changes as described above. Another factor may be the impairment of the methylation of ribosomal proteins, because we observed that the incorporation of [3H]methyl into ribosomal protein, 30 min after the injection of [3H]methyl-labeled methionine into the tail vain, was markedly decreased by ethionine treatment for 3.5 h (one-tenth of the control in the case of 60 S proteins and one-seventh in that of 40 S subunits). Further studies should be made to elucidate these points.
Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research (No. 448401 and No. 577140) from the Ministry of Education, Science and Culture of Japan.
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