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Cell-free synthesis of tubulin 1 and tubulin 2 on polysomes isolated from embryonic chick brain
321
Biochimica et Biophysica Acta, 414 ( 1 9 7 5 ) 321--325 © Elsevier Scientific Publishing Company, A m s t e r d a m -- Printed in The Netherlands
BBA 9 8 4 7 6
CELL-FREE SYNTHESIS OF TUBULIN 1 AND TUBULIN 2 ON POLYSOMES ISOLATED FROM EMBRYONIC CHICK BRAIN
ANNELISE OSTER JORGENSEN*
and S T U A R T M. H E Y W O O D
Genetics and Cell Biology Section, U-125, The University of Connecticut, Storrs, Conr~ 06268 (U.S.A.) (Received August 20th, 1975)
Summary Embryonic chick brain polysomes incubated in a cell-free, amino acid-incorporating system synthesize both tubulin subunits (tubulin 1 and tubulin 2) in nearly equal amounts.
In~oducfion The cell-free synthesis of tubulin on polysomes isolated from embryonic chick brain has been reported [1]. Tubulin synthesized in vitro was characterized by (1) its electrophoretic mobility on sodium dodecyl sulphate-acrylamide gels showing a molecular weight of 55 000, identical to the molecular weight of brain tubulin [2,3,4] and (2) its specific ability to participate in repeated cycles of polymerization and depolymerization in the in vitro microtubular assembly system. Since the newly synthesized tubulin was analyzed by an electrophoretic system [6], which does not resolve tubulin into its two components, it could not be concluded if one or both of the tubulin subunits was synthesized. In order to determine if both tubulin subunits are synthesized in the cell-free system, tubulin purified by two cycles of polymerization and depolymerization as well as by DEAE-Sephadex chromatography was analyzed on discontinuous sodium dodecyl sulphate-acrylamide gels [7]. This procedure resolves purified tubulin into its two components: tubulin 1 and tubulin 2, which are present in equimolar amounts (Rosenbaum, J.L., unpublished). The results presented here suggest that both tubulin 1 and tubulin 2 are synthesized in this cell-free system in a nearly equal amount.
* Present address: Biology Department, York University, Downsview. M3J IP3. Ontario, Canada
322 Methods The following procedures were carried out as described previously [1,9] : the isolation of polysomes from 14-day embryonic chick brain, the preparation of enzyme fraction (S-200) from 14-day embryonic chick leg muscle, and tRNA from 14-day chick embryos. The components of the cell-free incubation mixture; the subsequent purification of cell-free synthesized [3H]tubulin by (1) repeated cycles of polymerization and depolymerization in the in vitro microtubular assembly system and (2) by DEAE-Sephadex chromatography, and the extraction of 3H-labeled polypeptides from an acetone powder of the total product of the cell-free system with a low ionic strength buffer was also as previously described [9]. After reduction and carboxymethylation with iodoacetic acid [8] the radioactive products of the cell-free, amino acid-incorporating system were analyzed by sodium dodecyl sulphate-acrylamide gel electrophoresis as described by Laemmli [7]. The 1 × 8 cm, 7% gels were run for 8 h at 2 mA per tube. Determination of radioactivity was performed as previously described
[1]. Purified actin and in vivo-labeled [ 3 s S] tubulin were prepared as previously described [ 1 ]. Results and Discussion Brain polysomes were incubated in a cell-free, amino acid-incorporating system. After the addition of carrier tubulin, the newly synthesized tubulin was purified by two successive cycles of polymerization and depolymerization in the temperature-dependent, in vitro microtubular assembly system [5]. The purified [ 3H] tubulin as well as the total [ 3HI protein of the cell-free system was analyzed on discontinuous sodium dodecyl sulphate-acrylamide gels after carboxymethylation [7,8]. Under these conditions the tubulin separates into two components: tubulin 1 and tubulin 2 (T1 and T2), Fig. 1B (solid line). The tubulin subunits are present in equimolar amounts as judged by their staining with Coomassie Brilliant Blue (Fig. 18) [11]. In addition it can be seen that the in vitro-synthesized tubulin is also resolved into two similar components (T1 and T2) which migrate in an identical manner with in vivo 3 s S-labeled tubulin subunits. Furthermore, T1 and T2 appear to be synthesized in nearly equal amounts as judged by the amount of radioactivity migrating with the tubulin subunits during electrophoresis. We have previously reported that only a low degree of purification could be obtained by DEAE-Sephadex chromatography [1]. The estimation of purification was based on the assumption that the fraction of the total radioactively labeled product of the cell-free system which co-electrophoreses with purified tubulin is indeed tubulin. However, when the total [ 3HI protein synthesized in vitro is carboxymethylated and subsequently analyzed on discontinuous sodium dodecyl sulphate-acrylamide gels, additional radioactive peaks are resolved (T1, T2, and X, Fig. 2 A ) c o m p a r e d to our previous analysis (see Fig. 3A in reference 1). The radioactive peaks X, T1 and T2 were previously judged to be tubulin using the standard sodium dodecyl sulphate-acrylamide
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Fig. 1. E l e c t r o p h o r e t o g r a m s c o m p a r i n g t h e t o t a l 3 H - l a b e l e d p r o t e i n s y n t h e s i z e d in v i t r o o n b r a i n p o l y s o m e s ( A ) a n d t h e f r a c t i o n of t h e in v i t r o 3 H - l a b e l e d p r o t e i n w h i c h c o p u r i f i e d w i t h b r a i n t u b u l i n d u r i n g t w o successive c y c l e s of p o l y m e r i z a t i o n a n d d e p o l y m e r i z a t i o n as d e s c r i b e d in M e t h o d • (B). I n v l t r o - s y n thesized, 3H-labeled protein (e . . . . . . o). I n vivo 3 5 S - l a b e l e d t u b u l i n (o 0) p u r i f i e d b y D E A E - S e p h a dex c h r o m a t o g r a p h y a n d m i x e d w i t h in v i t r o - s y n t h e s l z e d 3 H - l a b e l e d p r o t e i n b e f o r e e l e c t r o p h o r e s i s . A parallel gel c o n t a i n i n g 50 fzg b r a i n t u b u l i n p u r i f i e d b y t h r e e c y c l e • o f p o l y m e r i z a t i o n a n d d e p o l y m e r i z a tion w a s r u n , s t a i n e d w i t h C o o m a s s i e Brilliant Blue a n d s c a n n e d a t 6 0 0 n m o n a G i l f o r d s p e c t r o p h o t o meter ( ). O n l y t w o s t a i n e d b a n d s w e r e o b s e r v e d a n d t h e p o s i t i o n o f t h e • e t u b u H n s u b u n i t s are i n d i c a t e d b y T1 a n d T 2 r e s p e c t i v e l y . All p r o t e i n s a m p l e s w e r e r e d u c e d a n d c a r b o x y m e t h y l a t e d p r i o r t o e l e c t r o p h o r e s e • as d e s c r i b e d in M e t h o d s . F i b 2. E l e c t x o p h o r e t o g r a m s c o m p a r i n g t h e t o t a l 3 H - l a b e l e d p r o t e i n s y n t h e s i z e d in v i t r o o n b r a i n p o l y s o m e s ( A ) a n d t h e f r a c t i o n o f t h e in v i t r o 3 H - l a b e l e d p r o t e i n w h i c h c o - c l t t o m a t o g r a p h s w i t h b r a i n t u b u l i n on a D E A E - S e p h a d e x c o l u m n (B) as d e s c r i b e d in M e t h o d s . I n v i t r o - • y n t h e s l z e d 3 H - l a b e l e d proteizt (e e). I n v i v o 3 6 S - l a b e l e d t u b u l i n (o o) p u r i f i e d b y D E A E - S e p h a d e x c h r o m a t o g r a p h y a n d m i x e d w i t h t h e in v i t r o 3 H - l a b e l e d p r o t e i n i m m e d i a t e l y b e f o r e e l e c t r o p h o r e s i s . All p r o t e i n s w e r e r e d u c e d a n d c a r b o x y m e t h y l a t e d p r i o r t o e l e c t r o p h o r e s i s as d e s c r i b e d in M e t h o d s .
gels. Therefore, at most two thirds of the radioactivity migrating with tubulin in the previous analysis [1] can be characterized as tubulin. These results also suggest that at least some of the tubulin synthesized in vitro has the characteristic chromatographic behavior on DEAE-Sephadex as purified brain tubulin (Fig. 2). The tubulin purified in this manner can subsequently be resolved into its subunits, T1 and T2, by acrylamide gel electrophoresis of the carboxymethylated protein. A similar analysis of in vitro synthesized tubulin again suggest that both T1 and T2 subunits are synthesized in almost equal amounts (Fig.
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2B). However, to be certain that tubulin 1 and tubulin 2 are synthesized in equal amounts in the cell-free protein synthesizing system, it will be necessary to show by peptide analysis that the in vitro synthesized [ 3H] proteins which copurify and co-electrophorese with the tubulins are indeed tubulin 1 and tubulin 2. In an attempt to purify tubulin using an acetone powder extraction of the cell-free system with a low ionic strength buffer [10], it is observed that the major product obtained electrophoreses as radioactive peak "X" and both tubulin 1 and 2 subunits are greatly diminished (Fig. 3). The same results are observed when leg muscle polysomes from embryonic chick were substituted for brain polysomes in the incubation mixture (unpublished results). This suggests that component "X" is not a cell-specific protein. We have not attempted to identify further this ubiquitous protein. The results presented here suggest that tubulin synthesized in vitro by brain polysomes and subsequently purified by repeated cycles of polymerization and depolymerization or by DEAE-Sephadex chromatography is composed of almost equal amounts of subunits 1 and 2. Assuming that during purification the tubulin subunits are recovered in equal amounts, it can be concluded that tubulin 1 a n d 2 are synthesized in nearly equal amounts in the cell-free amino acid incorporating system.
325
References 1 Jorgensen, A.O. and Heywood, S.M. (1974) Proc. Natl. Aead. Sci. U.S. 71, 4278--4282 2 Weisenberg, R.C., Borisy, G.G. and Taylor, E.W. (1968) Biochemistry 7, 4466--4479 3 Eipper, B.A. (1972) Proe. Natl. Aead. Sei. U.S. 69, 2283--2287 4 Bryan, J. and Wilson, L. (1971) Proc. Natl. Acad, Sci. U.S. 68, 2273--2277 5 0 l m s t e d , J.B. and Borisy, G.G. (1973) Biochemistry 12, 4282---4289 6 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 224, 4406--4412 V Laemmli, U.K. (1970) Nature 227, 680--685 8 Crestfield, A.M., Standford, M. and Stein, W.H. (1963) J. Biol. Chem. 238, 622--627 9 Rourke, A.W. and Heywood, S.M. (1970) Biochemistry 11; 2061--2066 10 Gibbons, I.E., Tilney, L.G. and Porter, K.R. (1969) J. Cell BioL 41, 201--226 11 Bibring, T. and Baxandall, J. (19"/4) Exp. Cell Res. 86, 120--126
Biochimica et Biophysica Acta, 414 ( 1 9 7 5 ) 321--325 © Elsevier Scientific Publishing Company, A m s t e r d a m -- Printed in The Netherlands
BBA 9 8 4 7 6
CELL-FREE SYNTHESIS OF TUBULIN 1 AND TUBULIN 2 ON POLYSOMES ISOLATED FROM EMBRYONIC CHICK BRAIN
ANNELISE OSTER JORGENSEN*
and S T U A R T M. H E Y W O O D
Genetics and Cell Biology Section, U-125, The University of Connecticut, Storrs, Conr~ 06268 (U.S.A.) (Received August 20th, 1975)
Summary Embryonic chick brain polysomes incubated in a cell-free, amino acid-incorporating system synthesize both tubulin subunits (tubulin 1 and tubulin 2) in nearly equal amounts.
In~oducfion The cell-free synthesis of tubulin on polysomes isolated from embryonic chick brain has been reported [1]. Tubulin synthesized in vitro was characterized by (1) its electrophoretic mobility on sodium dodecyl sulphate-acrylamide gels showing a molecular weight of 55 000, identical to the molecular weight of brain tubulin [2,3,4] and (2) its specific ability to participate in repeated cycles of polymerization and depolymerization in the in vitro microtubular assembly system. Since the newly synthesized tubulin was analyzed by an electrophoretic system [6], which does not resolve tubulin into its two components, it could not be concluded if one or both of the tubulin subunits was synthesized. In order to determine if both tubulin subunits are synthesized in the cell-free system, tubulin purified by two cycles of polymerization and depolymerization as well as by DEAE-Sephadex chromatography was analyzed on discontinuous sodium dodecyl sulphate-acrylamide gels [7]. This procedure resolves purified tubulin into its two components: tubulin 1 and tubulin 2, which are present in equimolar amounts (Rosenbaum, J.L., unpublished). The results presented here suggest that both tubulin 1 and tubulin 2 are synthesized in this cell-free system in a nearly equal amount.
* Present address: Biology Department, York University, Downsview. M3J IP3. Ontario, Canada
322 Methods The following procedures were carried out as described previously [1,9] : the isolation of polysomes from 14-day embryonic chick brain, the preparation of enzyme fraction (S-200) from 14-day embryonic chick leg muscle, and tRNA from 14-day chick embryos. The components of the cell-free incubation mixture; the subsequent purification of cell-free synthesized [3H]tubulin by (1) repeated cycles of polymerization and depolymerization in the in vitro microtubular assembly system and (2) by DEAE-Sephadex chromatography, and the extraction of 3H-labeled polypeptides from an acetone powder of the total product of the cell-free system with a low ionic strength buffer was also as previously described [9]. After reduction and carboxymethylation with iodoacetic acid [8] the radioactive products of the cell-free, amino acid-incorporating system were analyzed by sodium dodecyl sulphate-acrylamide gel electrophoresis as described by Laemmli [7]. The 1 × 8 cm, 7% gels were run for 8 h at 2 mA per tube. Determination of radioactivity was performed as previously described
[1]. Purified actin and in vivo-labeled [ 3 s S] tubulin were prepared as previously described [ 1 ]. Results and Discussion Brain polysomes were incubated in a cell-free, amino acid-incorporating system. After the addition of carrier tubulin, the newly synthesized tubulin was purified by two successive cycles of polymerization and depolymerization in the temperature-dependent, in vitro microtubular assembly system [5]. The purified [ 3H] tubulin as well as the total [ 3HI protein of the cell-free system was analyzed on discontinuous sodium dodecyl sulphate-acrylamide gels after carboxymethylation [7,8]. Under these conditions the tubulin separates into two components: tubulin 1 and tubulin 2 (T1 and T2), Fig. 1B (solid line). The tubulin subunits are present in equimolar amounts as judged by their staining with Coomassie Brilliant Blue (Fig. 18) [11]. In addition it can be seen that the in vitro-synthesized tubulin is also resolved into two similar components (T1 and T2) which migrate in an identical manner with in vivo 3 s S-labeled tubulin subunits. Furthermore, T1 and T2 appear to be synthesized in nearly equal amounts as judged by the amount of radioactivity migrating with the tubulin subunits during electrophoresis. We have previously reported that only a low degree of purification could be obtained by DEAE-Sephadex chromatography [1]. The estimation of purification was based on the assumption that the fraction of the total radioactively labeled product of the cell-free system which co-electrophoreses with purified tubulin is indeed tubulin. However, when the total [ 3HI protein synthesized in vitro is carboxymethylated and subsequently analyzed on discontinuous sodium dodecyl sulphate-acrylamide gels, additional radioactive peaks are resolved (T1, T2, and X, Fig. 2 A ) c o m p a r e d to our previous analysis (see Fig. 3A in reference 1). The radioactive peaks X, T1 and T2 were previously judged to be tubulin using the standard sodium dodecyl sulphate-acrylamide
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Fig. 1. E l e c t r o p h o r e t o g r a m s c o m p a r i n g t h e t o t a l 3 H - l a b e l e d p r o t e i n s y n t h e s i z e d in v i t r o o n b r a i n p o l y s o m e s ( A ) a n d t h e f r a c t i o n of t h e in v i t r o 3 H - l a b e l e d p r o t e i n w h i c h c o p u r i f i e d w i t h b r a i n t u b u l i n d u r i n g t w o successive c y c l e s of p o l y m e r i z a t i o n a n d d e p o l y m e r i z a t i o n as d e s c r i b e d in M e t h o d • (B). I n v l t r o - s y n thesized, 3H-labeled protein (e . . . . . . o). I n vivo 3 5 S - l a b e l e d t u b u l i n (o 0) p u r i f i e d b y D E A E - S e p h a dex c h r o m a t o g r a p h y a n d m i x e d w i t h in v i t r o - s y n t h e s l z e d 3 H - l a b e l e d p r o t e i n b e f o r e e l e c t r o p h o r e s i s . A parallel gel c o n t a i n i n g 50 fzg b r a i n t u b u l i n p u r i f i e d b y t h r e e c y c l e • o f p o l y m e r i z a t i o n a n d d e p o l y m e r i z a tion w a s r u n , s t a i n e d w i t h C o o m a s s i e Brilliant Blue a n d s c a n n e d a t 6 0 0 n m o n a G i l f o r d s p e c t r o p h o t o meter ( ). O n l y t w o s t a i n e d b a n d s w e r e o b s e r v e d a n d t h e p o s i t i o n o f t h e • e t u b u H n s u b u n i t s are i n d i c a t e d b y T1 a n d T 2 r e s p e c t i v e l y . All p r o t e i n s a m p l e s w e r e r e d u c e d a n d c a r b o x y m e t h y l a t e d p r i o r t o e l e c t r o p h o r e s e • as d e s c r i b e d in M e t h o d s . F i b 2. E l e c t x o p h o r e t o g r a m s c o m p a r i n g t h e t o t a l 3 H - l a b e l e d p r o t e i n s y n t h e s i z e d in v i t r o o n b r a i n p o l y s o m e s ( A ) a n d t h e f r a c t i o n o f t h e in v i t r o 3 H - l a b e l e d p r o t e i n w h i c h c o - c l t t o m a t o g r a p h s w i t h b r a i n t u b u l i n on a D E A E - S e p h a d e x c o l u m n (B) as d e s c r i b e d in M e t h o d s . I n v i t r o - • y n t h e s l z e d 3 H - l a b e l e d proteizt (e e). I n v i v o 3 6 S - l a b e l e d t u b u l i n (o o) p u r i f i e d b y D E A E - S e p h a d e x c h r o m a t o g r a p h y a n d m i x e d w i t h t h e in v i t r o 3 H - l a b e l e d p r o t e i n i m m e d i a t e l y b e f o r e e l e c t r o p h o r e s i s . All p r o t e i n s w e r e r e d u c e d a n d c a r b o x y m e t h y l a t e d p r i o r t o e l e c t r o p h o r e s i s as d e s c r i b e d in M e t h o d s .
gels. Therefore, at most two thirds of the radioactivity migrating with tubulin in the previous analysis [1] can be characterized as tubulin. These results also suggest that at least some of the tubulin synthesized in vitro has the characteristic chromatographic behavior on DEAE-Sephadex as purified brain tubulin (Fig. 2). The tubulin purified in this manner can subsequently be resolved into its subunits, T1 and T2, by acrylamide gel electrophoresis of the carboxymethylated protein. A similar analysis of in vitro synthesized tubulin again suggest that both T1 and T2 subunits are synthesized in almost equal amounts (Fig.
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2B). However, to be certain that tubulin 1 and tubulin 2 are synthesized in equal amounts in the cell-free protein synthesizing system, it will be necessary to show by peptide analysis that the in vitro synthesized [ 3H] proteins which copurify and co-electrophorese with the tubulins are indeed tubulin 1 and tubulin 2. In an attempt to purify tubulin using an acetone powder extraction of the cell-free system with a low ionic strength buffer [10], it is observed that the major product obtained electrophoreses as radioactive peak "X" and both tubulin 1 and 2 subunits are greatly diminished (Fig. 3). The same results are observed when leg muscle polysomes from embryonic chick were substituted for brain polysomes in the incubation mixture (unpublished results). This suggests that component "X" is not a cell-specific protein. We have not attempted to identify further this ubiquitous protein. The results presented here suggest that tubulin synthesized in vitro by brain polysomes and subsequently purified by repeated cycles of polymerization and depolymerization or by DEAE-Sephadex chromatography is composed of almost equal amounts of subunits 1 and 2. Assuming that during purification the tubulin subunits are recovered in equal amounts, it can be concluded that tubulin 1 a n d 2 are synthesized in nearly equal amounts in the cell-free amino acid incorporating system.
325
References 1 Jorgensen, A.O. and Heywood, S.M. (1974) Proc. Natl. Aead. Sci. U.S. 71, 4278--4282 2 Weisenberg, R.C., Borisy, G.G. and Taylor, E.W. (1968) Biochemistry 7, 4466--4479 3 Eipper, B.A. (1972) Proe. Natl. Aead. Sei. U.S. 69, 2283--2287 4 Bryan, J. and Wilson, L. (1971) Proc. Natl. Acad, Sci. U.S. 68, 2273--2277 5 0 l m s t e d , J.B. and Borisy, G.G. (1973) Biochemistry 12, 4282---4289 6 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 224, 4406--4412 V Laemmli, U.K. (1970) Nature 227, 680--685 8 Crestfield, A.M., Standford, M. and Stein, W.H. (1963) J. Biol. Chem. 238, 622--627 9 Rourke, A.W. and Heywood, S.M. (1970) Biochemistry 11; 2061--2066 10 Gibbons, I.E., Tilney, L.G. and Porter, K.R. (1969) J. Cell BioL 41, 201--226 11 Bibring, T. and Baxandall, J. (19"/4) Exp. Cell Res. 86, 120--126