- Email: [email protected]
chl) from spinach chloroplasts
Volume 146, number 1
FEBS LETTERS
September 1982
A rapid procedure for the purification of elongation factor Tu (EF-Tu/chl) from spinach chloroplasts Orsola T i b o n i a n d Orio Ciferri Institute of Microbiology and Plant Physiology, University of Pavia, P.O. Box 202, 2 7100 Pavia, Italy Received 20 July 1982 Elongation factor Tu
1. INTRODUCTION Chloroplasts contain protein factors responsible for peptide chain elongation (elongation factors) distinct from those present in the cell cytoplasm and in mitochondria [1]. In [21 we reported the purification of EF-Gchl, and EF-TUchl from spinach chloroplasts. The structure of a EF-Tu from a number of organisms has been the subject of considerable investigation [3-5]. Such analysis has been hampered so far in the case of EF-Tu from chloroplasts, by the lack of sufficient amounts of the purified protein. Here, we report a one-step procedure that allows the rapid purification with high yields of spinach EF-TUchl. This procedure could be used for the purification of EF-Tu from chloroplasts of other plants and may also be utilized with EF-Tu in mitochondria. 2. MATERIALS AND METHODS GDP-Sepharose was prepared by coupling GDP (Boehringer) to aminohexyl-Sepharose 4B (Pharmacia) exactly as in [6,7]. EF-Tu and EF-Ts from Escherichia coli were a generous gift of Dr G. Chinali. Standards of EFTUchl from spinach were prepared by the procedure in [2]. * To whom correspondence should be addressed t Dedicated to the memory of Mario Sacchi whose untimely death deprived the authors of his skilled and invaluable technical assistance
Spinach chloroplast
2.1. Chloroplast crude extract., Chloroplasts were isolated from leaves of commercially-grown spinach and a chloroplast extract was prepared as in [2]. The protein recovered by precipitation with ammonium sulphate between 40-70% saturation was dissolved in a solution containing 10 mM Tris-HCl (pH 7.4), 10 mM Mgacetate, 10 mM 2-mercaptoethanol, 100 mM KC1, 20% (v/v) glycerol (chloroplast-soluble protein) and used for adsorption to GDP-Sepharose. 2.2. Affinity chromatography In a typical experiment, 1 ml chloroplast soluble protein (78 mg protein, 2100 units EF-TUchl) was mixed with l ml packed GDP-Sepharose prewashed with a solution containing 0.05 M TrisHC1 (pH 8), 0.35 M NaC1, 0.01 M MgC12 and 1 mM dithiothreitol. After stirring in a Vortex stirrer for a few minutes, the suspension was transferred to a dialysis tube (A. Thomas) and dialysed for >1 16 h at room temperature against 1 liter of the above buffer containing 0.1 M phenylmethylsulphonylfluoride (PMSF). The GDP-Sepharose suspension was then transferred to a 0.6 × 2.5 cm column and the same buffer (without PMSF) was passed through the column until the A280 nm of the eluate reached the baseline (120-140 ml). One bed volume of the same buffer containing 0.1 mM GDP was then passed through the column and the flow arrested for 1 h. Finally the column was eluted with - 15 ml GDP-containing buffer and 1 ml fractions collected. On aliquots of the fractions, EF-Tu activity and protein concentration were determined. The peak fractions were pooled and kept frozen
Publishedby ElsevierBiomedicalPress 00145793/82/0000-0000/$2.75 © 1982Federationof European BiochemicalSocieties
197
Volume 146, number l
FEBS LETTERS
at -70°C. Alternatively, EF-TUchl was precipitated from these solutions by adding solid ammonium sulphate to give a final saturation of 80% at 0°C. 2.3. Assays Binding of [3H]GDP to EF-Tu, either from E. coli or chloroplasts, was assayed as in [2,8]. One unit of EF-Tu was defined as the amount of protein that catalyses the binding of 1 pmol [3H]GDP in 10 min at 30°C. Formation of the EF-Tu-EF-Ts complex was assayed as in [9] except that chromatography was performed on a 0.9 x 55 cm column of Sephadex G-100 (Pharmacia, fine) rather than on a column of Ultrogel Ac44. Protein concentration was measured spectrophotometrically [10]. 2.4. Electrophoresis Standard SDS-10% acrylamide gel electrophoresis was done as in [2].
E c o
September 1982
3. RESULTS AND DISCUSSION EF-Tuchl may be purified very easily by affinity chromatography on GDP-Sepharose (fig.l). The purified EF-Tuchl has the same mobility as an authentic sample of EF-TUchl purified by the laborious procedure in [2]. As shown by electrophoresis under denaturing conditions (fig.2), the preparations obtained by affinity chromatography appear to contain very little contamination by other proteins. In certain experiments, a slight contamination by ribulose-bisphosphate carboxylase accounted for < 5% of the total protein recovered. Ribulose-bisphosphate carboxylase may be removed by a second GDP-Sepharose step or by preliminarily passing the chloroplast extract (section 2) through a column of Sephadex G-100 that separates the carboxylase from the other proteins [2]. Attempts to purify EF-TUchl from total leaf extracts were unsuccessful, possibly due to the presence of other molecules binding aspecifically to GDP-Sepharose.
2.C
2.0
1.C
ID
o.
-6 g
CJ
Q
l °
I
0.5
5
I 10
15 Fraction
5
10
~
15
number
Fig.l. Purification of EF-TUchI by affinity chromatography. The column was prepared and run as in section 2. Protein (A280, o o); [3H]GDP bound by 25 ~1 aliquots of fractions (e ,). Only the relevant portion of the graph is depicted. 198
Volume 146, number 1
FEBS LETTERS
September 1982
20
10
o E
Ix a O
20
"1-
10
10
Fig.2. Gel electrophoresis of purified EF-Tuclai and E. coli EF-Tu. SDS-10% acrylamide gel electrophoresis
was run with the anode at the top. Bands were stained with Coomassie brilliant blue. (1) M r markers (phosphorylase B, 94000; bovine serum albumin, 68 000; ovalbumin, 45000; carbonic anhydrase, 30000; (2) chloroplast crude extract (156 ~g protein); (3) EF-TUchl purified by affinity chromatography (10~g); (4) EFTUchl purified as in [2] (6 ~g); (5) E. coli EF-Tu purified by affinity chromatography as in [6] (8 ~g); (6) E. coli crude extract (150/zg protein).
As measured by assaying the binding of GDP, purified EF-TUchl spec. act. was 1.8-2 × 104 units/mg protein, a value close to that reported for the same factor purified from E. coli [8]. This value is -5-fold higher than that found for the EFTUchl purified by the procedure in [2]. However, while the latter procedure leads to the purification of both active and inactive EF-Tuchl molecules, affinity chromatography allows us to purify only the active EF-TUchl molecules, that is those that bind GDP. As judged by the binding assay, the recovery of the EF-Tu purified by affinity chromatography
20
30
Fraction
number
40
50
Fig.3. Formation of an E. coli EF-Ts-EF-TUchl complex. Top: Purified EF-TUchl (200~g) was passed through a Sephadex G-100 column (section 2) equilibrated and eluted with a buffer containing 0.0! mM GDP" ( ~ ) position ofE. coli EF-T; ( ,,~ ) position of E. coli EF-Tu. Bottom: Purified EF-TUchl (200 jag) was mixed with E. coli EF-Ts (400/tg) and passed through the same column as in Top but using a buffer without GDP.
ranged from 40-60% of the activity present in crude extracts. Confirmation that the purified protein is indeed EF-TUchl was obtained by showing that, if the EFTUchI preparation is mixed with E. coli EF-Ts, a complex EF-T (composed of EF-TUchl and E. coli EF-Ts) is formed (fig.3). These data may be taken as a further indication of the similarity between elongation factors from chloroplasts and from prokaryotes since they show that it is possible to form in vitro a hybrid complex composed of 1 elongation factor from E. coli and 1 from chloroplasts. Another hybrid complex, active in vitro, was shown to occur between the 30 S ribosomal sub199
Volume 146, number 1
FEBS LETTERS
unit from Euglena gracilis chloroplasts and the 50S subunit from E. coli [11]. Further, E. coli elongation factors EF-T and E F - G were found to be functionally interchangeable with the corresponding elongation factors from spinach chloroplast on either E. coli or spinach ribosomes [12]. ACKNOWLEDGEMENT This work was supported by grants from Consiglio Nazionale delle Ricerche.
REFERENCES [1] Ciferri, O., Tiboni, O., Munoz-Calvo, M.L. and Camerino, G. (1977) in: Nucleic acids and protein synthesis in plants (Bogorad, L. and Weil, J.H. eds) pp. 155-166, Plenum, New York. [2] Tiboni, O., Pasquale, G. and Ciferri, O. (1978) Eur. J. Biochem. 92, 471-477.
200
September 1982
[3] Wittinghofer, A. and Leberman, R. (1976) Eur. J. Biochem. 92, 373-382. [4] Arai, K., Clark, B.F.C., Duffy, L., Jones, M.D., Kaziro, Y., Laursen, R.A., L'Italien, J., Miller, D.L., Nagarkatti, S., Nakamura, S., Nielsen, K.M., Petersen, T.E., Takahashi, K. and Wade, M. (1980) Proc. Natl. Acad. Sci. USA 77, 1326-1330. [5] Ferro-Luzzi Ames, G. and Makido, K. (1980) J. Biol. Chem. 254, 9947-9950. [6] Jacobson, G.R. and Rosenbusch, J.P. (1977) FEBS Lett. 79, 8-10. [7] Blumenthal, T., Saari, B., Van Der Meide, P.H. and Bosch, L. (1980) J. Biol. Chem. 25, 5300-5305. [8] Furano, A.V. (1975) Proc. Natl. Acad. Sci. USA 72, 4780-4784. [9] Chu, P., Miller, D.L., Schultz, T., Weissbach, H. and Brot, N. (1976) Biochem. Biophys. Res. Commun. 73,917-926. [10] Layne, E. (1957) Methods Enzymol. 2, 447-449. [11] Lee, S.G. and Evans, W.R. (1971) Science 173, 241-242. [12] Tiboni, O., Di Pasquale, G. and Ciferri, O. (1976) Plant Sci. Lett. 6, 419-429.
FEBS LETTERS
September 1982
A rapid procedure for the purification of elongation factor Tu (EF-Tu/chl) from spinach chloroplasts Orsola T i b o n i a n d Orio Ciferri Institute of Microbiology and Plant Physiology, University of Pavia, P.O. Box 202, 2 7100 Pavia, Italy Received 20 July 1982 Elongation factor Tu
1. INTRODUCTION Chloroplasts contain protein factors responsible for peptide chain elongation (elongation factors) distinct from those present in the cell cytoplasm and in mitochondria [1]. In [21 we reported the purification of EF-Gchl, and EF-TUchl from spinach chloroplasts. The structure of a EF-Tu from a number of organisms has been the subject of considerable investigation [3-5]. Such analysis has been hampered so far in the case of EF-Tu from chloroplasts, by the lack of sufficient amounts of the purified protein. Here, we report a one-step procedure that allows the rapid purification with high yields of spinach EF-TUchl. This procedure could be used for the purification of EF-Tu from chloroplasts of other plants and may also be utilized with EF-Tu in mitochondria. 2. MATERIALS AND METHODS GDP-Sepharose was prepared by coupling GDP (Boehringer) to aminohexyl-Sepharose 4B (Pharmacia) exactly as in [6,7]. EF-Tu and EF-Ts from Escherichia coli were a generous gift of Dr G. Chinali. Standards of EFTUchl from spinach were prepared by the procedure in [2]. * To whom correspondence should be addressed t Dedicated to the memory of Mario Sacchi whose untimely death deprived the authors of his skilled and invaluable technical assistance
Spinach chloroplast
2.1. Chloroplast crude extract., Chloroplasts were isolated from leaves of commercially-grown spinach and a chloroplast extract was prepared as in [2]. The protein recovered by precipitation with ammonium sulphate between 40-70% saturation was dissolved in a solution containing 10 mM Tris-HCl (pH 7.4), 10 mM Mgacetate, 10 mM 2-mercaptoethanol, 100 mM KC1, 20% (v/v) glycerol (chloroplast-soluble protein) and used for adsorption to GDP-Sepharose. 2.2. Affinity chromatography In a typical experiment, 1 ml chloroplast soluble protein (78 mg protein, 2100 units EF-TUchl) was mixed with l ml packed GDP-Sepharose prewashed with a solution containing 0.05 M TrisHC1 (pH 8), 0.35 M NaC1, 0.01 M MgC12 and 1 mM dithiothreitol. After stirring in a Vortex stirrer for a few minutes, the suspension was transferred to a dialysis tube (A. Thomas) and dialysed for >1 16 h at room temperature against 1 liter of the above buffer containing 0.1 M phenylmethylsulphonylfluoride (PMSF). The GDP-Sepharose suspension was then transferred to a 0.6 × 2.5 cm column and the same buffer (without PMSF) was passed through the column until the A280 nm of the eluate reached the baseline (120-140 ml). One bed volume of the same buffer containing 0.1 mM GDP was then passed through the column and the flow arrested for 1 h. Finally the column was eluted with - 15 ml GDP-containing buffer and 1 ml fractions collected. On aliquots of the fractions, EF-Tu activity and protein concentration were determined. The peak fractions were pooled and kept frozen
Publishedby ElsevierBiomedicalPress 00145793/82/0000-0000/$2.75 © 1982Federationof European BiochemicalSocieties
197
Volume 146, number l
FEBS LETTERS
at -70°C. Alternatively, EF-TUchl was precipitated from these solutions by adding solid ammonium sulphate to give a final saturation of 80% at 0°C. 2.3. Assays Binding of [3H]GDP to EF-Tu, either from E. coli or chloroplasts, was assayed as in [2,8]. One unit of EF-Tu was defined as the amount of protein that catalyses the binding of 1 pmol [3H]GDP in 10 min at 30°C. Formation of the EF-Tu-EF-Ts complex was assayed as in [9] except that chromatography was performed on a 0.9 x 55 cm column of Sephadex G-100 (Pharmacia, fine) rather than on a column of Ultrogel Ac44. Protein concentration was measured spectrophotometrically [10]. 2.4. Electrophoresis Standard SDS-10% acrylamide gel electrophoresis was done as in [2].
E c o
September 1982
3. RESULTS AND DISCUSSION EF-Tuchl may be purified very easily by affinity chromatography on GDP-Sepharose (fig.l). The purified EF-Tuchl has the same mobility as an authentic sample of EF-TUchl purified by the laborious procedure in [2]. As shown by electrophoresis under denaturing conditions (fig.2), the preparations obtained by affinity chromatography appear to contain very little contamination by other proteins. In certain experiments, a slight contamination by ribulose-bisphosphate carboxylase accounted for < 5% of the total protein recovered. Ribulose-bisphosphate carboxylase may be removed by a second GDP-Sepharose step or by preliminarily passing the chloroplast extract (section 2) through a column of Sephadex G-100 that separates the carboxylase from the other proteins [2]. Attempts to purify EF-TUchl from total leaf extracts were unsuccessful, possibly due to the presence of other molecules binding aspecifically to GDP-Sepharose.
2.C
2.0
1.C
ID
o.
-6 g
CJ
Q
l °
I
0.5
5
I 10
15 Fraction
5
10
~
15
number
Fig.l. Purification of EF-TUchI by affinity chromatography. The column was prepared and run as in section 2. Protein (A280, o o); [3H]GDP bound by 25 ~1 aliquots of fractions (e ,). Only the relevant portion of the graph is depicted. 198
Volume 146, number 1
FEBS LETTERS
September 1982
20
10
o E
Ix a O
20
"1-
10
10
Fig.2. Gel electrophoresis of purified EF-Tuclai and E. coli EF-Tu. SDS-10% acrylamide gel electrophoresis
was run with the anode at the top. Bands were stained with Coomassie brilliant blue. (1) M r markers (phosphorylase B, 94000; bovine serum albumin, 68 000; ovalbumin, 45000; carbonic anhydrase, 30000; (2) chloroplast crude extract (156 ~g protein); (3) EF-TUchl purified by affinity chromatography (10~g); (4) EFTUchl purified as in [2] (6 ~g); (5) E. coli EF-Tu purified by affinity chromatography as in [6] (8 ~g); (6) E. coli crude extract (150/zg protein).
As measured by assaying the binding of GDP, purified EF-TUchl spec. act. was 1.8-2 × 104 units/mg protein, a value close to that reported for the same factor purified from E. coli [8]. This value is -5-fold higher than that found for the EFTUchl purified by the procedure in [2]. However, while the latter procedure leads to the purification of both active and inactive EF-Tuchl molecules, affinity chromatography allows us to purify only the active EF-TUchl molecules, that is those that bind GDP. As judged by the binding assay, the recovery of the EF-Tu purified by affinity chromatography
20
30
Fraction
number
40
50
Fig.3. Formation of an E. coli EF-Ts-EF-TUchl complex. Top: Purified EF-TUchl (200~g) was passed through a Sephadex G-100 column (section 2) equilibrated and eluted with a buffer containing 0.0! mM GDP" ( ~ ) position ofE. coli EF-T; ( ,,~ ) position of E. coli EF-Tu. Bottom: Purified EF-TUchl (200 jag) was mixed with E. coli EF-Ts (400/tg) and passed through the same column as in Top but using a buffer without GDP.
ranged from 40-60% of the activity present in crude extracts. Confirmation that the purified protein is indeed EF-TUchl was obtained by showing that, if the EFTUchI preparation is mixed with E. coli EF-Ts, a complex EF-T (composed of EF-TUchl and E. coli EF-Ts) is formed (fig.3). These data may be taken as a further indication of the similarity between elongation factors from chloroplasts and from prokaryotes since they show that it is possible to form in vitro a hybrid complex composed of 1 elongation factor from E. coli and 1 from chloroplasts. Another hybrid complex, active in vitro, was shown to occur between the 30 S ribosomal sub199
Volume 146, number 1
FEBS LETTERS
unit from Euglena gracilis chloroplasts and the 50S subunit from E. coli [11]. Further, E. coli elongation factors EF-T and E F - G were found to be functionally interchangeable with the corresponding elongation factors from spinach chloroplast on either E. coli or spinach ribosomes [12]. ACKNOWLEDGEMENT This work was supported by grants from Consiglio Nazionale delle Ricerche.
REFERENCES [1] Ciferri, O., Tiboni, O., Munoz-Calvo, M.L. and Camerino, G. (1977) in: Nucleic acids and protein synthesis in plants (Bogorad, L. and Weil, J.H. eds) pp. 155-166, Plenum, New York. [2] Tiboni, O., Pasquale, G. and Ciferri, O. (1978) Eur. J. Biochem. 92, 471-477.
200
September 1982
[3] Wittinghofer, A. and Leberman, R. (1976) Eur. J. Biochem. 92, 373-382. [4] Arai, K., Clark, B.F.C., Duffy, L., Jones, M.D., Kaziro, Y., Laursen, R.A., L'Italien, J., Miller, D.L., Nagarkatti, S., Nakamura, S., Nielsen, K.M., Petersen, T.E., Takahashi, K. and Wade, M. (1980) Proc. Natl. Acad. Sci. USA 77, 1326-1330. [5] Ferro-Luzzi Ames, G. and Makido, K. (1980) J. Biol. Chem. 254, 9947-9950. [6] Jacobson, G.R. and Rosenbusch, J.P. (1977) FEBS Lett. 79, 8-10. [7] Blumenthal, T., Saari, B., Van Der Meide, P.H. and Bosch, L. (1980) J. Biol. Chem. 25, 5300-5305. [8] Furano, A.V. (1975) Proc. Natl. Acad. Sci. USA 72, 4780-4784. [9] Chu, P., Miller, D.L., Schultz, T., Weissbach, H. and Brot, N. (1976) Biochem. Biophys. Res. Commun. 73,917-926. [10] Layne, E. (1957) Methods Enzymol. 2, 447-449. [11] Lee, S.G. and Evans, W.R. (1971) Science 173, 241-242. [12] Tiboni, O., Di Pasquale, G. and Ciferri, O. (1976) Plant Sci. Lett. 6, 419-429.