Purification of chloroplast elongation factors

296

THE CHLOROPLAST

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[19] Purification of Chloroplast E l o n g a t i o n Factors

By ORSOLATIBON1 and ORIO CIFERRI Elongation factors are soluble proteins that participate in protein synthesis and, more exactly, in the elongation cycle. In bacteria three such factors have been isolated and characterized, elongation factor G (EF-G), elongation factor Tu (EF-Tu), and elongation factor Ts (EF-Ts).I EF-G is responsible for the translocation of peptidyl-tRNA on the ribosome whereas EF-Tu binds and carries to the ribosome the different aminoacyltRNAs. EF-Ts, involved in the detachement of GDP from the EF-Tu • GDP complex, may not be required in vitro whereas it is presumably necessary in vivo where it may exist in a stoichiometric complex with EFTu to give EF-T. Chloroplasts have been found to contain elongation factors structurally and functionally analogous to those present in bacteria. 2 Indeed, the assay systems for chloroplast elongation factors rely on the functional interchangeability of ribosomes and elongation factors from these organelles and those from Escherichia coli. It is worth recalling that chloroplast elongation factors are proteins different from those present in the cell cytoplasm as well as those present in mitochondria. We report here the procedures for the purification of EF-Tu and EF-G from isolated spinach chloroplasts and of ET-Tu starting from intact cells of Euglena gracilis.

Purification The following procedure for the purification of EF-Tu from spinach chloroplasts differs significantly from those previously reported3,4 whereas that for EF-G is essentially the same? The isolation of chloroplasts and the preparation of crude elongation factors are common to the purification of EF-Tu and EF-G. Since it is difficult to prepare amounts of intact chloroplasts of E. gracilis sufficient for the purification of EF-Tu, a procedure has been developed starting from intact cells of the alga. The first portion of this procedure has been adapted from that reported by Spremulli. 5 A. 20. 30. 40. 5 L.

Parmeggiani, G. Singer, and E. M. Gottschalk, this series, Vol. 20, p. 291. Tiboni, G. Di Pasquale, and O. Ciferri, Plant Sci. Lett. 6, 416 (1976). Tiboni, G. Di Pasquale, and O. Ciferri, Eur. J. Biochem. 92, 471 (1978). Tiboni and O. Ciferri, FEBS Lett. 146, 197 (1982). L. Spremulli, Arch. Biochem. Biophys. 214, 734 (1982).

METHODS IN ENZYMOLOGY, VOL. 118

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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297

Reagents Elongation factor T and ribosomes are prepared from E. coli as already described. 6 The washed ribosomes do not show any EF-T activity and are also essentially free of EF-G. L-[~4C]Phenylalanyl-tRNA is prepared according to the procedure of Kaji et al. 7 starting from stripped tRNA from Escherichia coli MRE600 (Boehringer). Sephadex G-100 medium and DEAE-Sephadex A-50 (Pharmacia). DEAE-cellulose, Cellex D, and hydroxyapatite (Bio-Rad Laboratories). Aminohexyl-Sepharose 4B (Pharmacia). GDP-Sepharose is prepared by coupling GDP to aminohexylSepharose 4B as previously described. 8 Uniformly labeled L-[14C]phenylalanine (sp. act. 436 mCi/mmol) and [3H]GDP (sp. act. 10.8 Ci/mmol) (The Radiochemical Center). Assay Methods Chloroplast EF-Tu. The activity of elongation factor Tu is assayed by measuring the amount of [3H]GDP bound to the protein. 9 The standard assay mixture (50/.d) contains 50 mM Tris-HC1 (pH 7.4), 10 mM magnesium acetate, 160 mM NH4C1, 5 mM DTT (dithiothreitol), 75/xg bovine serum albumin, and 1 /xCi [3H]GDP. The tubes are kept in ice and the reaction is initiated by adding EF-Tu. After incubation for 10 min at 30°, the reaction is terminated by addition of 1 ml of an ice-cold buffer containing 10 mM Tris-HC1 (pH 7.4), I0 mM magnesium acetate, and 160 mM NH4CI. The solution is filtered on nitrocellulose filters (Millipore HA, 0.45/~m), washed three times with 2 ml of the same buffer, dried, and counted in a liquid scintillation counter. One unit is defined as the amount of enzyme that catalyzes the binding of 1 nmol of [3H]GDP in 10 min at 30°. Chloroplast EF-G. The activity of elongation factor G is assayed by testing its ability to complement E. coli EF-T in the synthesis of polyphenylalanine directed by poly(U) on E. coli ribosomes. 1 The standard assay mixture (250/xl) contains 50 mM maleate buffer (pH 6.6), 12 mM magnesium acetate, 0.5 mM GTP, 2.5 mM 2-mercaptoethanol, 1 mM reduced glutathione, 5 mM creatine phosphate, 7/xg creatine phospho6 y . Nishizuka, F. Lipmann, and J. Lucas-Lenard, this series, Vol. 12, Part B, p. 708. 7 A. Kaji, H. Kaji, and G. D. Novelli, J. Biol. Chem. 240, 1185 (1965). 8 T. Blumenthal, B. Saari, P. H. van der Meide, and L. Bosch, J. Biol. Chem. 255, 5300 (1980). 9 A. V. Furano, Proc. Natl. Acad. Sci. U.S.A. 72, 4780 (1975).

298

THE CHLOROPLAST

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kinase, 10 ~g spermine, 20/zg poly(U), 15-30 ~g E. coli ribosomes, E. coli EF-T (crude preparation) corresponding to 20/zg of protein, and 10 pmol [14C]phenylalanyl-tRNA. The assay tubes are kept in ice and the reaction is initiated by adding EF-G. After incubation for 30 min at 30°, 1 ml of 10% (w/v) trichloroacetic acid and 0.1 ml of a 1% (w/v) solution of egg albumin are added to each assay mixture. The mixtures are then treated in a boiling water bath for 10 min and the insoluble precipitate collected by filtration on glass filters GF/C (Whatman), washed twice with 3 ml of cold 5% trichloroacetic acid, 2 ml of ethanol, dried, and counted in a liquid scintillation counter. One unit is defined as the amount of enzyme that polymerizes 1 nmol of phenylalanine in 30 min at 30° in the presence of saturating amounts of E. coli EF-T. Protein. Protein concentration is determined colorimetrically~° or by scanning Coomassie Blue-stained sodium dodecyl sulfate-polyacrylamide gels with an integrating scanning densitometer. Purification of EF-Tu and EF-G from Isolated Spinach Chloroplasts Buffers

Buffer A: 1.25 M NaC1, 50 mM Tris-HCl (pH 8.0), and 25 mM Na3EDTA (pH 8.0); 10 mM 2-mercaptoethanol and 0.1% (w/v) bovine serum albumin are added immediately before use. Buffer B: 1 M NH4C1, I0 mM Tris-HCl (pH 7.4), I0 mM magnesium acetate, 10 mM 2-mercaptoethanol, and 50/xM GTP. Buffer C: 0.35 M NaC1, 50 mM Tris-HCl (pH 8.0), 10 mM MgCI2, and I mM DTT; 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 50/zg/ml of soybean trypsin inhibitor (SBTI) are added immediately before use. Buffer D: 0.1 M KC1, 10 mM Tris-HCl (pH 7.4), 10 mM magnesium acetate, 10 mM 2-mercaptoethanol, and 20% glycerol (v/v). Buffer E: 10 mM Tris-HC1 (pH 7.4), 10 mM 2-mercaptoethanol, and 40% polyethylene glycol (w/v). Buffer F: 0.1 M KCI, 10 mM potassium phosphate buffer (pH 7.2), 10 mM 2-mercaptoethanol. Buffer G: 10 mM magnesium acetate, 10 mM Tris-HC1 (pH 7.4), and 10 mM 2-mercaptoethanol. Buffers H: 10 mM potassium phosphate buffer (pH 7.2) and 10 mM 2mercaptoethanol. H~, H2, H3 : 30, 32, 40 mM potassium phosphate buffer, respectively. ~00. H. Lowry, N. J. Rosenbrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265

0951).

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CHLOROPLAST ELONGATION FACTORS

299

Isolation of Chloroplasts Chloroplasts are prepared from commercially or greenhouse-grown spinach. Approximately 2 kg of leaves, 3-6 cm long, kept in the dark for one night in order to reduce the amount of starch, are washed first in tap water, then with 1% hypochlorite and finally with deionized water. From this point all operations are performed at 4 °. About 200 g of leaves in 500 ml of buffer A is homogenized by treating 3 times for 10 sec each in a Waring blender or similar apparatus at the highest speed. The homogenate is filtered through a double layer of muslin and then through a single layer of a 65/~m nylon gauze. Chloroplasts are collected from the filtrate by centrifuging for 2 min at 2000 g. The chloroplast sediment is gently suspended in one-third of the original volume of buffer A and centrifuged for 15 min at 7000 g. The chloroplast pellet may be stored at - 7 0 ° for several months without loss in the activity of the elongation factors. This procedure typically yields about 15 g of chloroplasts per kg of leaves.

Preparation of Crude Chloroplast Elongation Factors Frozen chloroplasts (about 30 g) are thawed and suspended in 200 ml of buffer B and blended at high speed in a Waring blender (3-4 times, 10 sec each) until the chloroplasts appear broken on microscopic observation. After centrifugation to remove cellular debris for 30 min at 30,000 g, glycerol is added to the supernatant to give a 10% (v/v) concentration and the solution is ultracentrifuged for 90 min at 150,000 g. To the clear supernatant solid ammonium sulfate is added to give a saturation of 40% at 0°. After standing in the cold for 15 min, the suspension is centrifuged for 15 min at 15,000 g and the resulting pellet is discarded. This step removes a large portion of ribulose-1,5-bisphosphate carboxylase without any significant loss in elongation factors. The supernatant is then brought to 70% saturation with solid ammonium sulfate. After standing in the cold for at least 2 hr, the precipitate is recovered by centrifuging (15 rain at 15,000 g) and dissolved in 10 ml of buffer C for the purification of EF-Tu or 10 ml of buffer D for the purification of EF-G. In general, this procedure yields 1000-2000 units of EF-G and 100-200 units of EF-Tu.

Purification of Elongation Factor Tu Step 1. Sephadex G-IO0 Chromatography. About 10 ml of a crude preparation of chloroplast elongation factors containing 50-70 units of EF-Tu is applied to a 2.5 x 90-cm column of Sephadex G-100 equilibrated with buffer C and eluted with the same buffer at a flow rate of approximately 25 ml/hr. Samples of approximately 7 ml are collected and EF-Tu activity assayed on aliquots (5-10/zl) of each fraction. The tubes contain-

300

THE CHLOROPLAST

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ing the highest activity (in general tubes 25 to 30) are pooled and solid ammonium sulfate is added to give a 75% saturation at 0°. After standing in the cold overnight, the precipitate is recovered by centrifuging for 15 min at 15,000 g and dissolved in 2 ml of buffer C. Step 2. Affinity Chromatography on GDP-Sepharose. The solution from step 1 is mixed with 2 ml of packed GDP-Sepharose prewashed with buffer C. From this point all operations are performed at room temperature. After stirring in a Vortex for a few minutes, the suspension is transferred to a dialysis tube and dialyzed overnight in a rotating bath against 1 liter of buffer C without SBTI. The GDP-Sepharose suspension is then transferred to a small column (0.6 cm diameter) and, after settling, the resin is washed first with 50 ml of buffer C, then with the same buffer without protease inhibitors until the effluent is free of protein as judged spetrophotometrically (50-80 ml). One bed volume of the same buffer containing 0.12 mM GDP is then passed through the column and the flow arrested for 1 hr. The bound EF-Tu is eluted with I0 ml of the GDPcontaining buffer C and 1 ml fractions are collected. EF-Tu activity is assayed on aliquots of the fractions and the fractions containing the highest activity, taking in account the dilution brought about by the unlabeled GDP, are further analyzed on 10% polyacrylamide gels to determine purity of EF-Tu and protein content. The fractions are then pooled and stored at - 7 0 °. Under these conditions, purified EF-Tu does not lose any activity after storage for several months. A summary of the purification procedure is given in Table I. As compared to the previously reported procedure, the present one requires much less time and results in the isolation of highly purified EF-Tu. The procedure has been utilized also for the purification of EF-Tu from chloroplasts of two species of Nicotiana and their somatic hybrid 11 as well as from the cyanobacterium Spirulina platensis. 12 Properties o f Chloroplast EF-Tu. EF-Tu purified by this procedure appears to be essentially devoid of other protein contaminants (Fig. 1, lane 3). By electrophoresis in SDS-10% polyacrylamide gels, the Mr appears to be -45,000, a value close to that reported for bacterial EF-Tu. Besides binding GDP, purified chloroplast EF-Tu promotes the poly(U)directed synthesis of phenylalanine by E. coli ribosomes in the presence of E. coil EF-G. Both reactions are affected by kirromycin, an antibiotic that specifically interacts with the EF-Tu from many eubacteria. Finally, chloroplast EF-Tu interacts with E. coli EF-Ts to give a hybrid EF-T. 4

S. M. Zhao, O. Tiboni, and O. Ciferri, Plant Sci. Lett. 32, 287 (1983). 12 O. Tiboni and O. Ciferri, Eur. J. Biochem. 136, 241 (1983). H

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FIG, 1. 10%'SDS-polyacrylamide gel electrophoresis of purified spinach chloroplast EFTu and EF-G. Lane 1: molecular weight markers (from top to bottom: phosphorylase B, Mr 92,500; bovine serum albumin, Mr 68,000; ovalbumin, Mr 45,000; carbonic anhydrase, M, 30,000); lane 2: crude extract from spinach chloroplasts; lane 3: purified EF-Tu; lane 4: purified EF-G; lane 5: crude extract from spinach chloroplasts.

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CHLOROPLAST ELONGATION FACTORS

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Purification of Elongation Factor G Step 1. Sephadex G-IO0 Chromatography. About 10 ml of a crude preparation of chloroplast elongation factors containing 1000-2000 units of EF-G are applied to a 5 x 95-cm column of Sephadex G-100 equilibrated with buffer D. The column is then eluted with the same buffer at a flow rate of - 2 5 ml/hr collecting samples of 12 ml. EF-G activity is assayed on the fractions and those containing the highest activity are pooled (in general tubes 48 to 60) and concentrated by dialysis for 6-10 hr against 2 liters of buffer E followed by overnight dialysis against 2 liters of buffer F. Step 2. DEAE-Cellulose Chromatography. The protein solution is applied to a 2.5 x 40 cm column of DEAE-cellulose equilibrated with buffer F. After a wash with 120 ml of the same buffer, EF-G is eluted with a linear gradient formed between 500 ml of buffer F and 500 ml of the same buffer containing 0.32 M KCI. Fractions of 12 ml are collected at a flow rate of about 120 ml/hr. The fractions containing the highest EF-G activity (in general tubes 60 to 70, 0.27 M KCI) are pooled and concentrated as reported in step 1 until the volume is reduced to - 5 ml (in general overnight). The solution is then dialyzed for 1 hr against 1 liter of buffer G. Step 3. Sephadex G-IO0 Chromatography. The dialyzed solution is layered on top of a 2.5 × 90-cm column of Sephadex G-100 equilibrated with buffer G. The same buffer is used for the elution at a flow rate of about 20 ml/hr and fractions of 3 ml are collected. EF-G activity elutes shortly after the excluded volume (V0 = 150 ml). The fractions endowed with highest EF-G activity are pooled and concentrated as reported in step 1. The concentrated solution ( - 5 ml) is then dialyzed against three 500 ml changes of buffer H allowing 30 rain for each change. Step 4. Hydroxyapatite Chromatography. The protein solution is loaded onto a 1.4 x 10 cm hydroxyapatite column equilibrated with buffer H. The column is then eluted stepwise with buffer HI (50 ml), H2 (50 ml), and H3 (100 ml) at a flow rate of 30 ml/hr collecting 3 ml fractions. EF-G is eluted with buffer H3 (40 mM potassium phosphate). The pooled fractions are concentrated to a final volume of 1 ml by dialysis against buffer E and stored at - 7 0 ° for several months without loss of activity. A summary of the purification procedure is given in Table II. Properties of Chloroplast EF-G. EF-G purified by this procedure appears to be approximately 90% pure for the presence of a few contaminating proteins with Mr lower than that of EF-G (Fig. 1, lane 4). By electrophoresis in SDS-10% polyacrylamide gels, the Mr is estimated to be -77,000, a value close to that reported for bacterial EF-G. Purified chloroplast EF-G forms a EF-G • GDP • ribosome complex in the presence of

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fusidic acid and, in the presence of saturating amounts of E. coli EF-T, promotes the poly(U)-directed synthesis of polyphenylalanine by E. coli ribosomes. Purification of Chloroplast EF-Tu from Cells of E. gracilis

Buffers Buffer A: 50 mM KCI, 50 mM Tris-HCl (pH 7.8), and 5 mM MgC12. Buffer B: 50 mM NH4CI, 25 mM Tris-HCl (pH 7.6), 5 mM MgCI2, 0.1 mM Na3EDTA, 2 mM DTT, 10 /xM GDP, and 25% (v/v) glycerol. Buffer C: 25 mM Tris-HCl (pH 7.6), 0.1 mM Na3EDTA, 6 mM 2mercaptoethanol, 25% glycerol containing 0.05 or 0.4 M NH4CI. Buffer D: the same of buffer C for spinach chloroplast EF-Tu.

Growth of E. gracilis Cells E. gracilis strain Z is grown in continuous light at 27° in a 6-liter flask containing 2 liters of a chemically defined medium 13 and the cells are harvested at a cell density of 1 x 106/ml. From this point all operations are performed at 4°. The cells are collected by centrifuging 5 min at 2500 g, washed in buffer A, and sedimented for 15 min at 12,000 g. The cell pellet is stored at - 7 0 °. Generally, 10-15 g of cells (wet weight) is obtained from 2 liters of culture. Preparation of Crude Extracts Frozen cells (30 g) are thawed and ground in a cold mortar for 20 min with twice the cell weight of acid-washed sea sand. The cell paste is extracted with 3 ml of buffer B/g of ceils and centrifuged I0 min at 10,000 g to remove sand and cellular debris. The supernatant is centrifuged first for 30 min at 30,000 g and then for 3 hr at 150,000 g. The top four-fifths of the supernatant is recovered and enough solid ammonium sulfate is added to give a saturation of 75% at 0 °. After standing overnight in the cold, the precipitate is collected by centrifuging for 15 min at 12,000 g. The sediment is dissolved in 5 ml of buffer C containing 0.05 M NHaCI and dialyzed against the same buffer for 90 min.

Purification of Chloroplast EF-Tu Step 1. DEAE-Sephadex Chromatography. The protein solution is applied to a 3 × 20 cm column of DEAE-Sephadex A-50 equilibrated with t3 W. Ortiz, E. M. Reardon, and C. A. Price, Plant Physiol. 66, 291 (1980).

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FIG. 2. 10% SDS-polyacrylamide gel electrophoresis of purified E. gracilis chloroplast EF-Tu. Lane 1: crude extract from E. gracilis cells; lane 2: partially purified chloroplast EFTu; lane 3: purified EF-Tu; lane 4: molecular weight markers (from top to bottom: bovine serum albumin, Mr 68,000; ovalbumin, Mr 45,000; carbonic anhydrase, Mr 30,000; soybean trypsin inhibitor, Mr 21,000).

308

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buffer C containing 0.05 M NH4CI. The column is washed with the same buffer until the A2s0 of the effluent is less than 0.1. The proteins are then eluted with a linear gradient formed between 100 ml of buffer C containing 0.05 M NH4C1 and 100 ml of the same buffer containing 0.4 M NH4C1 collecting 4.5 ml fractions at a flow rate of 10 ml/hr. EF-Tu activity is tested on aliquots of the fractions. This step separates chloroplast EF-Tu from the cytoplasmic counterpart (EF-1) that is not adsorbed onto the resin and is removed with the wash. 5 The chloroplast EF-Tu, eluting at ~0.29 M NH4CI, may be contaminated by a small amount of mitochondrial EF-Tu, the bulk of which is, however, eluted at 0.24 M NH4CI, just before the chloroplast peak. The assignment of the 0.29 M NH4C1 peak to the chloroplast EF-Tu is substantiated by the finding that this peak is practically absent from extracts obtained from cells grown heterotrophically in the dark and undetectable in extracts from an aplastidic mutant of Euglena gracilis. Fractions endowed with chloroplast EF-Tu activity are pooled and the protein precipitated by adding solid ammonium sulfate to give a final saturation of 75% at 0 °. After standing in the cold overnight, the protein precipitate is recovered by centrifuging for 15 rain at 12,000 g and the pellet dissolved in 2 ml of buffer D. (See Table III.) Step 2. Affinity Chromatography on GDP-Sepharose. This step is performed exactly as reported in step 2 for the purification of EF-Tu from spinach chloroplasts. Chloroplast EF-Tu is eluted within the first 2-3 ml of the GDP-containing buffer. The most active fractions are stored frozen at - 7 0 °. (See Table III.) Properties of Chloroplast EF-Tu. EF-Tu purified by this procedure appears to be essentially devoid of other protein contaminants (Fig. 2, lane 3). By electrophoresis in 10% SDS-polyacrylamide gels, the Mr is estimated to be -45,000, a value close to that reported for the EF-Tu isolated from bacteria as well as from chloroplasts from higher plants. 3,4,11 Acknowledgments This work was supportedby grants from ConsiglioNazionaledelle Ricerche.The purification of E. gracilisEF-Tu has been performedby Miss A. Orlandoni.