ANALYTICAL BIOCHEMISTRY 99, 434-440 (1979)
Purification of Eukaryotic Cytoplasmic Elongation Factor 2 and Organellar Elongation Factor G by an Affinity Binding Procedure CAROLINE A . BREITENBERGER, MARSHA N. MOORE, D A V I D W . RUSSELL, AND L I N D A L . SPREMULLI
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27514 Received May 4, 1979 A rapid affinity binding procedure for obtaining highly purified organellar elongation factor G (EF-G) and cytoplasmic elongation factor 2 (EF-2) in excellent yields from whole cell extracts is described. The procedure involves the addition of ribosomes, GTP, and fusidic acid to a crude cell extract. The fusidic acid stabilizes the formation of a translocaseGDP-ribosome complex which can be recovered from the extract by high-speed centrifugation. The translocase is then released from the ribosomes by a high salt wash. To purify organellar EF-G, 70 S ribosomes from Escherichia coli are used. If 80 S ribosomes such as those from wheat germ are used instead of 70 S ribosomes, EF-2 can be selectively purified from the cell extract. This procedure has been applied to the purification of both cytoplasmic and chloroplast translocases ofEuglena gracilis. Chloroplast EF-G and cytoplasmic EF-2 can be separated from each other and from the vast majority of the proteins in the postribosomal supernatant with yields in both cases of 60% or greater.
In eukaryotic ceils, proteins are synthe- chloroplasts. This approach requires that sized in several cellular compartments, each the organellar protein be separated from of which has its own distinct protein synthe- large amounts of cytoplasmic proteins, and sizing system. In animal cells, there are two in order to facilitate its purification we have such systems--one in the cell cytoplasm applied an affinity procedure useful in the and one in the mitochondria--while plant purification of Escherichia coli EF-G (2). cells have three sites of protein synthesis In addition, we have modified this affinity - - t h e cytoplasm, the mitochondria, and the procedure so that it can also be used for chloroplasts (1). Elongation factor 2 (EF-2) 1 the purification of cytoplasmic EF-2. The catalyzes the translocation step of protein procedure is based on the observation that synthesis on the 80 S ribosomes found in fusidic acid, a steroidal antibiotic, causes the cytoplasm. In cell organelles, a distinct the formation of a relatively stable E F - G translocase (EF-Gmit or EF-Gchl) catalyzes ribosome-GDP-fusidic acid complex. Chlotranslocation on the smaller (50 to 70 S) roplast EF-G will form this type of stable complex on E. coli ribosomes, which are organellar ribosome. We have been studying cytoplasmic EF-2 readily available in large amounts. The and chloroplast EF-G from Euglena gracilis complex can be isolated by high-speed cenand have chosen to purify both of these trifugation and chloroplast EF-G removed factors from whole cell extracts in order to by washing with high salt. Results presented avoid the large-scale preparation of isolated here also indicate that EF-2 can be substantially purified by this affinity binding procedure when wheat germ ribosomes are Abbreviations used: EF, elongation factor; phe, sdbstituted for E. coli ribosomes. phenylalanyl; SD.~, sodium dcdecyl sulfate. 0003-2697/79/160434-07502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any form reserved.
434
ELONGATION FACTOR G AND 2 AFFINITY PURIFICATION
MATERIALS AND METHODS
Materials. [a4C]Phenylalanine and E. coli tRNA were purchased from Schwarz/Mann. Poly(U) was obtained from Miles Laboratories; ATP and GTP from P-L. Laboratories. Sephadex G-200, Sephadex G-25, DEAE-Sephadex, phosphoenolpyruvate, and pyruvate kinase were from Sigma. Raw wheat germ was kindly supplied by Mr. J. M. de Rosier of International Multifoods Corporation. Sodium fusidate was a generous gift of Dr. Godtfredsen of Leo Pharmaceuticals. Growth conditions and preparation of whole cell extracts. Euglena gracilis Klebs var. bacillaris Cori (Euglena B) was grown on Hutner's pH 3.5 medium to late log phase (2 × 106 cells/ml) and was then transferred to pH 6.8 resting medium and light-induced as described previously (3,4). Cells were harvested after 36 h of exposure to 120 fc of white light. The preparation of the postribosomal supernatant of whole cell extracts was performed as described previously (4) except that the Sephadex G-25 step was replaced by an overnight dialysis. The final protein concentrations of the postribosomal supernatants ranged from 16 to 18 mg/ml as determined by the method of Lowry et al. (5), using bovine serum albumin as a standard.
Preparation of ribosomes, elongation factors, and [14C]phe-tRNA. Escherichia coli MRE 600 or W ribosomes were prepared and high salt washed as described previously (6) except that the first high salt wash contained 1 M NH4CI. They were freed of any residual EF-G contamination by a further 0.60 M NH4C1 wash and collected through a 20% sucrose cushion. Wheat germ high salt washed ribosomes were prepared as described previously (7) and freed from residual EF-2 by washing with 0.5% sodium deoxycholate followed by sedimentation through 10 and 20% sucrose cushions. A complex containing EF-Tu and EF-Ts was prepared from E. coli MRE 600 (8). This complex was free of E. coli EF-G. Wheat
435
germ extracts were prepared as described previously (7) and a 30-80% ammonium sulfate fraction of the postribosomal supernatant was used as a source of EF-1 and EF-2, which were then separated by chromatography on Sephadex G-200 (9). [14C]Phenylalanyl-tRNA (83-165 cpm/ pmol) was prepared as described by Ravel and Shorey (10), with synthetases prepared by the procedure of Muench and Berg (11). Affinity purification. Sodium fusidate and varying amounts of either E. coli or wheat germ ribosomes were added to a specified amount of the Euglena postribosomal supernatant in Buffer A (50 mM Tris-HC1, pH 7.8, 50 mM NH4C1, 5 mM MgClz, 0.1 mM EDTA, 6 mM fi-mercaptoethanol, and 10% glycerol). The magnesium concentration was increased to 20 mM by the addition of 1 M Mg-acetate and GTP was added to a final concentration of 20/zM. This mixture (5 ml) was layered onto a 2.0 ml cushion of Buffer B (44% sucrose, 50 mM Tris-HC1, pH 7.8, 10 mM NH4C1, 20 mM Mg-acetate, 6 mM fi-mercaptoethanol, 5 /zM GTP, and 1 mM fusidic acid) in a 7-cm 3 centrifuge tube. After centrifugation for 4 h at 120,000g in a Beckman type 50 rotor at 4°C, the supernatant was discarded and the ribosomal pellet was resuspended in Buffer C (50 mM Tris-HC1, pH 7.8, 1.0 M NH4C1, 20 mM Mg-acetate, 6 mM/3-mercaptoethanol, and 10% glycerol) and diluted to a final volume of 7 ml with Buffer C. The suspension was subjected to centrifugation for 3 h at 4°C at 120,000g. The top three-quarters of the supernatant was removed, placed in a dialysis bag and concentrated about twofold against solid polyethylene glycol. The sample was then dialyzed overnight against 100 vol of Buffer A, divided into small aliquots, fast frozen in an isopropyl alcohol-dry ice bath, and stored at -70°C. Assays. Assays for chloroplast EF-G and for cytoplasmic EF-2 were performed exactly as described previously (4) except that 2 mM phosphoenolpyruvate and 0.5 units pyruvate kinase were added as an energy
436
BREITENBERGER ET AL.
regenerating system in the EF-G assays. One unit of EF-G or EF-2 activity is defined as the incorporation of 1 pmol of [14C]phenylalanine into polypeptide under the assay conditions.
zlO0 uJ 8O rv>.F6O
RESULTS AND DISCUSSION
~ 4o F-
A general affinity binding procedure has been developed for use in the purification of protein synthesis translocases of both the cytoplasm (EF-2) and organelles (EF-G) of eukaryotic organisms. In this procedure the postribosomal supernatant of whole cell extracts was mixed with appropriate ribosomes, GTP, and the steroidal antibiotic fusidic acid, which inhibits the activity of most translocases by stabilizing the formation of a translocase-GDP-ribosome complex (12). The ribosomes were then separated from the extract by high-speed centrifugation and the bound proteins were dissociated from the ribosome by a high salt wash. The efficacy of this procedure in the purification of chloroplast EF-G and cytoplasmic EF-2 of E. gracilis is illustrated below. As reported previously (4) chloroplast EF-G is specific for 70 S ribosomes such as those from E. coli. Figure 1 indicates that the activity of chloroplast EF-G on E. coli ribosomes is extremely sensitive to inhibition by fusidic acid, approximately 10
Z W
~ 2o o
M o.I
013.
d~//--6 0.5 t.0 F U S I D I C ACID, mM
i 2.0
FIG. 1. Inhibition of chloroplast EF-G and cytoplasmic EF-2 by fusidic acid. Incubation mixtures were prepared as described previously (4) and contained increasing amounts of fusidic acid as indicated. Reaction mixtures contained 5.6/zg of the postribosomal supernatant (specific activity = 2.0 nnits//zg) to supply chloroplast EF-G or 16.7 /xg of the postribosomal supernatant (specific activity = 0.9 unit//xg) to supply EF-2. One hundred percent polymerization represents 11 pmol in the EF-G assays or 15 pmol for the EF-2 assays. A blank of radioactivity retained on the filter in the absence of EF-G (1.2 pmol) or EF-2 (0.5 pmol) has been subtracted from the appropriate values. (O) EF-2 activity; (O), EF-G activity.
/~M being sufficient to reduce its activity by 50%. Because of the ribosome specificity and fusidic acid sensitivity of this factor, it was possible to form a chloroplast EF-G complex on E. coli ribosomes in the presence of GTP and fusidic acid and to use the
TABLE 1 E F F E C T O F I N C R E A S I N G C O N C E N T R A T I O N S OF E. coli RIBOSOMES ON T H E P U R I F I C A T I O N OF CHLOROPLAST
EF-G
BY THE F U S I D I C A C I D P R O C E D U R E a
Sample
E. coli ribosomes (mg)
Protein (rag)
EF-G (units)
Specific activity (units/mg)
Percentage -recovery
n-Fold purification
Postribosomal supernatant I II III
-5.3 9.8 21.0
16.7 0.83 0.85 0.92
95,020 55,200 39,440 37,200
5,690 66,510 46,400 40,430
100 58 42 39
1.0 11.7 8.2 7.1
a The purification procedure was carried out as described under Materials and Methods. E. coli ribosomes were added in the indicated amounts with 1.0 ml (16.7 mg) ofEuglena postribosomal supernatant and 1.5 mM fusidic acid.
437
ELONGATION FACTOR G AND 2 AFFINITY PURIFICATION t
•1020 g
g8 4 W O~
~10 410 610 ~10 I00
POSTRI B O S O M AL SUPERNATANT, mg
F~G. 2. Effect of increased levels of Euglena postribosomal supernatant on the recovery of chloroplast EF-G. Incubation mixtures were prepared as described under Materials and Methods and contained 1.8 mM fusidic acid and the indicated amounts of Euglena postribosomal supernatant (16.7 mg/ml).
formation of this complex in the purification of chloroplast EF-G. Table 1 shows the effect of three levels ofE. coli ribosomes on the recovery of EF-G in the high salt wash following the fusidic acid affinity binding procedure. It was noted that the lowest level of E. coli ribosomes tested (5.3 mg) gave the best recovery and about a 12-fold purification of EF-G. More than 90% of the protein in the EF-G preparation was removed in this step. Smaller amounts of ribosomes could not conveniently be used because of the difficulty of working with a ribosome pellet of less than 5 mg. The lower apparent recovery obtained when higher levels ofE. coli ribosomes were used probably resulted from the removal of some proteins from the ribosomes themselves by
the I M NH4C1 wash. These proteins inhibited EF-G activity in the assay system used. Attempts to reduce this toxicity problem by removing the bound EF-G from the ribosome with 0.5 M NH4C1 rather than with 1 M NH4C1 resulted in lower yields of the factor. There was no detectable chloroplast EF-G activity remaining in the Euglena postribosomal supernatant following the fusidic acid purification step even when only 5 mg of ribosomes was used. Less than 5% of the cytoplasmic EF-2, which is specific for 80 S ribosomes, was recovered in this partially purified EF-G preparation. In an attempt to determine the limits of this purification procedure, the amount of postribosomal supernatant used was increased while holding the level of ribosomes constant at 5 mg. As indicated in Fig. 2, the units of EF-G recovered were still increasing linearly when the maximum capacity of the centrifuge tube was reached. This observation indicates that a very small amount of E. coli ribosomes can be used to recover a large number of units of chloroplast EF-G. If the postribosomal supernatant were first concentrated by ammonium sulfate precipitation, it might be possible to recover even larger amounts of EF-G with this level of E. coli ribosomes. Essentially no detectable activity was found in samples from which the postribosomal supernatant was omitted, indicating that the ribosomes used were free from E. coli EF-G contamination.
TABLE 2 EFFECT OF FUSIDIC ACID CONCENTRATION ON THE RECOVERY OF CHLOROPLAST E F - G a
Fusidic acid (mM)
Protein (rag)
EF-G recovered (units)
Specific activity (units/rag)
Percentage recovery
n-Fold purification
0.0 0.8 3.6
2.2 2.3 1.7
44,100 114,840 109,560
20,040 49,930 64,450
23 60 58
3.5 8.8 11.3
a Thirty-three milligrams ofEuglena postribosomal supernatant and 5.3 mg ofE. coli ribosomes were used under the conditions described under Materials and Methods.
438
B R E I T E N B E R G E R ET AL.
The chloroplast EF-G has a rather high affinity for E. coli ribosomes even in the absence of fusidic acid (Table 2). Between 20 and 25% of the EF-G in the postribosomal supernatant could be bound to and recovered from the E. coli ribosomes in the absence of fusidic acid. However, fusidic acid improved the yield to about 60% and resulted in a more highly purified EF-G preparation. Even low concentrations of fusidic acid (0.8 mM or less) resulted in high recoveries of chloroplast EF-G, though somewhat better purification was achieved when higher levels (2-4 mM) were used. SDS-Gel electrophoresis (Fig. 3) of the partially purified EF-G illustrates the effectiveness of the affinity binding procedure in the purification of this chloroplast protein. The first gel shows the pattern of proteins present in the postribosomal supernatant while the second illustrates the pattern of proteins present in the high salt wash following the affinity binding procedure. (The second gel has four times the number of units of EF-G found on the first gel.) Two major bands and several minor bands were present in the partially purified EF-G preparation. Further purification procedures have indicated that the upper major band (Mr =- 85,000) is chloroplast EF-G. The other major band was obtained when E. coli ribosomes were taken through the routine procedure without the Euglena extract (gel 3), indicating that it was being removed from the E. coli ribosomes by the high salt wash step. The use of this affinity procedure for EF-G purification followed by chromatography on DEAE-cellulose results in chloroplast EF-G of greater than 80% purity in just two steps. By contrast, Tiboni et al. (13) have recently reported the purification of EF-G from spinach chloroplasts. Their procedure required six steps and resulted in an EF-G preparation that was about 70% pure. The EF-G from mitochondria is also generally active on E. coli ribosomes and is inhibited by fusidic acid (14). The method
Mr x I0- 3 92.5
~-67
45
FIG. 3. SDS-Gel disc electrophoresis of EF-G preparations before and after the affinityprocedure. Gels (10% acrylamideand 0.3% bisacrylamide)were prepared and run as described by Weber and Osborn (22). Gel 1contains56/zgof the Euglena postribosomal supernatant (300 units of EF-G). Gel 2 contains20 p~g of the affinitypurifiedEF-G (1200 units). Gel 3 shows the pattern of proteins washed offorE. coli ribosomes alone duringthe procedure.
described here should be applicable to the purification of the mitochondrial translocases and its use should be especially advantageous since mitochondrial EF-G in most organisms is present in lower amounts than is chloroplast EF-G of light-grown Euglena gracilis, making the purification of the mitochondrial translocases by routine procedures an even more formidable task. Cytoplasmic EF-2 can also be substantially purified by the fusidic acid binding procedure if 80 S ribosomes are used in place of the 70 S E. coli ribosomes. Figure 1 illustrates that the activity of Euglena EF-2 on wheat germ 80 S ribosomes is sensitive to inhibition by fusidic acid, about 0.2 mM being sufficient to reduce its activity by 50%. Table 3 indicates that EF-2 could be substantially purified by the affinity binding procedure and that maxi-
ELONGATION FACTOR G AND 2 AFFINITY PURIFICATION
439
TABLE 3 RECOVERY OF EF-2 WITH THE AFFINITY PURIFICATION PROCEDUREa
Sample
Wheat germ ribosomes (rag)
Protein (mg)
Postribosomal supernatant I II III
-20 41 81
14.6 1.3 2.8 1.9
EF-2 recovered (units)
10,780 4,760 7,490 / 6,770
Specific activity (units/mg)
Percentage recovery
n-Fold purification
740 3,660 2,660 3,560
100 44 69 63
1.0 4.9 3.6 4.8
Fusidic acid concentration was 1.8 mM.
mal recovery was achieved when about 40 mg of wheat germ ribosomes were used for every 10,000 units of EF-2. Lower levels of ribosomes resulted in decreased yields. Recoveries were high, generally 65-70%, with
M r x 10- 5 92,5 6"7
45
12.5
FIG. 4. SDS-Gel disc electrophoresis of EF-2 preparations partially purified by the affinity procedure. Gels (10% acrylamide and 0.3% bisacrylamide) were prepared and run as described by Weber and Osborn (22). Gel 1 shows the pattern obtained with 17/zg of the Euglena postribosomal supernatant containing 12 units of EF-2. Gel 2 shows the protein profile obtained from 21 /~g of the partially purified EF-2 (80 units). Gel 3 shows the pattern of proteins (22 /~g) removed from the wheat germ ribosomes by the washing procedures.
a maximum purification of almost fivefold. No EF-2 was obtained from wheat germ ribosomes taken through this procedure without the Euglena extract. Less that 6% of the chloroplast EF-G in the postribosomal supernatant was found in the partially purified EF-2 preparation. Substantial amounts of EF-2 could be recovered from the wheat germ ribosomes even when fusidic acid was omitted, but the addition of 2-3 mM fusidic acid increased the recovery from about 40 to almost 70% (data not shown). SDS-Gel electrophoresis patterns of the partially purified EF-2 are shown in Fig. 4. Further purification has indicated that the protein band with a molecular weight of 89,000 is probably EF-2. A number of the low molecular weight proteins contaminating the EF-2 preparation are apparently wheat germ ribosomal proteins released by the high salt wash. The low molecular weights of most of the proteins contaminating this EF-2 preparation suggest that they can be removed by gel-filtration chromatography. EF-2 has been purified from a number of other sources (15-20). In general it is rather labile and relatively difficult to purify. Recently Lam and Heintz (21) have reported the use of NAD+-dependent ribosylation of EF-2 by diphtheria.toxin as an affinity purification. This procedure looks promising and gives about a 10-fold purification but reported yields are rather low (10%). Most EF-2 isolation schemes involve at least six or seven steps and give an overall yield of only
440
B R E I T E N B E R G E R ET AL.
about 10%. The affinity procedure we have applied here gives high yields (65-70%) of EF-2 and eliminates most of the proteins of the postribosomal supernatant. It will reduce the time required and losses encountered in most EF-2 preparation schemes and should prove to be a useful step in the purification of this factor from any eukaryotic source. ACKNOWLEDGMENTS This work was supported in part by funds from the Petroleum Research Fund administered by the American Chemical Society (Grant 9810-GM), the North Carolina Science and Technology Committee (Grant 882), and the National Institutes of Health (Grant GM 24963).
REFERENCES 1. Boulter, D., Ellis, R., and Yarwood, A. (1972)Biol. Rev. o f the Cambridge Phil. Soc. 47, 113-175. 2. Rohrbach, M. S., Dempsey, M. E., and Bodley, J. W. (1974)J. Biol. Chem. 249, 5094-5101. 3. Holowinsky, A., and Schiff, J. (1970)Plant Physiol. 45, 339-347. 4. Breitenberger, C. A., Graves, M. C., and SpremuUi, L. L. (1979) Arch. Biochem. Biophys. 194, 265-270. 5. Lowry, O. H., Rosebrough, N. J., Fan', A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 6. Remold-O'Donnell, E., and Thach, R. (1970) J. Biol. Chem. 245, 5737-5742. 7. Spremulli, L., Walthall, B.; Lax, S., and Ravel, J. (1977)Arch. Biochem. Biophys. 178, 565-575.
8. Ravel, J., Shorey, R., Froehner, S., and Shive, W. (1968) Arch. Biochem. Biophys. 125, 514-526. 9. Golinska, B., and Legocki, A. (1973) Biochim. Biophys. Acta 324, 156-170. 10. Ravel, J., and Shorey, R. (1971) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Part C, Vol. 20, 306-316, Academic Press, New York. 11. Muench, K., and Berg, P. (1966) in Procedures in Nucleic Acid Research (Cantoni, G., and Davies, D., eds.), Vol. 1, p. 375, Harper & Row, New York. 12. Brot, N., Spears, C., and Weissbach, H. (1969) Biochem. Biophys. Res. Commun. 34, 843. 13. Tiboni, O., DiPasquale, G., and Ciferri, O. (1978) Eur. J. Biochem. 92, 471-477. 14. Ciferri, O., and Tiboni, O. (1973)Nature New Biol. 245, 209-211. 15. Mizumoto, K., Iwasaki, K., Tanaka, M., and Kaziro, Y. (1974)J. Biochem. (Tokyo) 75, 1047-1056. 16. Merrick, W. C., Kemper, W. M., Kantor, J. A., and Anderson, W. F. (1975) J. Biol. Chem. 250, 2620-2625. 17. Raeburn, S., Collins, J. F., Moon, H. M., and Maxwell, E. S. (1971) J. Biol. Chem. 246, 1041-1048. 18. Taira, H., Ejiri, S., and Shimura, K. (1972) J. Biochem. (Tokyo) 72, 1527. 19. Twardowski, T., and Legocki, A. B. (1973) Biochim. Biophys. Acta 324, 171-183. 20. Richter, D., and Lipmann, F. (1970) Biochemistry 9, 5065. 21. Lam, K. S., and Heintz, R. L. (1978) Eur. J. Biochem. 88, 459-466. 22. Weber, K., and Osborn, M. (1969)J. Biol. Chem. 244, 4406-4412.
Purification of Eukaryotic Cytoplasmic Elongation Factor 2 and Organellar Elongation Factor G by an Affinity Binding Procedure CAROLINE A . BREITENBERGER, MARSHA N. MOORE, D A V I D W . RUSSELL, AND L I N D A L . SPREMULLI
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27514 Received May 4, 1979 A rapid affinity binding procedure for obtaining highly purified organellar elongation factor G (EF-G) and cytoplasmic elongation factor 2 (EF-2) in excellent yields from whole cell extracts is described. The procedure involves the addition of ribosomes, GTP, and fusidic acid to a crude cell extract. The fusidic acid stabilizes the formation of a translocaseGDP-ribosome complex which can be recovered from the extract by high-speed centrifugation. The translocase is then released from the ribosomes by a high salt wash. To purify organellar EF-G, 70 S ribosomes from Escherichia coli are used. If 80 S ribosomes such as those from wheat germ are used instead of 70 S ribosomes, EF-2 can be selectively purified from the cell extract. This procedure has been applied to the purification of both cytoplasmic and chloroplast translocases ofEuglena gracilis. Chloroplast EF-G and cytoplasmic EF-2 can be separated from each other and from the vast majority of the proteins in the postribosomal supernatant with yields in both cases of 60% or greater.
In eukaryotic ceils, proteins are synthe- chloroplasts. This approach requires that sized in several cellular compartments, each the organellar protein be separated from of which has its own distinct protein synthe- large amounts of cytoplasmic proteins, and sizing system. In animal cells, there are two in order to facilitate its purification we have such systems--one in the cell cytoplasm applied an affinity procedure useful in the and one in the mitochondria--while plant purification of Escherichia coli EF-G (2). cells have three sites of protein synthesis In addition, we have modified this affinity - - t h e cytoplasm, the mitochondria, and the procedure so that it can also be used for chloroplasts (1). Elongation factor 2 (EF-2) 1 the purification of cytoplasmic EF-2. The catalyzes the translocation step of protein procedure is based on the observation that synthesis on the 80 S ribosomes found in fusidic acid, a steroidal antibiotic, causes the cytoplasm. In cell organelles, a distinct the formation of a relatively stable E F - G translocase (EF-Gmit or EF-Gchl) catalyzes ribosome-GDP-fusidic acid complex. Chlotranslocation on the smaller (50 to 70 S) roplast EF-G will form this type of stable complex on E. coli ribosomes, which are organellar ribosome. We have been studying cytoplasmic EF-2 readily available in large amounts. The and chloroplast EF-G from Euglena gracilis complex can be isolated by high-speed cenand have chosen to purify both of these trifugation and chloroplast EF-G removed factors from whole cell extracts in order to by washing with high salt. Results presented avoid the large-scale preparation of isolated here also indicate that EF-2 can be substantially purified by this affinity binding procedure when wheat germ ribosomes are Abbreviations used: EF, elongation factor; phe, sdbstituted for E. coli ribosomes. phenylalanyl; SD.~, sodium dcdecyl sulfate. 0003-2697/79/160434-07502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any form reserved.
434
ELONGATION FACTOR G AND 2 AFFINITY PURIFICATION
MATERIALS AND METHODS
Materials. [a4C]Phenylalanine and E. coli tRNA were purchased from Schwarz/Mann. Poly(U) was obtained from Miles Laboratories; ATP and GTP from P-L. Laboratories. Sephadex G-200, Sephadex G-25, DEAE-Sephadex, phosphoenolpyruvate, and pyruvate kinase were from Sigma. Raw wheat germ was kindly supplied by Mr. J. M. de Rosier of International Multifoods Corporation. Sodium fusidate was a generous gift of Dr. Godtfredsen of Leo Pharmaceuticals. Growth conditions and preparation of whole cell extracts. Euglena gracilis Klebs var. bacillaris Cori (Euglena B) was grown on Hutner's pH 3.5 medium to late log phase (2 × 106 cells/ml) and was then transferred to pH 6.8 resting medium and light-induced as described previously (3,4). Cells were harvested after 36 h of exposure to 120 fc of white light. The preparation of the postribosomal supernatant of whole cell extracts was performed as described previously (4) except that the Sephadex G-25 step was replaced by an overnight dialysis. The final protein concentrations of the postribosomal supernatants ranged from 16 to 18 mg/ml as determined by the method of Lowry et al. (5), using bovine serum albumin as a standard.
Preparation of ribosomes, elongation factors, and [14C]phe-tRNA. Escherichia coli MRE 600 or W ribosomes were prepared and high salt washed as described previously (6) except that the first high salt wash contained 1 M NH4CI. They were freed of any residual EF-G contamination by a further 0.60 M NH4C1 wash and collected through a 20% sucrose cushion. Wheat germ high salt washed ribosomes were prepared as described previously (7) and freed from residual EF-2 by washing with 0.5% sodium deoxycholate followed by sedimentation through 10 and 20% sucrose cushions. A complex containing EF-Tu and EF-Ts was prepared from E. coli MRE 600 (8). This complex was free of E. coli EF-G. Wheat
435
germ extracts were prepared as described previously (7) and a 30-80% ammonium sulfate fraction of the postribosomal supernatant was used as a source of EF-1 and EF-2, which were then separated by chromatography on Sephadex G-200 (9). [14C]Phenylalanyl-tRNA (83-165 cpm/ pmol) was prepared as described by Ravel and Shorey (10), with synthetases prepared by the procedure of Muench and Berg (11). Affinity purification. Sodium fusidate and varying amounts of either E. coli or wheat germ ribosomes were added to a specified amount of the Euglena postribosomal supernatant in Buffer A (50 mM Tris-HC1, pH 7.8, 50 mM NH4C1, 5 mM MgClz, 0.1 mM EDTA, 6 mM fi-mercaptoethanol, and 10% glycerol). The magnesium concentration was increased to 20 mM by the addition of 1 M Mg-acetate and GTP was added to a final concentration of 20/zM. This mixture (5 ml) was layered onto a 2.0 ml cushion of Buffer B (44% sucrose, 50 mM Tris-HC1, pH 7.8, 10 mM NH4C1, 20 mM Mg-acetate, 6 mM fi-mercaptoethanol, 5 /zM GTP, and 1 mM fusidic acid) in a 7-cm 3 centrifuge tube. After centrifugation for 4 h at 120,000g in a Beckman type 50 rotor at 4°C, the supernatant was discarded and the ribosomal pellet was resuspended in Buffer C (50 mM Tris-HC1, pH 7.8, 1.0 M NH4C1, 20 mM Mg-acetate, 6 mM/3-mercaptoethanol, and 10% glycerol) and diluted to a final volume of 7 ml with Buffer C. The suspension was subjected to centrifugation for 3 h at 4°C at 120,000g. The top three-quarters of the supernatant was removed, placed in a dialysis bag and concentrated about twofold against solid polyethylene glycol. The sample was then dialyzed overnight against 100 vol of Buffer A, divided into small aliquots, fast frozen in an isopropyl alcohol-dry ice bath, and stored at -70°C. Assays. Assays for chloroplast EF-G and for cytoplasmic EF-2 were performed exactly as described previously (4) except that 2 mM phosphoenolpyruvate and 0.5 units pyruvate kinase were added as an energy
436
BREITENBERGER ET AL.
regenerating system in the EF-G assays. One unit of EF-G or EF-2 activity is defined as the incorporation of 1 pmol of [14C]phenylalanine into polypeptide under the assay conditions.
zlO0 uJ 8O rv>.F6O
RESULTS AND DISCUSSION
~ 4o F-
A general affinity binding procedure has been developed for use in the purification of protein synthesis translocases of both the cytoplasm (EF-2) and organelles (EF-G) of eukaryotic organisms. In this procedure the postribosomal supernatant of whole cell extracts was mixed with appropriate ribosomes, GTP, and the steroidal antibiotic fusidic acid, which inhibits the activity of most translocases by stabilizing the formation of a translocase-GDP-ribosome complex (12). The ribosomes were then separated from the extract by high-speed centrifugation and the bound proteins were dissociated from the ribosome by a high salt wash. The efficacy of this procedure in the purification of chloroplast EF-G and cytoplasmic EF-2 of E. gracilis is illustrated below. As reported previously (4) chloroplast EF-G is specific for 70 S ribosomes such as those from E. coli. Figure 1 indicates that the activity of chloroplast EF-G on E. coli ribosomes is extremely sensitive to inhibition by fusidic acid, approximately 10
Z W
~ 2o o
M o.I
013.
d~//--6 0.5 t.0 F U S I D I C ACID, mM
i 2.0
FIG. 1. Inhibition of chloroplast EF-G and cytoplasmic EF-2 by fusidic acid. Incubation mixtures were prepared as described previously (4) and contained increasing amounts of fusidic acid as indicated. Reaction mixtures contained 5.6/zg of the postribosomal supernatant (specific activity = 2.0 nnits//zg) to supply chloroplast EF-G or 16.7 /xg of the postribosomal supernatant (specific activity = 0.9 unit//xg) to supply EF-2. One hundred percent polymerization represents 11 pmol in the EF-G assays or 15 pmol for the EF-2 assays. A blank of radioactivity retained on the filter in the absence of EF-G (1.2 pmol) or EF-2 (0.5 pmol) has been subtracted from the appropriate values. (O) EF-2 activity; (O), EF-G activity.
/~M being sufficient to reduce its activity by 50%. Because of the ribosome specificity and fusidic acid sensitivity of this factor, it was possible to form a chloroplast EF-G complex on E. coli ribosomes in the presence of GTP and fusidic acid and to use the
TABLE 1 E F F E C T O F I N C R E A S I N G C O N C E N T R A T I O N S OF E. coli RIBOSOMES ON T H E P U R I F I C A T I O N OF CHLOROPLAST
EF-G
BY THE F U S I D I C A C I D P R O C E D U R E a
Sample
E. coli ribosomes (mg)
Protein (rag)
EF-G (units)
Specific activity (units/mg)
Percentage -recovery
n-Fold purification
Postribosomal supernatant I II III
-5.3 9.8 21.0
16.7 0.83 0.85 0.92
95,020 55,200 39,440 37,200
5,690 66,510 46,400 40,430
100 58 42 39
1.0 11.7 8.2 7.1
a The purification procedure was carried out as described under Materials and Methods. E. coli ribosomes were added in the indicated amounts with 1.0 ml (16.7 mg) ofEuglena postribosomal supernatant and 1.5 mM fusidic acid.
437
ELONGATION FACTOR G AND 2 AFFINITY PURIFICATION t
•1020 g
g8 4 W O~
~10 410 610 ~10 I00
POSTRI B O S O M AL SUPERNATANT, mg
F~G. 2. Effect of increased levels of Euglena postribosomal supernatant on the recovery of chloroplast EF-G. Incubation mixtures were prepared as described under Materials and Methods and contained 1.8 mM fusidic acid and the indicated amounts of Euglena postribosomal supernatant (16.7 mg/ml).
formation of this complex in the purification of chloroplast EF-G. Table 1 shows the effect of three levels ofE. coli ribosomes on the recovery of EF-G in the high salt wash following the fusidic acid affinity binding procedure. It was noted that the lowest level of E. coli ribosomes tested (5.3 mg) gave the best recovery and about a 12-fold purification of EF-G. More than 90% of the protein in the EF-G preparation was removed in this step. Smaller amounts of ribosomes could not conveniently be used because of the difficulty of working with a ribosome pellet of less than 5 mg. The lower apparent recovery obtained when higher levels ofE. coli ribosomes were used probably resulted from the removal of some proteins from the ribosomes themselves by
the I M NH4C1 wash. These proteins inhibited EF-G activity in the assay system used. Attempts to reduce this toxicity problem by removing the bound EF-G from the ribosome with 0.5 M NH4C1 rather than with 1 M NH4C1 resulted in lower yields of the factor. There was no detectable chloroplast EF-G activity remaining in the Euglena postribosomal supernatant following the fusidic acid purification step even when only 5 mg of ribosomes was used. Less than 5% of the cytoplasmic EF-2, which is specific for 80 S ribosomes, was recovered in this partially purified EF-G preparation. In an attempt to determine the limits of this purification procedure, the amount of postribosomal supernatant used was increased while holding the level of ribosomes constant at 5 mg. As indicated in Fig. 2, the units of EF-G recovered were still increasing linearly when the maximum capacity of the centrifuge tube was reached. This observation indicates that a very small amount of E. coli ribosomes can be used to recover a large number of units of chloroplast EF-G. If the postribosomal supernatant were first concentrated by ammonium sulfate precipitation, it might be possible to recover even larger amounts of EF-G with this level of E. coli ribosomes. Essentially no detectable activity was found in samples from which the postribosomal supernatant was omitted, indicating that the ribosomes used were free from E. coli EF-G contamination.
TABLE 2 EFFECT OF FUSIDIC ACID CONCENTRATION ON THE RECOVERY OF CHLOROPLAST E F - G a
Fusidic acid (mM)
Protein (rag)
EF-G recovered (units)
Specific activity (units/rag)
Percentage recovery
n-Fold purification
0.0 0.8 3.6
2.2 2.3 1.7
44,100 114,840 109,560
20,040 49,930 64,450
23 60 58
3.5 8.8 11.3
a Thirty-three milligrams ofEuglena postribosomal supernatant and 5.3 mg ofE. coli ribosomes were used under the conditions described under Materials and Methods.
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B R E I T E N B E R G E R ET AL.
The chloroplast EF-G has a rather high affinity for E. coli ribosomes even in the absence of fusidic acid (Table 2). Between 20 and 25% of the EF-G in the postribosomal supernatant could be bound to and recovered from the E. coli ribosomes in the absence of fusidic acid. However, fusidic acid improved the yield to about 60% and resulted in a more highly purified EF-G preparation. Even low concentrations of fusidic acid (0.8 mM or less) resulted in high recoveries of chloroplast EF-G, though somewhat better purification was achieved when higher levels (2-4 mM) were used. SDS-Gel electrophoresis (Fig. 3) of the partially purified EF-G illustrates the effectiveness of the affinity binding procedure in the purification of this chloroplast protein. The first gel shows the pattern of proteins present in the postribosomal supernatant while the second illustrates the pattern of proteins present in the high salt wash following the affinity binding procedure. (The second gel has four times the number of units of EF-G found on the first gel.) Two major bands and several minor bands were present in the partially purified EF-G preparation. Further purification procedures have indicated that the upper major band (Mr =- 85,000) is chloroplast EF-G. The other major band was obtained when E. coli ribosomes were taken through the routine procedure without the Euglena extract (gel 3), indicating that it was being removed from the E. coli ribosomes by the high salt wash step. The use of this affinity procedure for EF-G purification followed by chromatography on DEAE-cellulose results in chloroplast EF-G of greater than 80% purity in just two steps. By contrast, Tiboni et al. (13) have recently reported the purification of EF-G from spinach chloroplasts. Their procedure required six steps and resulted in an EF-G preparation that was about 70% pure. The EF-G from mitochondria is also generally active on E. coli ribosomes and is inhibited by fusidic acid (14). The method
Mr x I0- 3 92.5
~-67
45
FIG. 3. SDS-Gel disc electrophoresis of EF-G preparations before and after the affinityprocedure. Gels (10% acrylamideand 0.3% bisacrylamide)were prepared and run as described by Weber and Osborn (22). Gel 1contains56/zgof the Euglena postribosomal supernatant (300 units of EF-G). Gel 2 contains20 p~g of the affinitypurifiedEF-G (1200 units). Gel 3 shows the pattern of proteins washed offorE. coli ribosomes alone duringthe procedure.
described here should be applicable to the purification of the mitochondrial translocases and its use should be especially advantageous since mitochondrial EF-G in most organisms is present in lower amounts than is chloroplast EF-G of light-grown Euglena gracilis, making the purification of the mitochondrial translocases by routine procedures an even more formidable task. Cytoplasmic EF-2 can also be substantially purified by the fusidic acid binding procedure if 80 S ribosomes are used in place of the 70 S E. coli ribosomes. Figure 1 illustrates that the activity of Euglena EF-2 on wheat germ 80 S ribosomes is sensitive to inhibition by fusidic acid, about 0.2 mM being sufficient to reduce its activity by 50%. Table 3 indicates that EF-2 could be substantially purified by the affinity binding procedure and that maxi-
ELONGATION FACTOR G AND 2 AFFINITY PURIFICATION
439
TABLE 3 RECOVERY OF EF-2 WITH THE AFFINITY PURIFICATION PROCEDUREa
Sample
Wheat germ ribosomes (rag)
Protein (mg)
Postribosomal supernatant I II III
-20 41 81
14.6 1.3 2.8 1.9
EF-2 recovered (units)
10,780 4,760 7,490 / 6,770
Specific activity (units/mg)
Percentage recovery
n-Fold purification
740 3,660 2,660 3,560
100 44 69 63
1.0 4.9 3.6 4.8
Fusidic acid concentration was 1.8 mM.
mal recovery was achieved when about 40 mg of wheat germ ribosomes were used for every 10,000 units of EF-2. Lower levels of ribosomes resulted in decreased yields. Recoveries were high, generally 65-70%, with
M r x 10- 5 92,5 6"7
45
12.5
FIG. 4. SDS-Gel disc electrophoresis of EF-2 preparations partially purified by the affinity procedure. Gels (10% acrylamide and 0.3% bisacrylamide) were prepared and run as described by Weber and Osborn (22). Gel 1 shows the pattern obtained with 17/zg of the Euglena postribosomal supernatant containing 12 units of EF-2. Gel 2 shows the protein profile obtained from 21 /~g of the partially purified EF-2 (80 units). Gel 3 shows the pattern of proteins (22 /~g) removed from the wheat germ ribosomes by the washing procedures.
a maximum purification of almost fivefold. No EF-2 was obtained from wheat germ ribosomes taken through this procedure without the Euglena extract. Less that 6% of the chloroplast EF-G in the postribosomal supernatant was found in the partially purified EF-2 preparation. Substantial amounts of EF-2 could be recovered from the wheat germ ribosomes even when fusidic acid was omitted, but the addition of 2-3 mM fusidic acid increased the recovery from about 40 to almost 70% (data not shown). SDS-Gel electrophoresis patterns of the partially purified EF-2 are shown in Fig. 4. Further purification has indicated that the protein band with a molecular weight of 89,000 is probably EF-2. A number of the low molecular weight proteins contaminating the EF-2 preparation are apparently wheat germ ribosomal proteins released by the high salt wash. The low molecular weights of most of the proteins contaminating this EF-2 preparation suggest that they can be removed by gel-filtration chromatography. EF-2 has been purified from a number of other sources (15-20). In general it is rather labile and relatively difficult to purify. Recently Lam and Heintz (21) have reported the use of NAD+-dependent ribosylation of EF-2 by diphtheria.toxin as an affinity purification. This procedure looks promising and gives about a 10-fold purification but reported yields are rather low (10%). Most EF-2 isolation schemes involve at least six or seven steps and give an overall yield of only
440
B R E I T E N B E R G E R ET AL.
about 10%. The affinity procedure we have applied here gives high yields (65-70%) of EF-2 and eliminates most of the proteins of the postribosomal supernatant. It will reduce the time required and losses encountered in most EF-2 preparation schemes and should prove to be a useful step in the purification of this factor from any eukaryotic source. ACKNOWLEDGMENTS This work was supported in part by funds from the Petroleum Research Fund administered by the American Chemical Society (Grant 9810-GM), the North Carolina Science and Technology Committee (Grant 882), and the National Institutes of Health (Grant GM 24963).
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8. Ravel, J., Shorey, R., Froehner, S., and Shive, W. (1968) Arch. Biochem. Biophys. 125, 514-526. 9. Golinska, B., and Legocki, A. (1973) Biochim. Biophys. Acta 324, 156-170. 10. Ravel, J., and Shorey, R. (1971) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Part C, Vol. 20, 306-316, Academic Press, New York. 11. Muench, K., and Berg, P. (1966) in Procedures in Nucleic Acid Research (Cantoni, G., and Davies, D., eds.), Vol. 1, p. 375, Harper & Row, New York. 12. Brot, N., Spears, C., and Weissbach, H. (1969) Biochem. Biophys. Res. Commun. 34, 843. 13. Tiboni, O., DiPasquale, G., and Ciferri, O. (1978) Eur. J. Biochem. 92, 471-477. 14. Ciferri, O., and Tiboni, O. (1973)Nature New Biol. 245, 209-211. 15. Mizumoto, K., Iwasaki, K., Tanaka, M., and Kaziro, Y. (1974)J. Biochem. (Tokyo) 75, 1047-1056. 16. Merrick, W. C., Kemper, W. M., Kantor, J. A., and Anderson, W. F. (1975) J. Biol. Chem. 250, 2620-2625. 17. Raeburn, S., Collins, J. F., Moon, H. M., and Maxwell, E. S. (1971) J. Biol. Chem. 246, 1041-1048. 18. Taira, H., Ejiri, S., and Shimura, K. (1972) J. Biochem. (Tokyo) 72, 1527. 19. Twardowski, T., and Legocki, A. B. (1973) Biochim. Biophys. Acta 324, 171-183. 20. Richter, D., and Lipmann, F. (1970) Biochemistry 9, 5065. 21. Lam, K. S., and Heintz, R. L. (1978) Eur. J. Biochem. 88, 459-466. 22. Weber, K., and Osborn, M. (1969)J. Biol. Chem. 244, 4406-4412.
