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[26] Separation of cytoplasmic and mitochondrial elongation factors from yeast
[26]
SEPARATIONOF ELONGATION FACTORS FROM YEAST
245
Comments
Ribosomes complexed with E F 2 and guanosine nucleotide are not only inactive in accepting EF 1-dependent Phe-tRNA binding, but also inactive in accepting nonenzymatically bound Phe-tRNA at the elevated Mg 2+ concentration of 20 mM. The competition test described here can also be carried out for the reverse reaction. In this case, E F 1 is first bound to the ribosomes; the ribosomes are then assayed for E F 2-dependent GDP binding. In any event, the results of these experiments show that both enzymes have at least an overlapping region on the ribosomes, and that once bound to them, either one of the two factors inhibits the function of its counterpartf
[26]
Separation of Cytoplasmic and Mitochondrial Elongation Factors from Yeast B y DIETMAR RICHTER and FRITZ LIPMANN
Only relatively recently has it been firmly established that the eukaryotic organism contains two sets of protein-synthesizing apparatus: one, located in the cytoplasm, which represents the typical eukaryotic system, and another in mitochondria, which closely resembles the prokaryotic system. 1 .~ Functionally and in chromatographic behavior, the elongation factors from the cytoplasm and mitochondria are quite different.' They can be isolated in two ways: (A) mitochondria and cytoplasm are separated by discontinuous centrifugation, and then the mitochondria are lysed mechanically or by osmotic shock to yield mitochondrial factors; (B) cells are disrupted by high shear forces which cause the breakage not only of cell walls but also of the organelles. Thus, these homogenates contain cytoplasmic as well as mitochondrial elongation factors which can be separated by chromatography.' Method B is rapid and convenient since it avoids isolation of the organelles; method A is essential, however, for identification. This article describes both methods of isolation of mitochondrial and cytoplasmic elongation factors from yeast cells. A brief method for the isolation of mitochondrial ribosomes is also included. Since the isolation ' A. W. Linnane, J. M. Haslam, H. B. Lukins, and P. Nagley, Annu. Rev. Microbiol. 26, 163 (1972). 2p. Borst, Annu. Rev. Biochem. 41, 333 (1972). '~H. KiJntzel, Curr. Top. Microbiol. lmmunol. 54, 94 (1971). 'D. Richter and F. Lipmann, Biochemistry 9, 5065 (1970).
246
ELONGATION FACTORS IN PROTEIN SYNTHESIS
[26]
of the two cytoplasmic elongation factors from yeast has been published elsewhere, 5 we have put more emphasis on the separation of the two sets of elongation factors as well as on the isolation and purification of the mitochondrial elongation factors. Because of parallels between bacterial and mitochondrial elongation factors, the nomenclature for prokaryotic elongation factors is used for the latter: EF T is the aminoacyl-tRNA binding factor, and E F G is the peptidyl translocase. For the cytoplasmic elongation factors, EF 1 and EF 2 are used, as generally applied in eukaryotic protein synthesis. It appears that mitochondrial ribosomes and elongation factors are interchangeable with their bacterial, but not with their cytoplasmic, counterparts in the eukaryotic cell. 4,G-s The only case where a mitochondrial (or bacterial) elongation factor is compatible with eukaryotic ribosomes is EF T; however, its cytoplasmic counterpart, EF 1, does not react with mitochondrial or prokaryotic ribosomes. ',~,1° For analysis during isolation of mitochondrial factors it is preferable to complement them with the more easily available ribosomes and elongation factors from Escherichia coli. Reagents and Materials
Buffer 1 : 2 0 m M Tris.HCl, pH 7.4, 1 m M dithiothreitol Buffer 2 : 5 m M K phosphate, pH 7.2, 1 m M dithiothreitol. All buffers are adjusted at 4 ° . Hydroxylapatite (Clarkson Chemical Co., Hypatite C) DEAE-cellulose (Bio-Rad) [l'C]Phenylalanine, specific activity 460 mCi/mmole Dithiothreitol (RSA Corporation) Yeast tRNA (Miles Laboratories) Triton X-100 (Rohm and Haas) Unless otherwise stated, all operations are carried out at 4 ° . Strains Saccharomyces fragilis, ATCC No. 10022. Saccharomyces cerevisiae, strain 18A; the mitochondrial DNA-
5 D. Richter and F. Klink, this series, Vol. 20, p. 349. ' M. Grandi and H. Kiintzel, FEBS (Fed. Eur. Biochem. Soc.) Lett. 10, 25 (1970). 7A. Perani, O. Tiboni, and O. Ciferri, 1. Mol. Biol. 55, 107 (1971). SA. H. Scragg, FEBS (Fed. Eur. Biochem. Soc.) Lett. 17, 111 (1971). *I. Krisko, J. Gordon, and F. Lipmann, I. Biol. Chem. 244, 6117 (1969). I°D. Richter, Biochem. Biophys. Res. Commun. 38, 864 (1970).
[26]
SEPARATIONOF ELONGATION FACTORS FROM YEAST
247
depleted "petite" mutants II-1-40 and II1-1-7 can be obtained by treatment of the cells with ethidium bromide? ~'~2 General Procedures A s s a y M e t h o d ]or Polyphenylalanine Synthesis
The function of the two elongation factors from yeast cytoplasm can be studied with 80 S cytoplasmic ribosomes from yeast or mammals; mitochondrial elongation factors can be combined with either mitochondrial or bacterial ribosomes. Polyphenylalanine synthesis is measured by determination of 14C radioactivity incorporated into hot trichloroacetic acid-insoluble protein. A typical assay (125 /d) contains: 50 m M Tris.HC1, pH 7.4, 100 mM NH4C1, 60 mM KC1, 10 mM Mg(CH3COO)~, 1 mM dithiothreitol, 2 mM GTP, 100-150 ~g of ribosomes, 30 /~g of p o l y ( U ) , 10-20 pmoles of [14C]Phe-tRNA (350 pmoles of [14C]Phe per milligram of yeast t R N A ) , and 5 0 - 7 0 ~g of each of the enzymes. Incubation is carried out at 37 ° for 10 minutes; the radioactive protein is analyzed as described. ~ Protein concentration can be estimated by the method of Lowry et al. 1~ or of Warburg and Christian? * G r o w t h Conditions
The following yeast strains can be used: S. cerevisiae (wild-type 18A and two mutants ~1 lacking mitochondrial DNA, II-140 and III-1-7), and S. fragilis. The yeast cells are grown in a medium containing, per liter: 5 g of yeast extract, 10 g of peptone, 6 g of ( N H , ) z H P O , , 2 g of MgSO~, 9 g of KC1, and 33 ml of a 60% lactate syrup. The pH is adjusted to 4.5 with concentrated HCI, and the medium is sterilized at 15 psi for 20 minutes. A 10-liter carboy is inoculated with 300 ml of an overnight culture containing 7.0 A~0 (absorbance at 450 nm) units/ml. Cells are grown at 30 ° in a New Brunswick fermentor under aeration (2 liters of air per minute) with stirring (800 rpm). The culture is grown to a turbidity of 8.0 as measured from the 450-nm absorbance, and is quickly cooled by passing it through a copper cooling coil. Cells are harvested in a Sharpies continuous flow centrifuge. The "petite" mutants are grown 11E. S. Goldring, L. J. Grossman, D. Krupnick, D. R. Cryer, and J. Marmur, J. Mol. Biol. 52, 323 (1970). 12p. O. Slonimski, G. Perrodin, and J. H. Croft, Biochem. Biophys. Res. Commun. 30, 232 (1968). 1~O. H. Lowry, W. J. Rosebrough, A. L. Farr, and R. J. Randall, 1. Biol. Chem. 193, 265 (1951). I~O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941).
248
ELONGATION FACTORS IN PROTEIN SYNTHESIS
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under similar conditions except that lactate is replaced by 1.5% glucose. The yield for all strains varies between 7 and 12 g of cells (wet weight) per liter of medium. The well-packed cells are kept overnight at 4 ° without loss of activity of the factors. Isolation of the Elongation Factors
Method A Elongation Factors from Isolated Mitochondria. For the isolation of mitoehondria, spheroplasts from 1 kg of yeast cells (wet weight) are prepared 1~ and gently homogenized for 15 seconds in a Waring Blendor; the mitochondrial particles are isolated and washed according to the method of Mattoon and Balcavage. 16 Washed mitochondria are suspended in buffer 1 with 10 mM Mg(CH.~COO)._,, and yield 4-5 mg of mitochondrial protein per milliliter of solution. The mitochondrial suspension is passed through a French press at 6000 psi. The homogenate is centrifuged at 105,000 g for 2 hours, the resulting S100 fraction is treated with 43 g of (NH4)2SO4/100 ml, pH 6.9, stirred for 15 minutes, and centrifuged at 20,000 g for 15 minutes. The pellet is dissolved in 5 ml of buffer 2 and dialyzed against 2 liters of the same buffer. About 100 mg of protein are applied to a hydroxyapatite column (0.8 × 12 cm) previously equilibrated with buffer 2, and the column is washed with 100 ml of the same buffer. Mitochondrial EF G elutes at 30 mM and E F T at 70 mM phosphate buffer; all buffers contain 1 mM dithiothreitol, and the pH is adjusted to 7.2. Fractions containing either EF T or EF G are combined and concentrated using a Diaflo Model 50 ultrafiltration cell with a PM-10 membrane. Both factors can be stored for several months in liquid nitrogen. For further purification steps of the mitochondrial elongation factors see method B. Preparation of Mitochondrial Ribosomes. Washed mitochondria from 1000 g of yeast ceils (wet weight) are diluted with an equal volume of 40 mM Tris .HC1 buffer, pH 7.4, and 20 mM Mg(CH3COO)2 and lysed by adding 1/20 of the volume of a 20% Triton X-100 solution. The ribosomes are pelleted at 105,000 g for 2 hours and redissolved in 20 mM Tris .HCI (pH 7.4) and 10 mM Mg(CH:,COO)~. The A260:A2,~ of this preparation is 1.93; the yield is 10-15 mg of ribosomal protein. Method B Separation o/Cytoplasmic and Mitochondrial Elongation Factors ]rom Yeast Homogenates. Yeast cells (200 g wet weight) are resuspended in 15E. A. Duell, S. Inoue, and M. F. Utter, J. Bacteriol. 88, 1762 (1964). ~ J. R. Mattoon and W. X. Balcavage, this series, Vol. 10, p. 135.
[26]
SEPARATION OF ELONGATION FACTORS FROM YEAST
249
400 ml of buffer 1 and disrupted in a Manton-Gaulin mill. 17 The homogenate is centrifuged at 5000 g for 10 minutes, and the pellet is reextracted twice with 300 ml of buffer 1. The supernatant fractions are combined and further clarified by centrifugation at 18,000 g for 20 minutes. The pH of the supernatant fluid is readjusted with 1 M Tris.HC1 to 7.4. After centrifugation at 78,000 g for 2 hours, two-thirds of the supernatant fractions are collected; the combined fractions are referred to as S100. The yield is 1040 ml or 9310 mg of protein. Ammonium Sulfate Fractionation. The pH of the S100 fraction is adjusted to 6.8 with 10% acetic acid, and 70 g of ammonium sulfate/100 ml of fluid are added. The slurry is stirred for 1 hour, then centrifuged at 18,000 g for 1 hour. The supernatant fluid is decanted and the protein pellet is reextracted three times with 150 ml of 25% (step 1), three times with 22% (step 2), and finally three times with 18% (step 3) ammonium sulfate solutions (w/w). All solutions contain 1 mM dithiothreitol, and the pH is adjusted to 6.8 with NH,OH. The supernatant fractions of each step are combined and reprecipitated with 20 g of ammonium sulfate/100 ml of fluid. The precipitates are collected by centrifugation at 18,000 g for 15 minutes, dissolved in buffer 1, and dialyzed against 3 liters of the same buffer. The yield for step 1 is 20 ml, or 935 mg of protein; for step 2, 47 ml, or 2700 mg of protein; and for step 3, 56 ml, or 1860 mg of protein. Step 1 contains no mitochondrial elongation factors, but cytoplasmic EF 1 and some cytoplasmic EF 2. The second step contains most of the mitochondrial E F T but is almost free of its complementary factor, EF G; this step also contains the bulk of cytoplasmic EF 1 and EF 2. Mitochondrial EF G is found in step 3 together with some EF T. The latter step also contains some cytoplasmic EF 2. The ammonium sulfate fractions can be stored for several months in liquid nitrogen without loss of activity. Separation of Mitochondrial E F T and Cytoplasmic EF 1. The cytoplasmic EF 1 can be isolated from either step 1 or step 2 of the ammonium sulfate fractionation by gel filtration on Sephadex G-200 columns. The molecular weight of this EF 1 is high (220,000), and hence it can be separated rather easily from the rest of the elongation factors which are of lesser size (between 60,000 and 90,000). Figure 1 shows typical elution and activity profiles of cytoplasmic EF 1 compared with those of its mitochondrial counterpart, E F T . From the ammonium sulfate step 2, 80 mg of protein are layered on top of a Sephadex G-200 column (4.5 × 80 cm) equilibrated with buffer 1. Elution is carried out with the same buffer; 4.5 ml per fraction are collected. Aliquots (10 ~1) of the eluted fractions are assayed for polyphenylalanine synthesis with an excess of ribosomes ~ J. Gordon, J. Lucas-Lenard, and F. Lipmann, this series, Vol. 20, p. 281.
250
ELONGATION FACTORS IN PROTEIN SYNTHESIS
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I0
0.6 i_
8
3.5
"6 Q.
¢
g
'\
.._o rO.
I
3.4~ o
0.3(~ 4
"6 0.2 0
E
z 0. I
)
20
30
40
50
60
70
80
Frocfion number
FIG. 1. Separation of mitochondrial E F T
(Q--Q)
and cytoplasmic EF 1
(0--0). and complementary factors. The cytoplasmic EF 1 is free of cytoplasmic E F 2 and mitochondrial elongation factors. The mitochondrial E F T is contaminated by small amounts of cytoplasmic E F 2 and mitochondrial EF G. For further purification of the mitochondrial EF T, see below. Separation of Mitochondrial EF G and Cytoplasmic EF 2. The ammonium sulfate step 3 contains most of the mitochondrial EF G as well as some cytoplasmic EF 2. The molecular weights of both factors are very close and do not separate on a Sephadex column. However, they both have different affinities for DEAE-cellulose. Figure 2 shows that mitochondrial EF G is recovered from the column at about 0.2 M KC1, whereas the cytoplasmic EF 2 comes off at 0.3 M KC1. DEAE-cellulose columns (1.2 × 15 cm) are equilibrated with buffer 1 containing 0.1 M KC1. From the ammonium sulfate steps 2 or 3, 50-80 mg of protein are dialyzed against the same buffer and applied to the columns, which are then washed with 100 ml of the same KCI concentration. Linear gradients from 0.10.4 M KCI in buffer 1 (50 X 50 ml) are used; 3.5 ml per fraction are collected. Aliquots (10 ~d) of the eluted fractions are analyzed for polyphenylalanine activity in the presence of ribosomes and complementary factors as described above. Both cytoplasmic EF 2 and mitochondrial EF G are free of contamination by other elongation factors. Purification of Mitochondrial EF T. The ammonium sulfate step 2,
[26]
251
SEPARATION OF ELONGATION FACTORS FROM YEAST
,,F"O.I M K C L
,[-~ 0 . 1 M ~
0.4 /V/ KCL
06
12 i
!:~
_j
i~
j-o4
04
02
o
0 oO (-q
03
4
£3
02
OI
g ~[-~O.IM KCL
~['~ O . I M ~
04 MKCL
06 'S 1.0
/!
rt
/:
p
/~- 04 i
i
0.5
s~
02 0.2
'"....
F.1.¢~"~'-........~
. . !.
20
50 Fraction
o4
02
O,I i
I0
O gO if3 O
I
0.6
/
04
40
i
50
number
FIG. 2. Chromatography of mitochondrial EF G ( 0 - - 0 ) EF 2 ( 0 - - 0 ) on DEAE-cellulose columns.
and cytoplasmic
which has most of the mitochondrial EF T, is used for its further purification. This includes protamine sulfate treatment, stepwise chromatography on hydroxylapatite columns, anion-exchange chromatography on DEAEcellulose, and gel filtration on Sephadex G-200. The steps are summarized in Table I. Protamine Sulfate Step. Neutralized protamine sulfate solution, 0.8 ml, is added per 10 ml of protein solution (step 2 of the ammonium sulfate fractionation). The solution is stirred for 20 minutes and the resulting precipitate is removed by centrifugation at 15,000 g for 15 minutes. The supernatant fluid is dialyzed against 2 liters of buffer 2. The yield is 45 ml, or 2480 mg of protein (Table I). Hydroxylapatite Step. A hydroxylapatite column (4.5 x 9 cm) is packed and equilibrated with buffer 2. The protein fraction of the protamine sulfate step containing mitochondrial EF T is then applied to this column; the latter is washed with 500 ml each of 10 mM and 30 mM phos-
252
ELONGATION FACTORS IN PROTEIN SYNTHESIS
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phate buffers, p H 7.2, both of which contain 1 m M dithiothreitol. Mitochondrial E F T is eluted with 70 m M phosphate buffer with 1 m M dithiothreitol. Fractions with mitochondrial E F T activity are combined and 43 g of (NH4)2SO4/100 ml of solution are added. The slurry is stirred for 20 minutes and then centrifuged at 18,000g for 20 minutes. The precipitate is dissolved in buffer 1 and dialyzed against 2 liters of the same buffer. The yield is 12 ml, or 424 nag of protein (Table I ) . DEAE-Cellulose Step. A DEAE-cellulose column (1.3 × 23 cm) is equilibrated with buffer 1 containing 0.1 M KC1. The mitochondrial E F T from the hydroxylapatite step is applied to the column, which is then washed with 200 ml of buffer 1 containing 0.1 M KC1; the mitochondrial E F T is eluted with a linear gradient from 0.1 to 0.5 M KC1 in buffer 1 (200 X 200 ml), and 4 m l / t u b e are collected. Fractions containing mitochondrial E F T are combined and concentrated in a Diaflo Model 50 ultrafiltration cell with a U M - 1 0 membrane. The yield is 1.5 ml, or 31 mg of protein. Sephadex G-150 Step. Mitochondrial E F T is further purified by gel filtration through a Sephadex G-150 column (1.3 x 75 c m ) . Buffer 1 is used to equilibrate the column and to elute mitochondrial E F T; 2.5 m l / tube are collected. The tubes with mitochondrial E F T are combined and concentrated as described for the DEAE-cellulose step. The yield is 1.5 ml, or 12 mg of protein. TABLE I PURIFICATION STEPS OF THE MITOCttONDRIAL E F T AND E F G FROM Saccharomyces cerevisiae, STaAIN 18A a
Total protein (mg) T 1. 2. 3. 4. 5. 6.
S100 Ammonium sulfate Protamine sulfate Hydroxylapatite DEAE-cellulose Sephadex G-150
G
9310 2720 1860 2480 1815 424 211 31.1 8.4 12.1 3.2
Specific activity (nmoles/ 10 min/mg) T
G
0.100 0.187 0.250 0.190 0.252 0.420 0. 430 5.5 7.0 9.2 12.7
Total enzyme units (nmoles/ 10 min) T
G
931 509 465 471 457 178 90.7 171 58.8 111 40.6
a Activities for EF G and E F T are determined by polyphenylalanine synthesis in the presence of an excess of the complementary Escherichia coli factor and E. coli ribosomes [D. Richter and F. Lipmann, Biochemistry 9, 5065 (1970)]. The activities are derived from experiments with a linear dependence on either EF G or E F T concentration.
[26]
253
SEPARATION OF ELONGATION FACTORS F R O M YEAST
TABLE II PURIFICATIONOF MITOCHONDRIALEF T AND EF G FROMSaccharomyces cerevisiae STRAIN 18A AND THE "PETITE" MUTANTSII-1-40 AND III-1-7 Enzyme units (nmoles/10 min)/g of total S100 protein 18A
1. 2. 3. 4, 5. 6.
II-1-40
III-1-7
Purification steps
T
G
T
G
T
G
S100 Ammonium sulfate Protamine sulfate Hydroxylapatite DEAE-cellulose Sephadex G-150
100 55 51 19 18 12
100 50 49 10 6 4
76 45 39 12 10 6
94 47 45 8 5 3
65 37 32 ----
82 43 31 ----
Further Purification of Mitochondrial EF G. Step 3 of the ammonium sulfate fractionation is used to purify mitochondrial E F G, using a similar method to that described for mitochondrial E F T, but with the following exceptions: mitochondrial E F G is eluted from the hydroxyapatite column (4.5 × 6.0 cm) with 30 mM phosphate buffer, and DEAE-chromatography (column size 1.2 × 24 era) is carried out with a linear gradient from 0.1 to 0.4 M KC1 in buffer 1 (150 × 150 ml). The results of the various purification steps of mitochondrial E F T and E F G are summarized in Table I. Mitochondrial Elongation Factors from "Petite" Mutants Mitochondrial elongation factors can be obtained from various yeast strains including mutants lacking mitochondrial DNA. This shows that mitochondrial E F T and E F G are coded by nuclear, not by mitochondrial, DNA. 1~ Table II compares mitochondrial E F T and E F G from the wildtype strain 18A and from the two "petite" mutants II-1-40 and III-1-7. Mitochondrial elongation factors from all these strains are identical in their functional, protein chemical, and immunological properties. TM
18D. Richter, Biochemistry 10, 4422 (1971).
SEPARATIONOF ELONGATION FACTORS FROM YEAST
245
Comments
Ribosomes complexed with E F 2 and guanosine nucleotide are not only inactive in accepting EF 1-dependent Phe-tRNA binding, but also inactive in accepting nonenzymatically bound Phe-tRNA at the elevated Mg 2+ concentration of 20 mM. The competition test described here can also be carried out for the reverse reaction. In this case, E F 1 is first bound to the ribosomes; the ribosomes are then assayed for E F 2-dependent GDP binding. In any event, the results of these experiments show that both enzymes have at least an overlapping region on the ribosomes, and that once bound to them, either one of the two factors inhibits the function of its counterpartf
[26]
Separation of Cytoplasmic and Mitochondrial Elongation Factors from Yeast B y DIETMAR RICHTER and FRITZ LIPMANN
Only relatively recently has it been firmly established that the eukaryotic organism contains two sets of protein-synthesizing apparatus: one, located in the cytoplasm, which represents the typical eukaryotic system, and another in mitochondria, which closely resembles the prokaryotic system. 1 .~ Functionally and in chromatographic behavior, the elongation factors from the cytoplasm and mitochondria are quite different.' They can be isolated in two ways: (A) mitochondria and cytoplasm are separated by discontinuous centrifugation, and then the mitochondria are lysed mechanically or by osmotic shock to yield mitochondrial factors; (B) cells are disrupted by high shear forces which cause the breakage not only of cell walls but also of the organelles. Thus, these homogenates contain cytoplasmic as well as mitochondrial elongation factors which can be separated by chromatography.' Method B is rapid and convenient since it avoids isolation of the organelles; method A is essential, however, for identification. This article describes both methods of isolation of mitochondrial and cytoplasmic elongation factors from yeast cells. A brief method for the isolation of mitochondrial ribosomes is also included. Since the isolation ' A. W. Linnane, J. M. Haslam, H. B. Lukins, and P. Nagley, Annu. Rev. Microbiol. 26, 163 (1972). 2p. Borst, Annu. Rev. Biochem. 41, 333 (1972). '~H. KiJntzel, Curr. Top. Microbiol. lmmunol. 54, 94 (1971). 'D. Richter and F. Lipmann, Biochemistry 9, 5065 (1970).
246
ELONGATION FACTORS IN PROTEIN SYNTHESIS
[26]
of the two cytoplasmic elongation factors from yeast has been published elsewhere, 5 we have put more emphasis on the separation of the two sets of elongation factors as well as on the isolation and purification of the mitochondrial elongation factors. Because of parallels between bacterial and mitochondrial elongation factors, the nomenclature for prokaryotic elongation factors is used for the latter: EF T is the aminoacyl-tRNA binding factor, and E F G is the peptidyl translocase. For the cytoplasmic elongation factors, EF 1 and EF 2 are used, as generally applied in eukaryotic protein synthesis. It appears that mitochondrial ribosomes and elongation factors are interchangeable with their bacterial, but not with their cytoplasmic, counterparts in the eukaryotic cell. 4,G-s The only case where a mitochondrial (or bacterial) elongation factor is compatible with eukaryotic ribosomes is EF T; however, its cytoplasmic counterpart, EF 1, does not react with mitochondrial or prokaryotic ribosomes. ',~,1° For analysis during isolation of mitochondrial factors it is preferable to complement them with the more easily available ribosomes and elongation factors from Escherichia coli. Reagents and Materials
Buffer 1 : 2 0 m M Tris.HCl, pH 7.4, 1 m M dithiothreitol Buffer 2 : 5 m M K phosphate, pH 7.2, 1 m M dithiothreitol. All buffers are adjusted at 4 ° . Hydroxylapatite (Clarkson Chemical Co., Hypatite C) DEAE-cellulose (Bio-Rad) [l'C]Phenylalanine, specific activity 460 mCi/mmole Dithiothreitol (RSA Corporation) Yeast tRNA (Miles Laboratories) Triton X-100 (Rohm and Haas) Unless otherwise stated, all operations are carried out at 4 ° . Strains Saccharomyces fragilis, ATCC No. 10022. Saccharomyces cerevisiae, strain 18A; the mitochondrial DNA-
5 D. Richter and F. Klink, this series, Vol. 20, p. 349. ' M. Grandi and H. Kiintzel, FEBS (Fed. Eur. Biochem. Soc.) Lett. 10, 25 (1970). 7A. Perani, O. Tiboni, and O. Ciferri, 1. Mol. Biol. 55, 107 (1971). SA. H. Scragg, FEBS (Fed. Eur. Biochem. Soc.) Lett. 17, 111 (1971). *I. Krisko, J. Gordon, and F. Lipmann, I. Biol. Chem. 244, 6117 (1969). I°D. Richter, Biochem. Biophys. Res. Commun. 38, 864 (1970).
[26]
SEPARATIONOF ELONGATION FACTORS FROM YEAST
247
depleted "petite" mutants II-1-40 and II1-1-7 can be obtained by treatment of the cells with ethidium bromide? ~'~2 General Procedures A s s a y M e t h o d ]or Polyphenylalanine Synthesis
The function of the two elongation factors from yeast cytoplasm can be studied with 80 S cytoplasmic ribosomes from yeast or mammals; mitochondrial elongation factors can be combined with either mitochondrial or bacterial ribosomes. Polyphenylalanine synthesis is measured by determination of 14C radioactivity incorporated into hot trichloroacetic acid-insoluble protein. A typical assay (125 /d) contains: 50 m M Tris.HC1, pH 7.4, 100 mM NH4C1, 60 mM KC1, 10 mM Mg(CH3COO)~, 1 mM dithiothreitol, 2 mM GTP, 100-150 ~g of ribosomes, 30 /~g of p o l y ( U ) , 10-20 pmoles of [14C]Phe-tRNA (350 pmoles of [14C]Phe per milligram of yeast t R N A ) , and 5 0 - 7 0 ~g of each of the enzymes. Incubation is carried out at 37 ° for 10 minutes; the radioactive protein is analyzed as described. ~ Protein concentration can be estimated by the method of Lowry et al. 1~ or of Warburg and Christian? * G r o w t h Conditions
The following yeast strains can be used: S. cerevisiae (wild-type 18A and two mutants ~1 lacking mitochondrial DNA, II-140 and III-1-7), and S. fragilis. The yeast cells are grown in a medium containing, per liter: 5 g of yeast extract, 10 g of peptone, 6 g of ( N H , ) z H P O , , 2 g of MgSO~, 9 g of KC1, and 33 ml of a 60% lactate syrup. The pH is adjusted to 4.5 with concentrated HCI, and the medium is sterilized at 15 psi for 20 minutes. A 10-liter carboy is inoculated with 300 ml of an overnight culture containing 7.0 A~0 (absorbance at 450 nm) units/ml. Cells are grown at 30 ° in a New Brunswick fermentor under aeration (2 liters of air per minute) with stirring (800 rpm). The culture is grown to a turbidity of 8.0 as measured from the 450-nm absorbance, and is quickly cooled by passing it through a copper cooling coil. Cells are harvested in a Sharpies continuous flow centrifuge. The "petite" mutants are grown 11E. S. Goldring, L. J. Grossman, D. Krupnick, D. R. Cryer, and J. Marmur, J. Mol. Biol. 52, 323 (1970). 12p. O. Slonimski, G. Perrodin, and J. H. Croft, Biochem. Biophys. Res. Commun. 30, 232 (1968). 1~O. H. Lowry, W. J. Rosebrough, A. L. Farr, and R. J. Randall, 1. Biol. Chem. 193, 265 (1951). I~O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941).
248
ELONGATION FACTORS IN PROTEIN SYNTHESIS
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under similar conditions except that lactate is replaced by 1.5% glucose. The yield for all strains varies between 7 and 12 g of cells (wet weight) per liter of medium. The well-packed cells are kept overnight at 4 ° without loss of activity of the factors. Isolation of the Elongation Factors
Method A Elongation Factors from Isolated Mitochondria. For the isolation of mitoehondria, spheroplasts from 1 kg of yeast cells (wet weight) are prepared 1~ and gently homogenized for 15 seconds in a Waring Blendor; the mitochondrial particles are isolated and washed according to the method of Mattoon and Balcavage. 16 Washed mitochondria are suspended in buffer 1 with 10 mM Mg(CH.~COO)._,, and yield 4-5 mg of mitochondrial protein per milliliter of solution. The mitochondrial suspension is passed through a French press at 6000 psi. The homogenate is centrifuged at 105,000 g for 2 hours, the resulting S100 fraction is treated with 43 g of (NH4)2SO4/100 ml, pH 6.9, stirred for 15 minutes, and centrifuged at 20,000 g for 15 minutes. The pellet is dissolved in 5 ml of buffer 2 and dialyzed against 2 liters of the same buffer. About 100 mg of protein are applied to a hydroxyapatite column (0.8 × 12 cm) previously equilibrated with buffer 2, and the column is washed with 100 ml of the same buffer. Mitochondrial EF G elutes at 30 mM and E F T at 70 mM phosphate buffer; all buffers contain 1 mM dithiothreitol, and the pH is adjusted to 7.2. Fractions containing either EF T or EF G are combined and concentrated using a Diaflo Model 50 ultrafiltration cell with a PM-10 membrane. Both factors can be stored for several months in liquid nitrogen. For further purification steps of the mitochondrial elongation factors see method B. Preparation of Mitochondrial Ribosomes. Washed mitochondria from 1000 g of yeast ceils (wet weight) are diluted with an equal volume of 40 mM Tris .HC1 buffer, pH 7.4, and 20 mM Mg(CH3COO)2 and lysed by adding 1/20 of the volume of a 20% Triton X-100 solution. The ribosomes are pelleted at 105,000 g for 2 hours and redissolved in 20 mM Tris .HCI (pH 7.4) and 10 mM Mg(CH:,COO)~. The A260:A2,~ of this preparation is 1.93; the yield is 10-15 mg of ribosomal protein. Method B Separation o/Cytoplasmic and Mitochondrial Elongation Factors ]rom Yeast Homogenates. Yeast cells (200 g wet weight) are resuspended in 15E. A. Duell, S. Inoue, and M. F. Utter, J. Bacteriol. 88, 1762 (1964). ~ J. R. Mattoon and W. X. Balcavage, this series, Vol. 10, p. 135.
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SEPARATION OF ELONGATION FACTORS FROM YEAST
249
400 ml of buffer 1 and disrupted in a Manton-Gaulin mill. 17 The homogenate is centrifuged at 5000 g for 10 minutes, and the pellet is reextracted twice with 300 ml of buffer 1. The supernatant fractions are combined and further clarified by centrifugation at 18,000 g for 20 minutes. The pH of the supernatant fluid is readjusted with 1 M Tris.HC1 to 7.4. After centrifugation at 78,000 g for 2 hours, two-thirds of the supernatant fractions are collected; the combined fractions are referred to as S100. The yield is 1040 ml or 9310 mg of protein. Ammonium Sulfate Fractionation. The pH of the S100 fraction is adjusted to 6.8 with 10% acetic acid, and 70 g of ammonium sulfate/100 ml of fluid are added. The slurry is stirred for 1 hour, then centrifuged at 18,000 g for 1 hour. The supernatant fluid is decanted and the protein pellet is reextracted three times with 150 ml of 25% (step 1), three times with 22% (step 2), and finally three times with 18% (step 3) ammonium sulfate solutions (w/w). All solutions contain 1 mM dithiothreitol, and the pH is adjusted to 6.8 with NH,OH. The supernatant fractions of each step are combined and reprecipitated with 20 g of ammonium sulfate/100 ml of fluid. The precipitates are collected by centrifugation at 18,000 g for 15 minutes, dissolved in buffer 1, and dialyzed against 3 liters of the same buffer. The yield for step 1 is 20 ml, or 935 mg of protein; for step 2, 47 ml, or 2700 mg of protein; and for step 3, 56 ml, or 1860 mg of protein. Step 1 contains no mitochondrial elongation factors, but cytoplasmic EF 1 and some cytoplasmic EF 2. The second step contains most of the mitochondrial E F T but is almost free of its complementary factor, EF G; this step also contains the bulk of cytoplasmic EF 1 and EF 2. Mitochondrial EF G is found in step 3 together with some EF T. The latter step also contains some cytoplasmic EF 2. The ammonium sulfate fractions can be stored for several months in liquid nitrogen without loss of activity. Separation of Mitochondrial E F T and Cytoplasmic EF 1. The cytoplasmic EF 1 can be isolated from either step 1 or step 2 of the ammonium sulfate fractionation by gel filtration on Sephadex G-200 columns. The molecular weight of this EF 1 is high (220,000), and hence it can be separated rather easily from the rest of the elongation factors which are of lesser size (between 60,000 and 90,000). Figure 1 shows typical elution and activity profiles of cytoplasmic EF 1 compared with those of its mitochondrial counterpart, E F T . From the ammonium sulfate step 2, 80 mg of protein are layered on top of a Sephadex G-200 column (4.5 × 80 cm) equilibrated with buffer 1. Elution is carried out with the same buffer; 4.5 ml per fraction are collected. Aliquots (10 ~1) of the eluted fractions are assayed for polyphenylalanine synthesis with an excess of ribosomes ~ J. Gordon, J. Lucas-Lenard, and F. Lipmann, this series, Vol. 20, p. 281.
250
ELONGATION FACTORS IN PROTEIN SYNTHESIS
[26]
I0
0.6 i_
8
3.5
"6 Q.
¢
g
'\
.._o rO.
I
3.4~ o
0.3(~ 4
"6 0.2 0
E
z 0. I
)
20
30
40
50
60
70
80
Frocfion number
FIG. 1. Separation of mitochondrial E F T
(Q--Q)
and cytoplasmic EF 1
(0--0). and complementary factors. The cytoplasmic EF 1 is free of cytoplasmic E F 2 and mitochondrial elongation factors. The mitochondrial E F T is contaminated by small amounts of cytoplasmic E F 2 and mitochondrial EF G. For further purification of the mitochondrial EF T, see below. Separation of Mitochondrial EF G and Cytoplasmic EF 2. The ammonium sulfate step 3 contains most of the mitochondrial EF G as well as some cytoplasmic EF 2. The molecular weights of both factors are very close and do not separate on a Sephadex column. However, they both have different affinities for DEAE-cellulose. Figure 2 shows that mitochondrial EF G is recovered from the column at about 0.2 M KC1, whereas the cytoplasmic EF 2 comes off at 0.3 M KC1. DEAE-cellulose columns (1.2 × 15 cm) are equilibrated with buffer 1 containing 0.1 M KC1. From the ammonium sulfate steps 2 or 3, 50-80 mg of protein are dialyzed against the same buffer and applied to the columns, which are then washed with 100 ml of the same KCI concentration. Linear gradients from 0.10.4 M KCI in buffer 1 (50 X 50 ml) are used; 3.5 ml per fraction are collected. Aliquots (10 ~d) of the eluted fractions are analyzed for polyphenylalanine activity in the presence of ribosomes and complementary factors as described above. Both cytoplasmic EF 2 and mitochondrial EF G are free of contamination by other elongation factors. Purification of Mitochondrial EF T. The ammonium sulfate step 2,
[26]
251
SEPARATION OF ELONGATION FACTORS FROM YEAST
,,F"O.I M K C L
,[-~ 0 . 1 M ~
0.4 /V/ KCL
06
12 i
!:~
_j
i~
j-o4
04
02
o
0 oO (-q
03
4
£3
02
OI
g ~[-~O.IM KCL
~['~ O . I M ~
04 MKCL
06 'S 1.0
/!
rt
/:
p
/~- 04 i
i
0.5
s~
02 0.2
'"....
F.1.¢~"~'-........~
. . !.
20
50 Fraction
o4
02
O,I i
I0
O gO if3 O
I
0.6
/
04
40
i
50
number
FIG. 2. Chromatography of mitochondrial EF G ( 0 - - 0 ) EF 2 ( 0 - - 0 ) on DEAE-cellulose columns.
and cytoplasmic
which has most of the mitochondrial EF T, is used for its further purification. This includes protamine sulfate treatment, stepwise chromatography on hydroxylapatite columns, anion-exchange chromatography on DEAEcellulose, and gel filtration on Sephadex G-200. The steps are summarized in Table I. Protamine Sulfate Step. Neutralized protamine sulfate solution, 0.8 ml, is added per 10 ml of protein solution (step 2 of the ammonium sulfate fractionation). The solution is stirred for 20 minutes and the resulting precipitate is removed by centrifugation at 15,000 g for 15 minutes. The supernatant fluid is dialyzed against 2 liters of buffer 2. The yield is 45 ml, or 2480 mg of protein (Table I). Hydroxylapatite Step. A hydroxylapatite column (4.5 x 9 cm) is packed and equilibrated with buffer 2. The protein fraction of the protamine sulfate step containing mitochondrial EF T is then applied to this column; the latter is washed with 500 ml each of 10 mM and 30 mM phos-
252
ELONGATION FACTORS IN PROTEIN SYNTHESIS
[26]
phate buffers, p H 7.2, both of which contain 1 m M dithiothreitol. Mitochondrial E F T is eluted with 70 m M phosphate buffer with 1 m M dithiothreitol. Fractions with mitochondrial E F T activity are combined and 43 g of (NH4)2SO4/100 ml of solution are added. The slurry is stirred for 20 minutes and then centrifuged at 18,000g for 20 minutes. The precipitate is dissolved in buffer 1 and dialyzed against 2 liters of the same buffer. The yield is 12 ml, or 424 nag of protein (Table I ) . DEAE-Cellulose Step. A DEAE-cellulose column (1.3 × 23 cm) is equilibrated with buffer 1 containing 0.1 M KC1. The mitochondrial E F T from the hydroxylapatite step is applied to the column, which is then washed with 200 ml of buffer 1 containing 0.1 M KC1; the mitochondrial E F T is eluted with a linear gradient from 0.1 to 0.5 M KC1 in buffer 1 (200 X 200 ml), and 4 m l / t u b e are collected. Fractions containing mitochondrial E F T are combined and concentrated in a Diaflo Model 50 ultrafiltration cell with a U M - 1 0 membrane. The yield is 1.5 ml, or 31 mg of protein. Sephadex G-150 Step. Mitochondrial E F T is further purified by gel filtration through a Sephadex G-150 column (1.3 x 75 c m ) . Buffer 1 is used to equilibrate the column and to elute mitochondrial E F T; 2.5 m l / tube are collected. The tubes with mitochondrial E F T are combined and concentrated as described for the DEAE-cellulose step. The yield is 1.5 ml, or 12 mg of protein. TABLE I PURIFICATION STEPS OF THE MITOCttONDRIAL E F T AND E F G FROM Saccharomyces cerevisiae, STaAIN 18A a
Total protein (mg) T 1. 2. 3. 4. 5. 6.
S100 Ammonium sulfate Protamine sulfate Hydroxylapatite DEAE-cellulose Sephadex G-150
G
9310 2720 1860 2480 1815 424 211 31.1 8.4 12.1 3.2
Specific activity (nmoles/ 10 min/mg) T
G
0.100 0.187 0.250 0.190 0.252 0.420 0. 430 5.5 7.0 9.2 12.7
Total enzyme units (nmoles/ 10 min) T
G
931 509 465 471 457 178 90.7 171 58.8 111 40.6
a Activities for EF G and E F T are determined by polyphenylalanine synthesis in the presence of an excess of the complementary Escherichia coli factor and E. coli ribosomes [D. Richter and F. Lipmann, Biochemistry 9, 5065 (1970)]. The activities are derived from experiments with a linear dependence on either EF G or E F T concentration.
[26]
253
SEPARATION OF ELONGATION FACTORS F R O M YEAST
TABLE II PURIFICATIONOF MITOCHONDRIALEF T AND EF G FROMSaccharomyces cerevisiae STRAIN 18A AND THE "PETITE" MUTANTSII-1-40 AND III-1-7 Enzyme units (nmoles/10 min)/g of total S100 protein 18A
1. 2. 3. 4, 5. 6.
II-1-40
III-1-7
Purification steps
T
G
T
G
T
G
S100 Ammonium sulfate Protamine sulfate Hydroxylapatite DEAE-cellulose Sephadex G-150
100 55 51 19 18 12
100 50 49 10 6 4
76 45 39 12 10 6
94 47 45 8 5 3
65 37 32 ----
82 43 31 ----
Further Purification of Mitochondrial EF G. Step 3 of the ammonium sulfate fractionation is used to purify mitochondrial E F G, using a similar method to that described for mitochondrial E F T, but with the following exceptions: mitochondrial E F G is eluted from the hydroxyapatite column (4.5 × 6.0 cm) with 30 mM phosphate buffer, and DEAE-chromatography (column size 1.2 × 24 era) is carried out with a linear gradient from 0.1 to 0.4 M KC1 in buffer 1 (150 × 150 ml). The results of the various purification steps of mitochondrial E F T and E F G are summarized in Table I. Mitochondrial Elongation Factors from "Petite" Mutants Mitochondrial elongation factors can be obtained from various yeast strains including mutants lacking mitochondrial DNA. This shows that mitochondrial E F T and E F G are coded by nuclear, not by mitochondrial, DNA. 1~ Table II compares mitochondrial E F T and E F G from the wildtype strain 18A and from the two "petite" mutants II-1-40 and III-1-7. Mitochondrial elongation factors from all these strains are identical in their functional, protein chemical, and immunological properties. TM
18D. Richter, Biochemistry 10, 4422 (1971).