From the Department of Chemistry and Biotechnology,
School of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo
113, Japan, the § Department of Molecular Biology, Nagoya
University, Chikusa-ku, Nagoya 464-01, Japan, the
¶ Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya,
Machida-shi, Tokyo 194, Japan, and the
Department of Chemistry,
University of North Carolina,
Chapel Hill, North Carolina 27599-3290
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ABSTRACT |
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The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmt formylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria.
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INTRODUCTION |
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During the initiation of protein biosynthesis the initiator methionyl-tRNA is bound to the ribosomal P-site. In prokaryotes, this step is facilitated by initiation factor 2 (IF-2)1 while in the eukaryotic cytoplasm this step is mediated by eIF-2. In contrast, all other aminoacyl-tRNAs function as elongator tRNAs and enter the A-site of the ribosome in a complex with elongation factor Tu (EF-Tu) in prokaryotes or eEF-1 in the eukaryotic cell cytoplasm (1). In most organisms, the initiator tRNA has distinct features that ensure its selection during the initiation process and its exclusion from the steps of polypeptide chain elongation. In prokaryotes and eukaryotic organelles, such as mitochondria and chloroplasts, the methionine attached to the initiator tRNA undergoes formylation at its amino group through the action of the enzyme methionyl-tRNA transformylase (MTF) (2-4). In Escherichia coli, MTF discriminates strictly between the initiator tRNA and the tRNAs used for chain elongation by recognizing specific determinants in the initiator tRNA (5). Formylation of methionyl-tRNA is necessary for the interaction of the tRNA with IF-2. Formylation also eliminates any significant interaction with EF-Tu. In the yeasts and plants, the initiator tRNA is not formylated. However, Met-tRNAiMet is excluded from chain elongation by the presence of a 2'-O-ribosyl phosphate modification at position 64 of the initiator tRNA (6).
The translational system in animal mitochondria is thought to be more closely related to that of prokaryotes than to that of the eukaryotic cell cytoplasm (7, 8). This idea is based on the use of fMet-tRNA for initiation, on the antibiotic sensitivity of the ribosomes, and on the ability of the mammalian mitochondrial elongation factors to function on bacterial ribosomes. However, animal mitochondrial protein synthesis has a number of unusual features that distinguish it from other translational systems. In general, mitochondrial tRNAs are shorter than their prokaryotic or eukaryotic cytoplasmic counterparts (59-75 nucleotides in length). They display numerous primary structural differences from "normal" tRNAs. In some cases, they cannot be folded into the typical cloverleaf secondary structure and lack one or more of the invariant or semi-invariant residues found in other tRNAs (9). There are genes for 22 tRNAs in the mammalian mitochondrial genome (10). This number is sufficient to read the altered genetic code found in this organelle. There is a single tRNA for each amino acid except for leucine and serine for which two tRNAs are required. A single gene for tRNAMet is present and no tRNAs appear to be imported into mammalian mitochondria (11). It is unclear how a single tRNAMet species can play the dual roles of an initiator and an elongator tRNA. Translational initiation in mammal mitochondria requires fMet-tRNA for the IF-2mt-dependent binding to ribosomes. However, the unformylated form is required by EF-Tumt for chain elongation (12, 13). Thus, the single tRNAMet gene must give rise to two species of tRNA (fMet-tRNA and Met-tRNA). This process requires a mechanism to adjust the ratio of formylated to non-formylated Met-tRNAs to meet the needs of both initiation and elongation. As a first step toward the investigation of this process, we report here the purification, cloning, and characterization of bovine mitochondrial MTF (MTFmt).
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EXPERIMENTAL PROCEDURES |
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Materials
Folinic acid and CHAPS were purchased from Sigma. [35S]Methionine (37 TBq/mmol) and [14C]methionine (1.85 GBq/mmol) were obtained from Amersham. DEAE-Sepharose fast flow, Mono S (HR5/5), Hi Trap Blue, and Hi Trap Heparin columns were purchased from Pharmacia. An affinity column using an immobilized E. coli tRNA mixture was prepared as described (14).
Buffers
Buffer TG contains 20 mM Tris-HCl (pH 7.6), 1 mM MgCl2, 0.1 mM EDTA, 6 mM -mercaptoethanol, 10% glycerol, 0.1 mM
phenymethylsulfonyl fluoride. Buffer PG contains 20 mM
potassium phosphate (pH 6.8), 10% glycerol, 1 mM
dithiothreitol, 0.1 mM phenymethylsulfonyl fluoride, 0.5%
CHAPS.
Analytical Methods
Protein concentrations were determined by the Bio-Rad protein assay kit using bovine serum albumin as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli (15).
Preparation of Mitochondrial Methionyl-tRNAMet and E. coli Methionyl-tRNAsMet
Mitochondrial Met-tRNA synthetase (MetRSmt) was partially purified from mitochondrial extracts by chromatography on DEAE-Sepharose and ceramic hydroxyapatite (Bio-Rad). E. coli MetRS was partially purified from E. coli extracts by chromatography on DEAE-Sepharose and ceramic hydroxyapatite (Bio-Rad). Mitochondrial tRNAMet was purified by a solid-phase hybridization method using DNA probes complementary to the 30 bases at the 3'-end of the tRNA (16). E. coli tRNAfMet and tRNAmMet were purified as described (17, 18). Further purification was carried out on a 6% native polyacrylamide gel if necessary.
Aminoacylation of mitochondrial tRNAMet was carried out in reaction mixtures (100 µl) containing 100 mM Tris-HCl (pH 8.5), 14 mM Mg(OAc)2, 20 mM KCl, 2 mM dithiothreitol, 4 mM ATP, 1 mM spermine, 20 µM [35S]methionine (200 GBq/mmol), 2-4 µM tRNAMet, and saturating amounts of partially purified MetRSmt. The tRNA was extracted using phenol equilibrated at pH 5.0 and the remaining ATP and methionine were removed on a Hi Trap desalting gel (Pharmacia). Aminoacylation of E. coli tRNA was carried out with [35S] or [14C]methionine as described (19) and the Met-tRNAs were purified as described above.
Purification of Bovine Liver Mitochondrial MTF
The bovine liver mitochondria were prepared as described (20).
About 60 g of mitochondria were resuspended in 240 ml of Buffer TG
containing 0.005 M KCl (TG.005), and disrupted by
sonication using five 20-s bursts at 100 watts followed by 40-s cooling
periods. The homogenate was subjected to centrifugation at 100,000 × g for 180 min. The supernatant fraction (S100) was either
processed immediately or frozen quickly and stored at 70 °C.
Step 1: Chromatography on DEAE-Sepharose-- CHAPS was added to a final concentration of 0.2% (w/v) to all the buffers indicated below. The S100 (5,700 mg) was applied to a 100-ml DEAE-Sepharose fast flow column (17.5 × 2.7 cm) equilibrated with Buffer TG.005, at a flow rate of about 2.0 ml/min. The column was washed by Buffer TG.005 until the absorbance at 280 nm became less than 0.1 and the proteins bound to the column were eluted by a 1.0-liter linear gradient of 5-400 mM KCl in Buffer TG. Fractions (10 ml) were collected at a flow rate of 2.0 ml/min. Fractions containing MTFmt activity were pooled and concentrated by ammonium sulfate precipitation (45-60% saturation). The pellet was then dissolved in Buffer PG and dialyzed against Buffer PG containing 0.15 M KCl (PG.15) for 6 h with two changes of buffer.
Step 2: Chromatography on Mono S--
The sample (1,300 mg) was
applied to a Mono S column (0.5 × 5 cm) equilibrated in Buffer
PG.15 at a flow rate of 0.25 ml/min. The column was then washed with
Buffer PG.15 and developed with a 10-ml linear gradient from 0.15 to
0.4 M KCl in Buffer PG. Fractions (0.25 ml) were collected
at the flow rate of 0.25 ml/min. The fractions with MTFmt
activity were pooled and diluted with Buffer PG until the concentration
of KCl was less than 0.25 M. The sample was immediately
frozen and stored at 70 °C.
Step 3: Chromatography on Hi Trap Blue--
The sample (1.6 mg)
was applied at a flow rate of 0.25 ml/min to a Hi Trap Blue column (1 ml) equilibrated in Buffer PG containing 0.25 M KCl
(PG.25). After washing with Buffer PG.25, bound proteins were eluted
with a 10-ml linear gradient from 0.25 to 0.75 M KCl in
Buffer PG. Fractions (0.25 ml) were collected at the flow rate of 0.25 ml/min. The fractions with MTFmt activity were pooled and
diluted with Buffer PG to decrease the KCl concentration to less than
0.10 M. The sample was then frozen quickly and stored at
70 °C.
Step 4: Chromatography on a Column Carrying Immobilized
tRNA--
E. coli tRNA was immobilized on CNBr-activated
Sepharose 4B (Pharmacia). The partially purified sample (0.24 mg)
containing MTFmt activity was applied to the column
(0.5 × 2.1 cm) which had been equilibrated in Buffer PG
containing 0.1 M KCl (PG.10). The column was developed with
a linear gradient (0.10 to 0.60 M KCl in Buffer PG).
Fractions (0.1 ml) were collected at the flow rate of 0.1 ml/min. The
fractions showing MTFmt activity were diluted with Buffer
PG to reduce the concentration of KCl to less than 0.25 M
and stored at 70 °C.
Step 5: Chromatography on Hi Trap Heparin--
The sample (0.13 mg) was applied to a Hi Trap Heparin column (1 ml) equilibrated with
Buffer PG.25. The column was washed with Buffer PG.25 and developed
with a 10-ml linear gradient from 0.25 to 0.75 M KCl in
Buffer PG. Fractions of 0.25 ml were collected at the flow rate of 0.25 ml/min. Fractions with MTFmt activity were pooled and
dialyzed against buffer containing 20 mM Tris-HCl (pH 7.6),
10 mM KCl, 5 mM MgCl2, 10%
glycerol, 1 mM dithiothreitol, and 0.5% CHAPS. The sample
was divided into small aliquots, fast-frozen, and stored at
70 °C.
Assays of Bovine MTFmt Activity
The assay of the formylation activity was carried out according to Ref. 21 with a slight modification as follows. Reaction mixtures (50 µl) contained 20 mM Tris-HCl (pH 7.6), 10 mM KCl, 5 mM MgCl2, 0.1 mg/ml bovine serum albumin, 0.5% (w/v) CHAPS, 1 mM dithiothreitol, 1 µM E. coli [14C]Met-tRNAfMet, 0.3 mM N10-formyltetrahydrofolate, and the indicated amounts of MTFmt.
Determination of the Amino Acid Sequence of MTFmt
The partially purified sample containing MTFmt was subjected to SDS-PAGE and blotted onto a siliconized glass-fiber membrane (22). The band believed to be MTFmt (based on the correlation between the intensity of this band and the activity of MTFmt) was excised. The amino-terminal sequence was obtained on an Applied Biosystems 477A/120A protein sequencer. The sequences of internal peptides were obtained according to Cleveland et al. (23) with modifications indicated in Ref. 24.
Screening of cDNA Libraries and DNA Sequencing
Approximately 1 × 106 plaques from a bovine
heart cDNA library (Uni-ZAPTM XR, Stratagene) were
screened by hybridization with a putative human MTFmt
cDNA probe labeled by random priming (25). Hybridizations were
carried out at 65 °C with 6 × SSC buffer containing 20 mM NaH2PO4 and 0.4% (w/v) SDS
(26). Positive plaques were isolated and the pBluescript SK() plasmid
clones were excised in vivo according to the manufacturer's
instructions (Stratagene). Plasmid DNA was subjected to
autosequencing using a HITACHI SQ-5500 sequencer (27).
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RESULTS AND DISCUSSION |
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Purification of MTFmt-- The initiation of protein synthesis in mitochondria requires the use of the formylated initiator tRNA (fMet-tRNA). Hence, this organelle must possess a factor equivalent to the bacterial methionyl-tRNA transformylase. When extracts of bovine mitochondria were tested for a factor that could carry out the formylation of E. coli Met-tRNAfMet, a small amount of activity could be detected. The partial purification of this activity (MTFmt) was carried out by successive column chromatography as described under "Experimental Procedures" (Table I).
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Amino Acid Sequence Determination of Peptides Derived from MTFmt and cDNA Cloning-- In order to obtain cDNA clones of MTFmt, partial peptide sequences were determined. MTFmt was first subjected to NH2-terminal Edman degradation. Second, for the determination of internal amino acid sequences, peptides resulting from digestion with endoproteinase V8 were purified by polyacrylamide gel electrophoresis and subjected to Edman degradation. Three peptide sequences were obtained (Table II).
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Characterization of the Sequence of Bovine MTFmt-- The mature form of MTFmt is 357 amino acids in length and has a molecular weight of 40,017. This value is consistent with the molecular weight of the band identified as MFTmt on SDS-PAGE. The amino acid sequence of MTFmt is about 30% identical to the corresponding prokaryotic factors (Fig. 3A) (29-33). It is interesting to note that the sequence of bovine MFTmt is also only 28% identical to that of yeast MTFmt (34) (Table III).
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Characterization of Substrate Specificity of MTFmt-- The initiation of translation in most prokaryotic organisms requires the formylation of the initiator Met-tRNA by MTF. In E. coli, the formyl group is a positive determinant for the specific interaction of IF-2 with the initiator tRNA. It also serves as a negative determinant that nearly eliminates the binding of EF-Tu to the initiator tRNA (5). Bacterial MTF will not formylate the Met-tRNAmMet species used for chain elongation. The strict substrate specificity of E. coli MTF is essential to ensure the accuracy and the efficiency of the initiation process. In contrast to all other systems, animal mitochondria do not contain two distinct methionyl-tRNA species that are used exclusively for the initiation or elongation phases of protein synthesis. Mammalian mitochondria have a single tRNAMet gene which is encoded in the organelle genome (10). There is no evidence that cytoplasmic tRNAs are imported into animal mitochondria (11). Thus, the single tRNAMet gene must, in some unknown manner, give rise to both an initiator tRNA (fMet-tRNA) and an elongator tRNA (Met-tRNA) (35, 19).
The unique presence of a single Met-tRNA species in mammalian mitochondria made it of considerable interest to address the substrate specificity of MTFmt. The kinetic parameters governing the formylation of three native tRNA molecules, bovine mitochondrial Met-tRNA, E. coli Met-tRNAfMet, and E. coli Met-tRNAmMet, were measured (Table IV). The results of these experiments indicated that MTFmt is clearly able to use E. coli Met-tRNAfMet with a Vmax that is about 3-fold higher than that observed with the mitochondrial Met-tRNAMet. The Km observed with the E. coli initiator tRNA is a little over 3-fold higher than with the mitochondrial tRNAMet. The net result is that the relative Vmax/Km for these two tRNAs are essentially the same. Surprisingly, MTFmt was also able to formylate the E. coli elongator Met-tRNAmMet. This tRNA is never a substrate for formylation by the homologous E. coli MTF (38). The Km value observed with Met-tRNAmMet is essentially the same as that observed for E. coli Met-tRNAfMet while the Vmax is about 3-fold lower than that observed with the mitochondrial Met-tRNA. MTFmt clearly does not discriminate between the bacterial initiator and elongator Met-tRNAs. This observation is compatible with the fact that there is a single Met-tRNA species in mammalian mitochondria.
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ACKNOWLEDGEMENTS |
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We are indebted to our previous co-workers, Dr. T. Yokogawa (Gifu University), Dr. C. Takemoto (University of Tokyo), T. Koike (National Institute of Genetics), and Dr. L. Benkowski (University of North Carolina), for their experimental supports and critical discussions. We thank S. Yoshida, Mitsubishikasei Institute of Life Science, for technical assistance in peptide sequence determination.
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FOOTNOTES |
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* This work is supported by a Grant-in-Aid for Scientific Research on Priority Area from Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB004316.
** To whom correspondence should be addressed. Fax: 81-3-5800-6950; E-mail: kw{at}kwl.t.u-tokyo.ac.jp.
1 The abbreviations used are: IF-2, initiation factor-2; PAGE, polyacrylamide gel electrophoresis; MTF, methionyl-tRNA transformylase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; bp, base pair; THF, tetrahydrofuran.
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REFERENCES |
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