Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver

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 coliand yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmtformylates 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.

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)(3)(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-tRNA iMet is excluded from chain elongation by the presence of a 2Ј-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 tRNA Met is present and no tRNAs appear to be imported into mammalian mitochondria (11). It is unclear how a single tRNA Met species can play the dual roles of an initiator and an elongator tRNA. Translational initiation in mammal mitochondria requires fMet-tRNA for the IF-2 mt -dependent binding to ribosomes. However, the unformylated form is required by EF-Tu mt for chain elongation (12,13). Thus, the single tRNA Met 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 (MTF mt ).

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-tRNA Met and E. coli Methionyl-tRNAs Met
Mitochondrial Met-tRNA synthetase (MetRS mt ) 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 tRNA Met was purified by a solid-phase hybridization method using DNA probes complementary to the 30 bases at the 3Ј-end of the tRNA (16). E. coli tRNA fMet and tRNA mMet were purified as described (17,18). Further purification was carried out on a 6% native polyacrylamide gel if necessary.
Aminoacylation of mitochondrial tRNA Met was carried out in reaction mixtures (100 l) containing 100 mM Tris-HCl (pH 8.  (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 ϫ g for 180 min. The supernatant fraction (S100) was either processed immediately or frozen quickly and stored at Ϫ70°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 ϫ 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 MTF mt 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 ϫ 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 MTF mt 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 Ϫ70°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 MTF mt 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 Ϫ70°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 MTF mt activity was applied to the column (0.5 ϫ 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 MTF mt activity were diluted with Buffer PG to reduce the concentration of KCl to less than 0.25 M and stored at Ϫ70°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 MTF mt activity were pooled and dialyzed against buffer containing 20 mM Tris-HCl (pH 7.6), 10 mM KCl, 5 mM MgCl 2 , 10% glycerol, 1 mM dithiothreitol, and 0.5% CHAPS. The sample was divided into small aliquots, fast-frozen, and stored at Ϫ70°C.

Assays of Bovine MTF mt Activity
The assay of the formylation activity was carried out according to

Determination of the Amino Acid Sequence of MTF mt
The partially purified sample containing MTF mt was subjected to SDS-PAGE and blotted onto a siliconized glass-fiber membrane (22). The band believed to be MTF mt (based on the correlation between the intensity of this band and the activity of MTF mt ) 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 ϫ 10 6 plaques from a bovine heart cDNA library (Uni-ZAP TM XR, Stratagene) were screened by hybridization with a putative human MTF mt cDNA probe labeled by random priming (25). Hybridizations were carried out at 65°C with 6 ϫ SSC buffer containing 20 mM NaH 2 PO 4 and 0.4% (w/v) SDS (26). Positive plaques were isolated and the pBluescript SK(Ϫ) 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).

RESULTS AND DISCUSSION
Purification of MTF mt -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-tRNA fMet , a small amount of activity could be detected. The partial purification of this activity (MTF mt ) was carried out by successive column chromatography as described under "Experimental Procedures" ( Table I).
The purification scheme resulted in a 2,200-fold purification of MTF mt with an overall yield of about 0.04%. Throughout the purification scheme, the recovery of MTF mt was significantly improved by the addition of the detergent CHAPS to all of the buffers used. This observation suggests that there are hydrophobic patches on MTF mt that lead to the absorption of this factor on the matrices of various resins or that reduce its solubility resulting in substantial losses of activity. Analysis of the partially purified preparation of MTF mt on SDS-PAGE (Fig. 1B) showed the presence of three major polypeptide bands. The band with a molecular mass of about 40,000 daltons was tentatively identified as MTF mt . The intensity of this band correlated with the amount of MTF mt activity observed. In addition, the transformylase would be expected to be about this size. This polypeptide represented about 25% of the protein in the partially purified sample. About 2.5 g of MTF mt were obtained from 2 kg of bovine liver.
Amino Acid Sequence Determination of Peptides Derived from MTF mt and cDNA Cloning-In order to obtain cDNA clones of MTF mt , partial peptide sequences were determined. MTF mt was first subjected to NH 2 -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).
These sequences were then used to search the data bases. The partial sequence of one human cDNA (GenBank number 108908) included the sequence EVVTVPSPSP found as an internal peptide in bovine MTF mt . The nucleotide sequence of this cDNA contained a 400-bp region which was homologous to E. coli MTF. This cDNA clone was, thus, predicted to encode a portion of human MTF mt . Probes prepared from this putative human MTF mt cDNA hybridized with the E. coli MTF gene (data not shown). A bovine heart Uni-ZAP TM XR cDNA library was screened using the human cDNA clone as a probe. Five positive plaques were isolated among 1 ϫ 10 6 plaques, and the  plasmids carrying the cDNA inserts of interest were excised in vivo. The largest clone characterized carried a 1355-bp insert (Fig. 2). Sequence analysis indicated that this clone contained the entire coding region for the mature form of MTF mt (1071 bp) and a portion of a putative mitochondrial import signal (45 bp). The 3Ј-untranslated region was 220 bp in length and contained a conventional polyadenylation signal (AAUAAA) 17 nucleotides upstream of the poly(A) tail (Fig. 2) (28).
Characterization of the Sequence of Bovine MTF mt -The mature form of MTF mt 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 MFT mt on SDS-PAGE. The amino acid sequence of MTF mt is about 30% identical to the corresponding prokaryotic factors (Fig. 3A) (29 -33). It is interesting to note that the sequence of bovine MFT mt is also only 28% identical to that of yeast MTF mt (34) (Table III).
It should be noted that, while the NH 2 -terminal amino acid sequence obtained by peptide sequencing was ASPGWED, the cDNA sequence obtained gave the sequence ASPPWED when translated into the amino acid sequence. This apparent dis-crepancy could arise if there is more than one copy of MTF mt gene in the bovine nuclear genome with slightly different sequences.
The crystal structure of E. coli MTF has recently been determined (35). Analysis of this structure indicates that MTF contains two domains, NH 2 -terminal and COOH-terminal domains. NH 2 -terminal domain carries the tetrahydrofolate (THF)-binding site in which the THF binding motif (SLLP motif) is contained, as well as a Rossman fold (35). The longest stretch of conserved residues among the MTFs from various sources is located in the THF-binding site proposed for E. coli MTF (35) (Fig. 3). The COOH-terminal domain provides a positively charged surface oriented toward the active center of the enzyme and is, presumably, involved in positioning the Met-tRNA substrate for the formylation reaction. The THF binding motif is present as SCLP in bovine MTF mt , and the Phe at position 14, which is assumed to be responsible for the interaction of E. coli MTF with the 3Ј-end of the Met-tRNA (35), is also conserved in the bovine mitochondrial counterpart (Phe-29) (Fig. 3). The COOH-terminal domain of E. coli MTF is Saccharomyces cerevisiae mitochondria (Yeast (mt)), and bovine liver mitochondria (Bovine(mt)). Multiple sequence alignment of MTF polypeptides was performed with the CLUSTAL V program. The positions strictly conserved in the seven compared sequences are marked with an asterisk below the sequence. Positions with conservative replacements are designated by a dot. The THF-binding site containing the THF binding motif (SLLP motif) and the residues thought to interact with the 3Ј-end of tRNA Met in the case of E. coli MTF are conserved (gray box) (19,35). The secondary structure of E. coli MTF is designated with red (␣-helix) and blue (␤-strand) lines above the sequence (35). The region that is speculated to correlate with the unique substrate specificity of bovine MTF mt is shown with bold letters. This region corresponds to the insertion sequence in the Rossman fold which is usually variable in length in the class I aminoacyl-tRNA synthetases (41). characterized by an oligonucleotide binding fold termed the OB fold. The OB fold is formed by two orthogonal sheets consisting of five antiparallel strands folded into a ␤-barrel surrounded by ␣-helices (35). Secondary structure predictions using the Chou-Fasman or Robson methods (36,37) have not able been to clearly identify a similar structure in MTF mt . The Lys at position 207 in the linker region between the two domains of E. coli MTF is thought to be involved in the interaction with the 3Ј-end of the tRNA (19). This residue (Lys-214) has been conserved in the spacer region between the domains of MTF mt (Fig. 3).
Characterization of Substrate Specificity of MTF mt -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-tRNA mMet 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 tRNA Met 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 tRNA Met 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 MTF mt . The kinetic parameters governing the formylation of three native tRNA molecules, bovine mitochondrial Met-tRNA, E. coli Met-tRNA fMet , and E. coli Met-tRNA mMet , were measured (Table IV). The results of these experiments indicated that MTF mt is clearly able to use E. coli Met-tRNA fMet with a V max that is about 3-fold higher than that observed with the mitochondrial Met-tRNA Met . The K m observed with the E. coli initiator tRNA is a little over 3-fold higher than with the mitochondrial tRNA Met . The net result is that the relative V max /K m for these two tRNAs are essentially the same. Surprisingly, MTF mt was also able to formylate the E. coli elongator Met-tRNA mMet . This tRNA is never a substrate for formylation by the homologous E. coli MTF (38). The K m value observed with Met-tRNA mMet is essentially the same as that observed for E. coli Met-tRNA fMet while the V max is about 3-fold lower than that observed with the mitochondrial Met-tRNA. MTF mt 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.
E. coli MTF is known to have some affinity for many different tRNAs that is mediated through an interaction between the 3Ј-end of the acceptor stem of the tRNA and regions surrounding Lys-207 in MTF (19) (Fig. 4). However, only Met-tRNA fMet is a substrate for the actual formylation process. It has been speculated that the formylation reaction is triggered by the melting of the acceptor stem of Met-tRNA fMet which is facilitated in the initiator tRNA by the unstable acceptor stem found in this tRNA (19,39). Several residues in MTF are thought to be involved in this melting process, such as basic residues in the loop I, and Phe-14 (35,40) (Fig. 4). Phe-14 corresponds to Phe-29 in MTF mt , but Arg-42 in the loop I could not be identified clearly in MTF mt . A comparison of the sequences of prokaryotic MTF and yeast MTF mt with the sequence of bovine MTF mt does not provide a clear rationale for the structural basis for the Met-tRNA specificities of these enzymes. However, it should be noted that the region termed the "insertion sequence" in the Rossman fold, which is usually variable in   length in class I aminoacyl-tRNA synthetases (41), is shorter in the case of bovine MTF mt than in prokaryotic MTF (Figs. 3 and  4). The difference in this distance would be expected to place a number of residues in somewhat different positions in MTF mt . This difference might permit the mitochondrial factor to form the transition state with the elongator Met-tRNA easily. Hence it might be able to formylate the elongator Met-tRNA without the necessity for melting the acceptor stem. Further experiments will be designed to evaluate this idea. How the single tRNA Met gene gives rise to both an initiator and an elongator tRNA Met species still remains unknown at this time. One possibility is that the formylation of Met-tRNA converts it from the elongator to the initiator tRNA since formylation increases its affinity for IF-2 mt and diminishes its ability to form a complex with EF-Tu mt . The ratio of formylated to the non-formylated Met-tRNA might be regulated by a competition between MTF mt and EF-Tu mt for Met-RNA. The purification and characterization of MTF mt as described here will allow us to investigate this issue by using purified IF-2 mt and EF-Tu mt . Alternatively, the activity of MTF mt might be regulated by an unknown mechanism in vivo in conjunction with the initiation of mitochondrial protein synthesis. An investigation of this possibility must await the development of a better understanding of the initiation of mitochondrial protein synthesis.