Title: Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs
Abstract: Human mitochondrial translational initiation factor 3 (IF3mt) has been identified from the human expressed sequence tag data base. Using consensus sequences derived from conserved regions of the bacterial IF3, several partially sequenced cDNA clones were identified, and the complete sequence was assembled in silico from overlapping clones. IF3mt is 278 amino acid residues in length. MitoProt II predicts a 97% probability that this protein will be localized in mitochondria and further predicts that the mature protein will be 247 residues in length. The cDNA for the predicted mature form of IF3mt was cloned, and the protein was expressed inEscherichia coli in a His-tagged form. The mature form of IF3mt has short extensions on the N and C termini surrounding a region homologous to bacterial IF3. The region of IF3mt homologous to prokaryotic factors ranges between 21–26% identical to the bacterial proteins. Purified IF3mt promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2mt), [35S]fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA. IF3mtshifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation. In addition, the ability ofE. coli initiation factor 1 to stimulate initiation complex formation on E. coli 70 S and mitochondrial 55 S ribosomes was investigated in the presence of IF2mt and IF3mt. Human mitochondrial translational initiation factor 3 (IF3mt) has been identified from the human expressed sequence tag data base. Using consensus sequences derived from conserved regions of the bacterial IF3, several partially sequenced cDNA clones were identified, and the complete sequence was assembled in silico from overlapping clones. IF3mt is 278 amino acid residues in length. MitoProt II predicts a 97% probability that this protein will be localized in mitochondria and further predicts that the mature protein will be 247 residues in length. The cDNA for the predicted mature form of IF3mt was cloned, and the protein was expressed inEscherichia coli in a His-tagged form. The mature form of IF3mt has short extensions on the N and C termini surrounding a region homologous to bacterial IF3. The region of IF3mt homologous to prokaryotic factors ranges between 21–26% identical to the bacterial proteins. Purified IF3mt promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2mt), [35S]fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA. IF3mtshifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation. In addition, the ability ofE. coli initiation factor 1 to stimulate initiation complex formation on E. coli 70 S and mitochondrial 55 S ribosomes was investigated in the presence of IF2mt and IF3mt. Mammalian mitochondria synthesize 13 polypeptides that are essential for oxidative phosphorylation. These 13 proteins are translated from nine monocistronic and two dicistronic mRNAs with overlapping reading frames (1Anderson S. de Brujin M. Coulson A. Eperon I. Sanger F. Young I. J. Mol. Biol. 1982; 156: 683-717Crossref PubMed Scopus (1193) Google Scholar, 2Wolstenholme D. Wolstenholme D. Jeon K. Mitochondrial Genomes. Academic Press, New York1992: 173-216Google Scholar). The protein-synthesizing system of mammalian mitochondria has a number of interesting features not observed in prokaryotes or the cell cytoplasm (3Pel H. Grivell L. Mol. Biol. Rep. 1994; 19: 183-194Crossref PubMed Scopus (53) Google Scholar). The mRNAs in this organelle have an almost complete lack of 5′- and 3′-untranslated nucleotides. The start codon is generally located within three nucleotides of the 5′ end of the mRNA (1Anderson S. de Brujin M. Coulson A. Eperon I. Sanger F. Young I. J. Mol. Biol. 1982; 156: 683-717Crossref PubMed Scopus (1193) Google Scholar, 4Montoya J. Ojala D. Attardi G. Nature. 1981; 290: 465-470Crossref PubMed Scopus (258) Google Scholar). Thus, mammalian mitochondrial ribosomes do not recognize the start codon using the Shine/Dalgarno interaction between the mRNA and the 16 S rRNA as observed in prokaryotes. Further, this system does not use a cap-binding and scanning mechanism such as observed in the eukaryotic cytoplasm. Three translational initiation factors, IF1, IF2, and IF3, 1The abbreviations used are: IF, initiation factor; IF2mt, mitochondrial IF2; IF3mt, mitochondrial IF3; EST, expressed sequence tag; Ni-NTA, nickel-nitrilotriacetic acid; CoII, cytochrome oxidase subunit II; N domain, N-terminal domain; C domain, C-terminal domain. are required for the initiation of protein synthesis in bacteria (5Van Knippenberg P. Hill W. Dahlberg A. Garrett R. Moore P. Schlessinger D. Warner J. The Ribosome: Structure, Function and Evolution. American Society for Microbiology, Washington, D.C.1990: 265-274Google Scholar, 6Gualerzi C. Pon C. Biochemistry. 1990; 29: 5881-5889Crossref PubMed Scopus (405) Google Scholar, 7Gualerzi C.O. Brandi L. Caserta E. Teana A. Spurio R. Tomsic J. Pon C.L. Garrett R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. American Society for Microbiology, Washington, D.C.2000: 477-494Google Scholar). Prior to the present report, the homolog of only one of these factors, IF2mt, had been identified, cloned, and characterized in mammalian mitochondria (8Liao H.-X. Spremulli L.L. J. Biol. Chem. 1991; 266: 20714-20719Abstract Full Text PDF PubMed Google Scholar, 9Liao H.-X. Spremulli L.L. J. Biol. Chem. 1990; 265: 13618-13622Abstract Full Text PDF PubMed Google Scholar, 10Ma L. Spremulli L.L. J. Biol. Chem. 1995; 270: 1859-1865Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 11Ma J. Farwell M. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1261: 321-324Crossref PubMed Scopus (20) Google Scholar, 12Ma J. Spremulli L.L. J. Biol. Chem. 1996; 271: 5805-5811Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Similar to its prokaryotic counterpart, IF2mt promotes the binding of fMet-tRNA to the small subunit of mitochondrial ribosomes in response to synthetic polynucleotides such as poly(A,U,G). The current report describes the identification and initial characterization of the mammalian mitochondrial factor equivalent to IF3. In prokaryotes IF3 has a number of roles in the initiation of protein synthesis. IF3 binds to the 30 S subunit and inhibits its association with the 50 S subunit, thus ensuring a supply of 30 S subunits for initiation (13Dottavio-Martin D. Suttle D.P. Ravel J.M. FEBS Lett. 1979; 97: 105-110Crossref PubMed Scopus (15) Google Scholar, 14Paci M. Pon C. Lammi M. Gualerzi C. J. Biol. Chem. 1984; 259: 9628-9634Abstract Full Text PDF PubMed Google Scholar). IF3 also promotes an adjustment of the position of the mRNA on the 30 S subunit facilitating codon-anticodon interactions between the AUG codon and fMet-tRNA in the P site (15Teana A. Gualerzi C. Brimacombe R. RNA. 1995; 1: 772-782PubMed Google Scholar, 16Hartz D. Binkley J. Hollingsworth T. Gold L. Genes Dev. 1990; 4: 1790-1800Crossref PubMed Scopus (109) Google Scholar, 17Sussman J. Simons E. Simons R. Mol. Microbiol. 1996; 21: 347-360Crossref PubMed Scopus (110) Google Scholar, 18Canonaco M. Gualerzi C. Pon C. Eur. J. Biochem. 1989; 182: 501-506Crossref PubMed Scopus (37) Google Scholar). IF3 acts to switch the decoding preference of the small ribosomal subunit from elongator tRNAs to the initiator tRNA in the P site, thus playing a proofreading role in initiation (19Hartz D. McPheeters D. Gold L. Genes Dev. 1989; 3: 1899-1912Crossref PubMed Scopus (192) Google Scholar, 20Berkhout B. van der Laken C.J. van Knippenberg P.H. Biochim. Biophys. Acta. 1986; 866: 144-153Crossref PubMed Scopus (19) Google Scholar, 21Shapkina T.G. Dolan M.A. Babin P. Wollenzien P. J. Mol. Biol. 2000; 299: 615-628Crossref PubMed Scopus (29) Google Scholar). IF3 is a small protein of 180 amino acids that folds into two distinct domains separated by a long flexible linker. The C-terminal domain is thought to carry out most of the direct functions of this factor, whereas the N-terminal domain stabilizes the interaction of IF3 with the 30 S subunit (22Petrelli D. LaTeana A. Garofalo C. Spurio R. Pon C.L. Gualerzi C.O. EMBO J. 2001; 20: 4560-4569Crossref PubMed Scopus (95) Google Scholar). No factor equivalent to IF1 has been observed in the mitochondria from any system nor can an EST for this protein be identified in the human EST data bases. A gene for IF1 is, however, apparent in many chloroplast genomes. This small protein (less than 90 residues) binds to the 30 S subunit around helix 44 in the region that will become the A site (23Carter A.P. Clemons J. Brodersen D.E. Morgan-Warren R.J. Hartsch T. Wimberly B.T. Ramakrishnan V. Science. 2001; 291: 498-501Crossref PubMed Scopus (309) Google Scholar). By binding to this site, IF1 is postulated to prevent accidental initiation from the A site and to promote the correct positioning of fMet-tRNA in the P site (24Moazed D. Samaha R.R. Gualerzi C. Noller H.F. J. Mol. Biol. 1995; 248: 207-210PubMed Google Scholar, 25Brock S. Skaradkiewicz K. Sprinzl M. Mol. Microbiol. 1998; 29: 409-417Crossref PubMed Scopus (64) Google Scholar). In the current report, IF3mt has been identified and characterized, and the effects of bacterial IF1 on the function of IF2mt and IF3mt have been investigated. Bovine mitochondria and 55 S ribosomes were prepared as described previously (26Matthews D.E. Hessler R.A. Denslow N.D. Edwards J.S. O'Brien T.W. J. Biol. Chem. 1982; 257: 8788-8794Abstract Full Text PDF PubMed Google Scholar). Escherichia coli ribosomes were prepared as described (27Graves M. Breitenberger C. Spremulli L.L. Arch. Biochem. Biophys. 1980; 204: 444-454Crossref PubMed Scopus (26) Google Scholar, 28Graves M. Spremulli L.L. Arch. Biochem. Biophys. 1983; 222: 192-199Crossref PubMed Scopus (22) Google Scholar), and tight couples were collected from a sucrose gradient in the presence of 5 mm Mg2+ (29Hapke B. Noll H. J. Mol. Biol. 1976; 105: 97-109Crossref PubMed Scopus (72) Google Scholar). Bovine IF2mt, yeast [35S]fMet-tRNA, and E. coli initiation factors were prepared as described (12Ma J. Spremulli L.L. J. Biol. Chem. 1996; 271: 5805-5811Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 28Graves M. Spremulli L.L. Arch. Biochem. Biophys. 1983; 222: 192-199Crossref PubMed Scopus (22) Google Scholar).E. coli IF2 was also prepared from an expression construct providing a mixture of the α and β forms of IF2 (kindly provided by Angela Coursey, University of North Carolina). The genes for E. coli IF3 and IF1 (kindly provided by Drs. Roberto Spurio and Claudio Gualerzi, University of Camerino, Italy, and Dr. Rebecca Alexander, Wake Forest University, respectively) were also amplified by PCR and cloned into pET-21(c). The constructs carryingE. coli IF3 and IF1 were transformed into an E. coli BL21(DE3) strain that also carried the plasmid pArgU218 (kindly provided by Dr. Yamada, Mitsubishi Chemical Corp., Yokohama, Japan). The His-tagged forms of the E. coli initiation factors were purified on Ni-NTA resins as described below. EST and genomic data base searches for human IF3mtwere performed using BLAST (National Center for Biotechnology Information) and the sequences of various prokaryotic IF3s as virtual probes (30Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nuc. Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). Sequence analysis was done using the GCG DNA analysis software package (Wisconsin Package, version 10, Genetics Computer Group, Madison WI), Vector NTI (Informax Inc.), and Biology WorkBench 3.2. The results were displayed using BOXSHADE (written by K. Hofmann and M. Baron). Prediction of the cleavage sites for the mitochondrial signal sequence was carried out using PSort and MitoProt II (31Nakai K. Kanehisa M. Genomics. 1992; 14: 897-911Crossref PubMed Scopus (1368) Google Scholar, 32Claros M.G. Vincens P. Eur. J. Biochem. 1996; 241: 770-786Crossref Scopus (1386) Google Scholar). Protein secondary and tertiary structures were predicted using Internet-based software, PHDsec and SWISS-Model, respectively (33Rost B. Sander C. Schneider R. Comput. Appl. Sci. 1994; 10: 53-60PubMed Google Scholar,34Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar). A full-length cDNA clone in vector pT7T3D-Pac carrying the human mitochondrial IF3mt cDNA was obtained from the American Culture Type Collection (number 526483). The region predicted to be present in the mature form of human IF3mt(residues 32–278) was amplified by PCR using the full-length cDNA as a template. The portion of the IF3mt cDNA predicted to correspond to the mature protein was cloned between theNdeI and XhoI sites of pET-21(c) using the forward primer 5′-CGCGGATCCAATTCATATGGCTGCTTTTTCT-3′ and the reverse primer 5′-CGCGGATCCGCTCGAGCTGATGCAGAACAT-3′. This vector provides a His tag at the C terminus. The construct carrying the human IF3mt was transformed into E. coli BL21(DE3) carrying the plasmid pArgU218 (Dr. Yamada, Mitsubishi Chemical Corp., Yokohama, Japan), which provides the gene for the isoacceptor of tRNAArg recognizing the AGA and AGG codons. Induction of IF3mt with 50 μm isopropylthiogalactoside was carried out for 5 h at 37 °C after the cell density had reached 0.5 at OD600. The cells were harvested by low speed centrifugation, and IF3mt was purified through a Ni-NTA column as described (35Woriax V. Burkhart W. Spremulli L.L. Biochim. Biophys. Acta. 1995; 1264: 347-356Crossref PubMed Scopus (78) Google Scholar). Protein concentrations were determined by the Micro-Bradford method (Bio-Rad). Because of the presence of the 19-kDa form of IF3mt found in the Ni-NTA column preparations, a second step of purification was carried out using high performance liquid chromatography. In this procedure, the partially purified IF3mt preparation prepared from 2 liters of cell culture (2 mg/ml, 6 mg) was dialyzed against 100-fold excess of Buffer A (20 mm HEPES-KOH, pH 7.6, 10 mmMg2Cl, 6 mm β-mercaptoethanol, 225 mm KCl, and 10% glycerol) for 1.5 h. The dialyzed sample was applied at a flow rate of 0.5 ml/min to a TSKgel SP-5PW column (7.5 × 75 mm, TosoHas Inc., Japan) that had been equilibrated in Buffer A except that the KCl was adjusted to 240 mm. The column was washed until the absorbance at 280 nm returned to base line. The column was then developed with a linear gradient (50 ml) from 0.24 to 0.30 m KCl in Buffer A at a flow rate of 0.5 ml/min. The fractions (0.5 ml) were collected. The fractions containing IF3mt and its major degradation product were pooled separately and fast-frozen in a dry ice isopropyl alcohol bath and stored at −70 °C. The N-terminal sequences of the expressed forms of IF3mt were determined using a Perkin Elmer/ABI Procise model 492 protein/peptide sequencer. The previously described clone carrying the bovine cytochrome oxidase subunit II (CoII) gene (36Liao H.-X. Spremulli L.L. J. Biol. Chem. 1989; 264: 7518-7522Abstract Full Text PDF PubMed Google Scholar) was modified to provide a sequence of 30 A residues at the 3′ end. The transcript prepared from this vector mimics mitochondrial mRNAs produced in vivo, which generally have poly(A) stretches up to 70 residues added following transcription and processing (37Kisselev O.I. Gaitskhoki V.S. Neifakh S.A. Nucleic Acids Res. 1977; 4: 4411-4424Crossref PubMed Scopus (5) Google Scholar). In vitro transcripts were prepared as described previously (36Liao H.-X. Spremulli L.L. J. Biol. Chem. 1989; 264: 7518-7522Abstract Full Text PDF PubMed Google Scholar). The activity of IF3mt in promoting initiation complex formation withE. coli and mitochondrial ribosomes was assayed using conditions basically described previously (8Liao H.-X. Spremulli L.L. J. Biol. Chem. 1991; 266: 20714-20719Abstract Full Text PDF PubMed Google Scholar, 9Liao H.-X. Spremulli L.L. J. Biol. Chem. 1990; 265: 13618-13622Abstract Full Text PDF PubMed Google Scholar). Reaction mixtures (100 μl) contained 0.5 OD260 units of E. coli70 S or 0.075–0.15 OD260 units of mitochondrial 55 S tight couples, 12.5 μg of poly(A,U,G), or 10 pmol of the CoII mRNA, 3.8 pmol of [35S]fMet-tRNA, and the indicated amounts of various initiation factors. All of the initiation complex formation assays were incubated at 37 °C for 15 min and analyzed as described (8Liao H.-X. Spremulli L.L. J. Biol. Chem. 1991; 266: 20714-20719Abstract Full Text PDF PubMed Google Scholar). The reaction mixtures (100 μl) were prepared containing 25 mm Tris-HCl, pH 7.6, 2 mmMg2+, 100 mm KCl, 0.5 mm EDTA, 1 mm dithiothreitol, 5% glycerol, 0.2 OD260units of 55 S ribosomes, and variable amounts of IF3mt(0–1.72 μg). The reactions were incubated for 15 min at 37 °C. The Mg2+ concentration was then adjusted to 7 mm by the addition of 2.5 μl of 0.1 mMgCl2, and the samples were analyzed for 28, 39, and 55 S particles on 10–30% (w/v) linear sucrose gradients prepared in the buffer described above containing 7 mm Mg2+ and analyzed as described previously (38Spremulli L.L. Kraus B. Biochem. Biophys. Res. Commun. 1987; 147: 1077-1081Crossref PubMed Scopus (9) Google Scholar). Although the mammalian mitochondrial ribosome has a low percentage of rRNA and a high protein content compared with bacterial ribosomes, portions of the rRNA where IF3 is thought to bind are present (39Dallas A. Noller H.F. Mol. Cell. 2001; 8: 855-864Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Further, the ribosomal proteins with which IF3 interacts (S7, S11, and S18) have homologs in the 28 S subunit. Hence, it was logical to postulate that mammalian mitochondria contain a homolog of bacterial IF3. Probing the human ESTs with the amino acid sequence of E. coli or most other IF3 species fails to provide any convincing evidence for a mammalian mitochondrial homolog of IF3. However, extensive data base searches with the sequences of IF3 from the Mycoplasma and the IF3 homology domain of Euglena gracilis chloroplast IF3 provide a hit in both the human and mouse EST data bases. The sequence detected by this search encodes a 278-amino acid protein (Fig. 1 A). MitoProt II gives this protein a 97% probability to be localized in mitochondria and predicts that the mature protein will be 247 residues in length. The mature form of IF3mt is predicted to have an N-terminal extension of about 30 residues (Fig. 1, A andB) that can form a coiled region followed by an α-helix. An N-terminal extension of about 150 residues has been noted onE. gracilis chloroplast IF3 (IF3chl) (40Lin Q., Ma, L. Burkhart W. Spremulli L.L. J. Biol. Chem. 1994; 269: 9436-9444Abstract Full Text PDF PubMed Google Scholar). IF3mt also has a C-terminal extension just over 30 residues long. Overall, it is quite hydrophilic and highly charged having nine acidic and five basic residues. The C-terminal extension, like the N-terminal extension, is predicted to have significant helical content. A 63-residue acidic C-terminal extension on E. gracilis IF3chl has been shown to reduce the activity on the chloroplast factor in initiation complex formation and may serve as a potential regulatory region (41Yu N.-J. Spremulli L.L. J. Biol. Chem. 1998; 273: 3871-3877Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar).Figure 1Sequence and domain organization of IF3mt. A, domain organization of prokaryotic and mitochondrial IF3. B, amino acid sequence of human IF3mt and alignment with its prokaryotic homologs from B. stearothermophilus (B.st) and E. coli. The predicted site of cleavage following import into mitochondria is indicated by the arrow (→). The location of the proteolytic cleavage observed in a portion of the factor during expression in E. coli is indicated by the arrow(↑). The asterisks indicate residues implicated in the binding of bacterial IF3 to 30 S subunits. C, alignment of the amino acid sequence of human IF3mt with its homologs from Bos taurus (Bovine), Mus musculus(Mouse), F. rubripes (Fugu), andD. melanogaster (Drosophila). The alignment was done with the CLUSTALW program in Biology Workbench, and the results are displayed in BOXSHADE. The full sequence of the F. rubripes IF3mt is not shown for convenience.D, alignment of human IF3mt with the putative IF3mt from S. pombe.View Large Image Figure ViewerDownload (PPT) Alignment of IF3mt with prokaryotic and chloroplast IF3 indicates that the mitochondrial factor has diverged considerably from other IF3s (Table I). Overall, it has only 20.8% identity to E. coli IF3, which explains the failure of data base searches with the sequence of E. coliIF3 to locate the corresponding mitochondrial factor. IF3mtis 25.9% and 23.7% identical to Bacillus stearothermophilus IF3 and to E. gracilis chloroplast IF3, respectively (40Lin Q., Ma, L. Burkhart W. Spremulli L.L. J. Biol. Chem. 1994; 269: 9436-9444Abstract Full Text PDF PubMed Google Scholar). Alignment of the sequence of IF3mtwith the bacterial factors indicates that regions of identity are rather scattered throughout the structure (Fig. 1 B). Residues that are responsible for the binding of IF3 to the small subunit are thought to be located primarily in the C-terminal domain. Crystallography experiments place the C-terminal domain of IF3 on the solvent side of the platform on the 30 S subunit (42Pioletti M. Schlunzen F. Harms J. Zarivach R. Gluhmann M. Avila H. Bashan A. Bartels H. Auerbach T. Jacobi C. Hartsch T. Yonath A. Franceschi F. EMBO J. 2001; 20: 1829-1839Crossref PubMed Scopus (436) Google Scholar), whereas cryo electron microscopy and footprinting suggest that it is located on the interface side (39Dallas A. Noller H.F. Mol. Cell. 2001; 8: 855-864Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 43McCutcheon J. Agrawal R. Philips S.M. Grassucci R. Gerchman S. Clemons W.M. Ramakrishnan V. Frank J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4301-4306Crossref PubMed Scopus (114) Google Scholar). Important regions of IF3 include residues 99–116, 127–137, 145–155, and 168 (E. coli numbering) as indicated by NMR experiments, mutagenesis, and structural studies (39Dallas A. Noller H.F. Mol. Cell. 2001; 8: 855-864Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 42Pioletti M. Schlunzen F. Harms J. Zarivach R. Gluhmann M. Avila H. Bashan A. Bartels H. Auerbach T. Jacobi C. Hartsch T. Yonath A. Franceschi F. EMBO J. 2001; 20: 1829-1839Crossref PubMed Scopus (436) Google Scholar, 44Sette M. Spurio R. VanTilborg P. Gualerzi C. Boelens R. RNA. 1999; 5: 82-92Crossref PubMed Scopus (38) Google Scholar). A number of the residues in these regions are conserved or are conservative replacements in the C-terminal domain of human IF3mt.Table IPercentage of identity of human mitochondrial IF3 homologsOrganismIdentityLengthAccession number%B. stearothermophilus25.9172P03000Mycoplasma genitalium24.6184P47438Mycoplasma pneumoniae21.5201NP_109803E. coli20.8180P02999E. gracilis (chloroplast)23.7538L23760A. thaialana (chloroplast)24.4312NP_179984S. pombe aPutative mitochondrial initiation factor 3.20.9233T39948S. cerevisiae aPutative mitochondrial initiation factor 3.16.2370NP_011356B. taurus(mitochondrial)69.5273cDNAsbcDNA sequences encoding for bovine IF3mt: AW658739 (nucleotides 316–582, aa residues 1–89), AW445348 (nucleotides 1–408, aa residues 26–161), BM106922(nucleotides 270–112, aa residues 149–201), and BM255983 (nucleotides 79–360, aa residues 180–273).Mouse (mitochondrial)66.0276BAB28438Drosophila (mitochondrial)22.6226AAF58534F. rubripes(mitochondrial)30.9362JGI_17846The percentage identity between human IF3mt and the bacterial factors is reported for the homology region only.a Putative mitochondrial initiation factor 3.b cDNA sequences encoding for bovine IF3mt: AW658739 (nucleotides 316–582, aa residues 1–89), AW445348 (nucleotides 1–408, aa residues 26–161), BM106922(nucleotides 270–112, aa residues 149–201), and BM255983 (nucleotides 79–360, aa residues 180–273). Open table in a new tab The percentage identity between human IF3mt and the bacterial factors is reported for the homology region only. One of the major roles of prokaryotic IF3 is the discrimination of the initiation codon (AUG or occasionally GUG or UUG) from other codons. This property can be observed in the isolated C-terminal domain of bacterial IF3 (22Petrelli D. LaTeana A. Garofalo C. Spurio R. Pon C.L. Gualerzi C.O. EMBO J. 2001; 20: 4560-4569Crossref PubMed Scopus (95) Google Scholar) but is strongly affected by conserved residues in the linker region (17Sussman J. Simons E. Simons R. Mol. Microbiol. 1996; 21: 347-360Crossref PubMed Scopus (110) Google Scholar, 45de Cock E. Springer M. Dardel F. Mol. Microbiol. 1999; 32: 193-202Crossref PubMed Scopus (31) Google Scholar, 46Sacerdot C. de Cock E. Engst K. Graffe M. Dardel F. Springer M. J. Mol. Biol. 1999; 288: 803-810Crossref PubMed Scopus (22) Google Scholar). Interestingly, these highly conserved residues, Tyr70, Gly71, and Tyr75, in prokaryotic IF3s are not conserved in human IF3mt. In mammalian mitochondria, both AUG and AUA (normally an isoleucine codon) serve as initiation codons. Consequently, the proofreading properties of human IF3mt could be quite different from those of the bacterial factors. Analysis of the mouse and bovine EST data bases indicates the presence of mammalian homologs of human IF3mt that are 65–70% identical to the human factor (Table I and Fig. 1 C). In addition, BLAST searches indicate the presence of IF3mt inFugu rubripes (puffer fish) and in Drosophila melanogaster (Table I and Fig. 1 C). No homolog can be detected in Caenorhabditis elegans. It is quite reasonable to assume that this organism will have a corresponding factor. However, IF3mt does not appear to be highly conserved throughout the animal kingdom, and it may be difficult to detect using BLAST searches. The IF3mt species detected in animals generally have N- and C-terminal extensions that surround a central section that has homology to the bacterial IF3s. The N-terminal extension on puffer fish IF3mt is considerably longer than that observed on the mammalian factors (Fig. 1 C). D. melanogasterIF3mt has a very short N-terminal extension. The mammalian and puffer fish IF3mts all have C-terminal extensions of around 30 residues compared with the bacterial IF3s. However, D. melanogaster IF3mt again lacks a significant extension at the C terminus. The linker regions of the mitochondrial factors are charged as observed for the prokaryotic proteins. Two potential homologs of IF3mt can be found inArabidopsis thaliana. One of these genes probably encodes the chloroplast factor, whereas the other encodes the mitochondrial factor. These two forms (NP-179984 and NP-174696) differ considerably in length (312 and 574 residues, respectively). Alignments of these two species with E. gracilis chloroplast IF3 suggests that the shorter form is more likely to encode the chloroplast factor based on the percentage of identity. However, the shorter form also has a higher percentage of identity to human IF3mt than the longer form, making it difficult to assign these two species clearly to one or the other compartments. Cyberprobing of the recently completed genome ofSchizosaccharomyces pombe allows the tentative identification of IF3mt in this organism (47Wood V. Gwilliam R. Rajandream M.A. Lyne M. Lyne R. Stewart A. Sgouros J. Peat N. Hayles J. Baker S. Basham D. Bowman S. Brooks K. Brown D. Brown S. Chillingworth T. Churcher C. Collins M. Connor R. Cronin A. Davis P. Feltwell T. Fraser A. Gentles S. Goble A. Hamlin N. Harris D. Hidalgo J. Hodgson G. Holroyd S. Hornsby T. Howarth S. Huckle E.J. Hunt S. Jagels K. James K. Jones L. Jones M. Leather S. McDonald S. McLean J. Mooney P. Moule S. Mungall K. Murphy L. Niblett D. Odell C. Oliver K. O'Neil S. Pearson D. Quail M.A. Rabbinowitsch E. Rutherford K. Rutter S. Saunders D. Seeger K. Sharp S. Skelton J. Simmonds M. Squares R. Squares S. Stevens K. Taylor K. Taylor R.G. Tivey A. Walsh S. Warren T. Whitehead S. Woodward J. Volckaert G. Aert R. Robben J. Grymonprez B. Weltjens I. Vanstreels E. Rieger M. Schafer M. Muller-Auer S. Gabel C. Fuchs M. Fritzc C. Holzer E. Moestl D. Hilbert H. Borzym K. Langer I. Beck A. Lehrach H. Reinhardt R. Pohl T.M. Eger P. Zimmermann W. Wedler H. Wambutt R. Purnelle B. Goffeau A. Cadieu E. Dreano S. Gloux S. Lelaure V. Mottier S. Galibert F. Aves S.J. Xiang Z. Hunt C. Moore K. Hurst S.M. Lucas M. Rochet M. Gaillardin C. Tallada V.A. Garzon A. Thode G. Daga R.R. Cruzado L. Jimenez J. Sanchez M. del Rey F. Benito J. Dominguez A. Revuelta J.L. Moreno S. Armstrong J. Forsburg S.L. Cerrutti L. Lowe T. McCombie W.R. Paulsen I. Potashkin J. Shpakovski G.V. Ussery D. Barrell B.G. Nurse P. Nature. 2002; 415: 871-880Crossref PubMed Scopus (1251) Google Scholar). The protein encoded by this gene is 20.9% identical to human IF3mt and 25% identical to E. coli IF3 (Table I and Fig.1 D). If the predicted import signal is cleaved from S. pombe IF3mt, no N-terminal extension would be present. IF3mt from S. pombe does not have any observable C-terminal extension. Searching the genome ofSaccharomyces cerevisiae with the sequence of human IF3mt fails to indicate the presence of a yeast homolog. However, a possible candidate can be detected that is 23.9% identical to the S. pombe factor over the IF3 region. The S. cerevisiae protein is considerably longer than traditional IF3s, and its classificati