Mitochondrial Translation Initiation Research Articles

A Single Mammalian Mitochondrial Translation Initiation Factor Functionally Replaces Two Bacterial Factors

The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2(mt)) complements E. coli containing a deletion of the IF2 gene (E. coli DeltainfB). We find that IF1 is no longer essential in an IF2(mt)-supported E. coli DeltainfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2(mt) substitutes for the function of IF1. Deletion of this insertion from IF2(mt) supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2(mt)) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2.

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3 mt

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3 mt

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3 mt

Mammalian mitochondrial initiation factor 3 (IF3mt) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3mt to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The Kd for the binding of IF3mt to 28S subunits is ∼30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3mt has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (Kd = 1.5 μM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (Kd = 0.12−0.25 μM). IF3mt does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3mt is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3mt from the 28S subunits upon 39S subunit joining.

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNα-induced apoptosis

Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2'-5' oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2'-5' oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNalpha-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNalpha-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNalpha and partially reverses IFNalpha-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt.

Role of the insertion region in mammalian mitochondrial initiation factor 2 in translational initiation

Prokaryotic protein synthesis requires three essential initiation factors (IFs). IF2 promotes the binding of the initiator tRNA to the small subunit of the ribosome. IF1 prevents incorrect initiation and stimulates the activities of IF2 and IF3. The mammalian mitochondrial translation initiation system is known to have two of the traditional translation initiation factors, IF2mt and IF3mt. No mitochondrial homolog of IF1 has been detected biochemically or by database searching. IF2mt has an insertion of 37 amino acids, when aligned with E. coli IF2, that is modeled to interact with the mitochondrial ribosome in the same region where IF1 is known to associate in the prokaryotic ribosome. This observation lead to the idea that the insertion might play the role of IF1 in the mitochondrial system. To study this possibility the insertion in IF2mt was removed and replaced with the truncated homologous region from E. coli IF2 (IF2mt{\Delta}eco). The IF2mt insertion was also incorporated into E. coli IF2 in the same manner as above (IF2eco{ \Delta }mt). When tested on E. coli ribosomes IF2mt{\Delta}eco was about 20 % as active in promoting $[^{35}S]$fMet-tRNA incorporation as IF2mt when tested in the presence of IF1. IF2mt{\Delta}eco was about 30 % as active as IF2mt when assayed without IF1. As predicted, IF1 stimulated initiation complex formation on prokaryotic ribosomes with E. coli IF2 and IF2mt{\Delta}eco, 5 and 4 fold, respectively, but did not stimulate IF2mt whose insertion plays the role of IF1.

Specificity and functional role of mammalian mitochondrial translational initiation factor 3 in initiation of protein synthesis. A comprehensive study of C and N domains with and without linker

Specificity and functional role of mammalian mitochondrial translational initiation factor 3 in initiation of protein synthesis. A comprehensive study of C and N domains with and without linker

Translation Initiation inSaccharomyces cerevisiaeMitochondria: Functional Interactions Among Mitochondrial Ribosomal Protein Rsm28p, Initiation Factor 2, Methionyl-tRNA-Formyltransferase and Novel Protein Rmd9p

Rsm28p is a dispensable component of the mitochondrial ribosomal small subunit in Saccharomyces cerevisiae that is not related to known proteins found in bacteria. It was identified as a dominant suppressor of certain mitochondrial mutations that reduced translation of the COX2 mRNA. To explore further the function of Rsm28p, we isolated mutations in other genes that caused a synthetic respiratory defective phenotype together with rsm28Delta. These mutations identified three nuclear genes: IFM1, which encodes the mitochondrial translation initiation factor 2 (IF2); FMT1, which encodes the methionyl-tRNA-formyltransferase; and RMD9, a gene of unknown function. The observed genetic interactions strongly suggest that the ribosomal protein Rsm28p and Ifm1p (IF2) have similar and partially overlapping functions in yeast mitochondrial translation initiation. Rmd9p, bearing a TAP-tag, was localized to mitochondria and exhibited roughly equal distribution in soluble and membrane-bound fractions. A small fraction of the Rmd9-TAP sedimented together with presumed monosomes, but not with either individual ribosomal subunit. Thus, Rmd9 is not a ribosomal protein, but may be a novel factor associated with initiating monosomes. The poorly respiring rsm28Delta, rmd9-V363I double mutant did not have a strong translation-defective phenotype, suggesting that Rmd9p may function upstream of translation initiation, perhaps at the level of localization of mitochondrially coded mRNAs.

Mitochondrial translation initiation factor 3 gene polymorphism associated with Parkinson's disease

Mitochondrial dysfunction occurs early in late-onset sporadic Parkinson's disease (PD), but the mitochondrial protein network mediating PD pathogenesis is largely unknown. Mutations in the mitochondrial serine-threonine kinase PINK1 have recently been shown to cause the early-onset autosomal recessive PARK6 variant of PD. We have now tested a candidate interactor protein of PINK1, the mitochondrial translation initiation factor 3 (MTIF3) for involvement in PD pathogenesis. In two independent case-control collectives, the c.798C>T polymorphism of the MTIF3 gene showed allelic association with PD, with a maximal significance of p = 0.0073. An altered function of variant MTIF3 may affect the availability of mitochondrial encoded proteins, lead to oxidative stress and create vulnerability for PD.

Nuclear respiratory factor 2 activates transcription of human mitochondrial translation initiation factor 2 gene

We studied the transcriptional regulation of the human mitochondrial translation initiation factor 2 (IF2mt) gene. The minimal promoter region for the human IF2mt gene contains binding sites for Nuclear Respiratory Factor 2 (NRF-2), which is often involved in the transcription of mitochondrial-related genes. Electrophoresis mobility shift assay (EMSA) analyses indicated that NRF-2α/β binds to the IF2mt promoter. Reporter assays, where HEK293T cells were co-transfected with an NRF-2α/β-expressing vector and/or an IF2mt promoter reporter vector, revealed that NRF-2 trans-activates the IF2mt promoter. NRF-2 sites were also found in the promoters of several other mitochondrial translation factors, which suggests NRF-2 may play a key role in the regulation of mitochondrial protein synthesis.

Mutational, structural and functional studies of the mitochondrial translational initiation factor 2 on the mammalian mitochondrial ribosome

The mammalian mitochondrial ribosome translates only 13 gene products from the mitochondrial genome. Initiation of translation of these proteins requires mitochondrial translational initiation factor 2 (IF-2mt), a G-protein. Mutational analysis of the X residue (Cys residue 290) in the NKXD guanine ring binding motif in the GTPase center of IF-2mt yielded significant functional changes. The mutation cysteine to serine (C290S) had very little effect on the function of this protein but the C290A and C290I mutations both resulted in a protein that was unable to fold and subsequently degraded. Interestingly, although the X residue of this motif is not thought to participate in the specific binding and GTPase activity of IF-2mt, mutation to even the most highly conserved residue in IF-2 at the X position, Ile, was detrimental to the protein’s structure and function. IF-2mt is known to specifically bind to the 28 S small subunit of the 55 S mammalian mitochondrial ribosome with nanomolar affinity. CryoEM studies on the 55 S mitochondrial ribosome indicate that the small subunit has a number of interesting features. Proteomic studies have shown that there are 29 proteins in the small subunit only half of which have homologs in bacteria. The binding site of IF-2mt on the 28 S subunit is being examined using dimethyl suberimidate (DMS) to crosslink ribosomal proteins to IF-2mt. The crosslinked proteins will be identified using mass spectrometry. These studies will yield important clues to the structure and mechanism of action of the mammalian mitochondrial ribosome. Funding: NIH GM32734.

Role of the N- and C-terminal extensions on the activity of mammalian mitochondrial translational initiation factor 3

Mammalian mitochondrial translational initiation factor 3 (IF3mt) promotes initiation complex formation on mitochondrial 55S ribosomes in the presence of IF2mt, fMet-tRNA and poly(A,U,G). The mature form of IF3mt is predicted to be 247 residues. Alignment of IF3mt with bacterial IF3 indicates that it has a central region with 20–30% identity to the bacterial factors. Both the N- and C-termini of IF3mt have extensions of ∼30 residues compared with bacterial IF3. To examine the role of the extensions on IF3mt, deletion constructs were prepared in which the N-terminal extension, the C-terminal extension or both extensions were deleted. These truncated derivatives were slightly more active in promoting initiation complex formation than the mature form of IF3mt. Mitochondrial 28S subunits have the ability to bind fMet-tRNA in the absence of mRNA. IF3mt promotes the dissociation of the fMet-tRNA bound in the absence of mRNA. This activity of IF3mt requires the C-terminal extension of this factor. Mitochondrial 28S subunits also bind mRNA independently of fMet-tRNA or added initiation factors. IF3mt has no effect on the formation of these complexes and cannot dissociate them once formed. These observations have lead to a new model for the function of IF3mt in mitochondrial translational initiation.

The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit

Bovine mitochondrial translational initiation factor 2 (IF-2 mt) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III–VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2 mt were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2 mt was strong ( K d∼10–20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2 mt to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2 mt can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2 mt variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2 mt. IF-2 mt contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA(Met) present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA(Met) carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA(Met). In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA(Met). Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA(Met), suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knock-down of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyl-tRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.

Role of the N- and C-terminal extensions on the activity of mammalian mitochondrial translational initiation factor 3

Role of the N- and C-terminal extensions on the activity of mammalian mitochondrial translational initiation factor 3

The human mitochondrial translation initiation factor 2 gene ( MTIF2): transcriptional analysis and identification of a pseudogene

Mitochondrial translation initiation factor 2 ( MTIF2) is nuclear-encoded and functions in mitochondria to initiate the translation of proteins encoded by the mitochondrial genome. To gain insight into mechanisms that regulate MTIF2 gene expression, the genomic copy and the 5′ and 3′ flanking regions of MTIF2 were isolated using a combination of genomic library screening and polymerase chain reaction (PCR). MTIF2 is ∼33.5-kb long and contains 16 exons, confirming data from the Human Genome Project. With RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we mapped the transcription start point in human heart tissue to a cytosine residue 296 bp upstream from the translation initiation site. The region surrounding the transcription start point contains consensus binding sites for transcription factors Sp1, nuclear respiratory factor 2 (NRF-2) and estrogen receptor, while enhancer binding sites were identified upstream. Promoter constructs were prepared in a luciferase reporter vector and transiently transfected into 293T cells. The minimal promoter gave an expression level 3.5× higher than the SV40 control ( P=0.001), while the construct containing the minimal promoter plus the enhancer region gave a 3.8× higher level of expression compared to the control ( P<0.001). We also discovered a pseudogene of MTIF2 and mapped it to chromosome 1p13-12.

Evolution of monoblepharidalean fungi based on complete mitochondrial genome sequences.

We have determined the complete mitochondrial DNA (mtDNA) sequences of three chytridiomycete fungi, Monoblepharella15, Harpochytrium94 and Harpochytrium105. Our phylogenetic analysis based on concatenated mitochondrial protein sequences confirms the placement of Mono blepharella15 together with Harpochytrium spp. and Hyaloraphidium curvatum within the taxonomic order Monoblepharidales, with overwhelming support. These four mtDNA sequences encode the standard fungal mitochondrial gene complement and, like certain other chytridiomycete fungi, encode a reduced complement of 7-9 tRNAs, some of which require 5'-tRNA editing to be functional. Highly conserved sequence elements were identified upstream of almost all protein-coding genes in the mtDNAs of Monoblepharella15 and both Harpochytrium species. Finally, a guanosine residue is conserved upstream of the predicted ATG or GTG start codons of almost every protein-coding gene in these genomes. The appearance of this G residue correlates with the presence of a non-canonical cytosine residue at position 37 in the anticodon loop of the mitochondrial initiator tRNAs. Based on the unorthodox features in these four genomes, we propose that a 4 bp interaction between the CAUC anticodon of these tRNAs and GAUG/GGUG codons is involved in translation initiation in monoblepharidalean mitochondria. Intriguingly, a similar interaction may also be involved in mitochondrial translation initiation in the sea anemone Metridium senile.

Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs

Human mitochondrial translational initiation factor 3 (IF3(mt)) 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. IF3(mt) 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 IF3(mt) was cloned, and the protein was expressed in Escherichia coli in a His-tagged form. The mature form of IF3(mt) has short extensions on the N and C termini surrounding a region homologous to bacterial IF3. The region of IF3(mt) homologous to prokaryotic factors ranges between 21-26% identical to the bacterial proteins. Purified IF3(mt) promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2(mt)), [(35)S]fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA. IF3(mt) shifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation. In addition, the ability of E. 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 IF2(mt) and IF3(mt).

Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs

Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs

Folylpoly-gamma-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity.

A single human gene encodes both mitochondrial and cytosolic isoforms of the enzyme. The major mRNA species in human cells encodes the mitochondrial isoform but alternate translation initiation at a downstream in-frame ATG also generates the cytosolic isoform. Cytosolic FPGS may also be generated by use of alternate transcription initiation start sites 3' to the start ATG of the mitochondrial FPGS. Three additional human FPGS mRNAs differing in exon 1 have been identified. One of these is a major species in HEP-G2 cells and other tissue culture cells, and can encode a protein lacking the first 8 amino acids of cytosolic FPGS. A protein of the predicted size is observed in coupled transcription/translation systems. However, expression of this protein in E. coli does not generate an active enzyme. Mutagenesis studies indicate that Tyr-3 of the missing N terminal residues is required for enzyme activity. The major cellular folate pools are in the cytosol and mitochondria and FPGS activity is normally distributed in both compartments. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, and cells lacking this isozyme are auxotrophic for glycine. Overexpression of cytosolic FPGS does not complement the lack of mitochondrial activity. Cells expressing FPGS activity solely in the mitochondria are glycine prototrophs, but also possess cytosolic folylpolyglutamates and are prototrophic for thymidine and purines, products of cytosolic one carbon metabolism. Although cytosolic folylpolyglutamates cannot enter the mitochondrion, mitochondrial folylpolyglutamates are released intact into the cytosolic compartment. Cellular accumulation of some antifolates and their cytotoxic efficacy is highly responsive to the level of FPGS activity. Polyglutamylation of methotrexate (MTX) has little affect on its affinity for dihydrofolate reductase, its target enzyme, but does affect the cellular accumulation of the drug. The sensitivity of model cells, expressing a range of FPGS activities similar to that observed in leukemia blasts, to MTX varied over four orders of magnitude. MTX toxicity was dependent on cytosolic FPGS activity as this drug does not enter the mitochondria, and cells expressing very high levels of FPGS solely in the mitochondria were resistant to MTX. The cytotoxic efficacy of other folate antagonists that are transported into the mitochondria was enhanced by mitochondrial FPGS activity, even when their loci of inhibition was a cytosolic enzyme. Mitochondrial metabolism of these drugs increased cytosolic drug levels. Compartmentalization of antifolate metabolism has to be considered in evaluating mechanisms for increased drug cytotoxicity and for the development of acquired resistance to these agents.

Differential expression of mitochondrial genes between queens and workers during caste determination in the honeybee Apis mellifera.

The nourishment received by female honeybee larvae determines their differentiation into queens or workers. In this study, we report the first molecular analysis of differences that occur between queens and workers during the caste-determination process. RNA-differential display experiments identified a clone that encodes for a gene that is homologous to the nuclear-encoded mitochondrial translation initiation factor (AmIF-2mt). Semi-quantitative analysis by reverse transcriptase/polymerase chain reaction (RT-PCR) throughout honeybee development detected a higher level of expression of this gene in queen larvae than in worker larvae. Analysis of two other genes encoding mitochondrial proteins, cytochrome oxidase subunit 1 (COX-1; mitochondrial-encoded) and cytochrome c (cyt c; nuclear-encoded) also showed differential expression of these two genes between queens and workers. In particular, the cyt c transcript is more abundant in queen larvae and throughout the metamorphosis of the queen. These results indicate that the higher respiratory rate previously documented in queen larvae is accomplished through a higher level of expression of both nuclear- and mitochondrial-encoded genes for mitochondrial proteins.