Mitochondrial Initiation Factor Research Articles

Mitoribosomal small subunit maturation involves formation of initiation-like complexes.

Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.

MtIF3 is locally translated in axons and regulates mitochondrial translation for axonal growth

BackgroundThe establishment and maintenance of functional neural connections relies on appropriate distribution and localization of mitochondria in neurites, as these organelles provide essential energy and metabolites. In particular, mitochondria are transported to axons and support local energy production to maintain energy-demanding neuronal processes including axon branching, growth, and regeneration. Additionally, local protein synthesis is required for structural and functional changes in axons, with nuclear-encoded mitochondrial mRNAs having been found localized in axons. However, it remains unclear whether these mRNAs are locally translated and whether the potential translated mitochondrial proteins are involved in the regulation of mitochondrial functions in axons. Here, we aim to further understand the purpose of such compartmentalization by focusing on the role of mitochondrial initiation factor 3 (mtIF3), whose nuclear-encoded transcripts have been shown to be present in axonal growth cones.ResultsWe demonstrate that brain-derived neurotrophic factor (BDNF) induces local translation of mtIF3 mRNA in axonal growth cones. Subsequently, mtIF3 protein is translocated into axonal mitochondria and promotes mitochondrial translation as assessed by our newly developed bimolecular fluorescence complementation sensor for the assembly of mitochondrial ribosomes. We further show that BDNF-induced axonal growth requires mtIF3-dependent mitochondrial translation in distal axons.ConclusionWe describe a previously unknown function of mitochondrial initiation factor 3 (mtIF3) in axonal protein synthesis and development. These findings provide insight into the way neurons adaptively control mitochondrial physiology and axonal development via local mtIF3 translation.

Muscle denervation reduces mitochondrial biogenesis and mitochondrial translation factor expression in mice

The mitochondrial translation process, in which mitochondrial DNA (mtDNA)-encoded genes are translated into their corresponding proteins, is crucial for mitochondrial function, biogenesis, and integrity. This process is divided into four phases—initiation, elongation, termination, and mitoribosome recycling—which are regulated by specific translation factors, including mitochondrial initiation factor 2 and 3 (mtIF2 and mtIF3), mitochondrial elongation factor Tu, Ts, and G1 (mtEFTu, mtEFTs, and mtEFG1), mitochondrial translational release factor 1-like (mtRF1L), and mitochondrial recycling factor 1 and 2 (mtRRF1 and mtRRF2). Muscle denervation downregulates mitochondrial biomass and induces skeletal muscle atrophy. However, it is unknown whether denervation affects the expression of mitochondrial translation factors in skeletal muscle. In this study, we hypothesized that denervation decreases the expression of mitochondrial translation factors. Therefore, we investigated the effect of muscle denervation on mitochondrial protein and mitochondrial translation factor expression in soleus muscle after surgery. Denervation induced muscle atrophy and activated the ubiquitin-proteasome pathway in soleus muscle. Additionally, muscle denervation decreased the expression of mitochondrial translation factors as well as nuclear DNA and mtDNA-encoded mitochondrial proteins in soleus muscle. Further, a correlation was found between the expression of mitochondrial translation factors and mtDNA-encoded proteins three and seven days after denervation. Taken together, these results demonstrated that the denervation-induced decrease in mitochondrial biogenesis corresponded with changes in mitochondrial translation factors in murine skeletal muscle, providing novel molecular-level insight into the effects of muscle denervation on the mitochondrial translation process.

Initiation Factor 3 is Dispensable For Mitochondrial Translation in Cultured Human Cells

The initiation of protein synthesis in bacteria is ruled by three canonical factors: IF1, IF2, and IF3. This system persists in human mitochondria; however, it functions in a rather different way due to specialization and adaptation to the organellar micro-environment. We focused on human mitochondrial IF3, which was earlier studied in vitro, but no knock-out cellular models have been published up to date. In this work, we generated human HeLa cell lines deficient in the MTIF3 gene and analyzed their mitochondrial function. Despite the lack of IF3mt in these cells, they preserved functional mitochondria capable of oxygen consumption and protein synthesis; however, the translation of ATP6 mRNA was selectively decreased which compromised the assembly of ATP synthase. Together with the analogous results obtained earlier for baker’s yeast mitochondrial IF3, our findings point to a functional divergence of mitochondrial initiation factors from their bacterial ancestors.

Msc6p is required for mitochondrial translation initiation in the absence of formylated Met-tRNAfMet.

Mitochondrial translation normally requires formylation of the initiator tRNA-met, a reaction catalyzed by the enzyme formyltransferase, Fmt1p and MTFMT in Saccharomyces cerevisiae and human mitochondria, respectively. Yeast fmt1 mutants devoid of Fmt1p, however, can synthesize all mitochondrial gene products by initiating translation with a non-formylated methionyl-tRNA. Yeast synthetic respiratory-deficient fmt1 mutants have uncovered several factors suggested to play a role in translation initiation with non-formylated methionyl-tRNA. Here, we present evidence that Msc6p, a member of the pentatricopeptide repeat (PPR) motif family, is another essential factor for mitochondrial translation in fmt1 mutants. The PPR motif is characteristic of RNA-binding proteins found in chloroplasts and plant and fungal mitochondria, and is generally involved in RNA stability and transport. Moreover, in the present study, we show that the respiratory deficiency of fmt1msc6 double mutants can be rescued by overexpression of the yeast mitochondrial initiation factor mIF-2, encoded by IFM1. The role of Msc6p in translational initiation is further supported by pull-down assays showing that it transiently interacts with mIF-2. Altogether, our data indicate that Msc6p is an important factor in mitochondrial translation with an auxiliary function related to the mIF-2-dependent formation of the initiation complex.

Exercise-induced mitochondrial biogenesis coincides with the expression of mitochondrial translation factors in murine skeletal muscle.

The process of mitochondrial translation, in which mitochondrial (mt)DNA‐encoded genes are translated into proteins, is crucial for mitochondrial function and biogenesis. In each phase, a series of mitochondrial translation factors is required for the synthesis of mtDNA‐encoded mitochondrial proteins. Two mitochondrial initiation factors (mtIF2 and mtIF3), three mitochondrial elongation factors (mtEFTu, mtEFTs, and mtEFG1), one mitochondrial release factor (mtRF1L), and two mitochondrial recycling factors (mtRRF1 and mtRRF2) are mitochondrial translation factors that coordinate each translational phase. Exercise increases both nuclear DNA‐ and mtDNA‐encoded mitochondrial proteins, resulting in mitochondrial biogenesis in skeletal muscles. Therefore, mitochondrial translation factors are likely regulated by exercise; however, it is unclear whether exercise affects mitochondrial translation factors in the skeletal muscles. We investigated whether exercise training comprehensively increases this series of mitochondrial translation factors, as well as mtDNA‐encoded proteins, in the skeletal muscle. Mice were randomly assigned to either the sedentary or exercise group and housed in standard cages with or without a running wheel for 1 and 8 weeks. The expression levels of mitochondrial translation factors in the plantaris and soleus muscles were then measured. Exercise training concomitantly upregulated mitochondrial translation factors and mitochondrial proteins in the plantaris muscle. However, in the soleus muscle, these comprehensive upregulations were not detected. These results indicate that exercise‐induced mitochondrial biogenesis coincides with the upregulation of mitochondrial translation factors.

60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3.

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, the ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of the ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with large subunits. In this work, we have exchanged IF3 in Escherichia coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E. coli ribosomes into subunits. Instead, it fixes a state of ribosomes characterized by sedimentation coefficient about 60S which is not a stable structure but rather reflects a shift of dynamic equilibrium between associated and dissociated states of the ribosome. Mitochondria-specific terminal extensions of Aim23p are necessary for “60S state” formation, and molecular modeling results point out that these extensions might stabilize the position of the protein on the bacterial ribosome.

Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM.

Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation1,2. The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals3,4. Furthermore, only one type of methionyl transfer RNA (tRNAMet) is used for both initiation and elongation4,5, necessitating that the initiation factor specifically recognizes the formylated version of tRNAMet (fMet-tRNAMet). Lastly, most mitochondrial mRNAs do not possess 5' leader sequences to promote mRNA binding to the ribosome2. There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNAMet and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.

Fidelity of translation in the presence of mammalian mitochondrial initiation factor 3

Initiation factor 3 (IF3) is a conserved translation factor. Mutations in mitochondrial IF3 (IF3mt) have been implicated in disease pathology. Escherichia coli infCΔ55, compromised for IF3 activity, has provided an excellent heterologous system for IF3mt structure-function analysis. IF3mt allowed promiscuous initiation from AUA, AUU and ACG codons but avoided initiation with initiator tRNAs lacking the conserved 3GC pairs in their anticodon stems. Expression of IF3mt N-terminal domain, or IF3mt devoid of its typical N-, and C-terminal extensions improved fidelity of initiation in E. coli. The observations suggest that the IF3mt terminal extensions relax the fidelity of translational initiation in mitochondria.

Computational Exploration of Structural Hypotheses for an Additional Sequence in a Mammalian Mitochondrial Protein

BackgroundProteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2mt) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2mt to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.ResultsThe present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2mt using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2mt predictions differ from the observed IF2mt cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed.ConclusionsBy hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2mt models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2mt cryo-EM map to get reasonable quasi-atomic IF2mt models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2mt models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine.

Preferential Selection of the 5′-Terminal Start Codon on Leaderless mRNAs by Mammalian Mitochondrial Ribosomes

Mammalian mitochondrial mRNAs are basically leaderless, having few or no untranslated nucleotides prior to the 5'-start codon. We demonstrate here that mammalian mitochondrial 55 S ribosomes preferentially form initiation complexes at a 5'-terminal AUG codon over an internal AUG. The preferential use of the 5'-start codon is also seen on mitochondrial 28 S small subunits, which suggests that mitochondrial translation initiation on leaderless mRNAs does not require the large ribosomal subunit. The selection of the 5'-AUG is dependent on the presence of fMet-tRNA and is enhanced by the presence of the mitochondrial initiation factor IF2(mt). In prokaryotes, IF3 is believed to antagonize initiation on leaderless mRNAs. However, IF3(mt) stimulates initiation complex formation on leaderless mRNAs when tested with 55 S ribosomes. The addition of even a few nucleotides 5' to the AUG codon significantly reduces the efficiency of initiation, highlighting the importance of the leaderless or nearly leaderless nature of mitochondrial mRNAs. In addition, very few initiation complexes could form on a hybrid mRNA construct consisting of tRNA(Met) attached at the 5'-end of a mitochondrial protein-coding sequence. This observation demonstrates that post-transcriptional processing must occur prior to translation in mammalian mitochondria.

The effect of spermine on the initiation of mitochondrial protein synthesis

Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2mt) and mitochondrial initiation factor 3 (IF3mt). This observation indicates that IF2mt and IF3mt, while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3mt dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2mt. The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.

Roles of the N- and C-Terminal Domains of Mammalian Mitochondrial Initiation Factor 3 in Protein Biosynthesis

Bacterial initiation factor 3 (IF3) is organized into N- and C-domains separated by a linker. Mitochondrial IF3 (IF3mt) has a similar domain organization, although both domains have extensions not found in the bacterial factors. Constructs of the N- and C-domains of IF3mt with and without the connecting linker were prepared. The Kd values for the binding of full-length IF3mt and its C-domain with and without the linker to mitochondrial 28S subunits are 30, 60, and 95 nM, respectively, indicating that much of the ribosome binding interactions are mediated by the C-domain. However, the N-domain binds to 28S subunits with only a 10-fold lower affinity than full-length IF3mt. This observation indicates that the N-domain of IF3mt has significant contacts with the protein-rich small subunit of mammalian mitochondrial ribosomes. The linker also plays a role in modulating the interactions between the 28S subunit and the factor; it is not just a physical connector between the two domains. The presence of the two domains and the linker may optimize the overall affinity of IF3mt for the ribosome. These results are in sharp contrast to observations with Escherichia coli IF3. Removal of the N-domain drastically reduces the activity of IF3mt in the dissociation of mitochondrial 55S ribosomes, although the C-domain itself retains some activity. This residual activity depends significantly on the linker region. The N-domain alone has no effect on the dissociation of ribosomes. Full-length IF3mt reduces the binding of fMet–tRNA to the 28S subunit in the absence of mRNA. Both the C-terminal extension and the linker are required for this effect. IF3mt promotes the formation of a binary complex between IF2mt and fMet–tRNA that may play an important role in mitochondrial protein synthesis. Both domains play a role promoting the formation of this complex.

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.

Roles of the N- and C-Terminal Domains of Mammalian Mitochondrial Initiation Factor 3 in Protein Biosynthesis

Roles of the N- and C-Terminal Domains of Mammalian Mitochondrial Initiation Factor 3 in Protein Biosynthesis

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.

The γ-core motif correlates with antimicrobial activity in cysteine-containing kaliocin-1 originating from transferrins

Kaliocin-1 is a 31-residue peptide derived from human lactoferrin, and with antimicrobial properties that recapitulate those of its 611 amino acid parent holoprotein. As kaliocin-1 is a cysteine-stabilized peptide, it was of interest to determine whether it contained a multidimensional γ-core signature recently identified as common to virtually all classes of disulfide-stabilized antimicrobial peptides. Importantly, sequence and structural analyses identified an iteration of this multidimensional antimicrobial signature in kaliocin-1. Further, the γ-core motif was found to be highly conserved in the transferrin family of proteins across the phylogenetic spectrum. Previous studies suggested that the mechanism by which kaliocin-1 exerts anti-candidal efficacy depends on mitochondrial perturbation without cell membrane permeabilization. Interestingly, results of a yeast two-hybrid screening analysis identified an interaction between kaliocin-1 and mitochondrial initiation factor 2 in a Saccharomyces cerevisiae model system. Taken together, these data extend the repertoire of antimicrobial peptides that contain γ-core motifs, and suggest that the motif is conserved within large native as well as antimicrobial peptide subcomponents of transferrin family proteins. Finally, these results substantiate the hypothesis that antimicrobial activity associated with host defense effector proteins containing a γ-core motif may correspond to targets common to fungal mitochondria or their bacterial ancestors.

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.

Mapping Functional Regions of Mitochondrial Initiation Factor 3

Mapping Functional Regions of Mitochondrial Initiation Factor 3

Characterization of the C2 subdomain of yeast mitochondrial initiation factor 2

The COOH-terminal part of the yeast mitochondrial initiation factor 2 (ymIF2), containing the C2 subdomain, was expressed and purified as a histidine-tagged polypeptide of 137 amino acids. Like the recombinant full-length protein, the C2 subdomain binds both formyl - Met - tRNA f Met and unformylated Met - tRNA f Met with only a small preference for the former species. Formation of a binary complex between the C2 subdomain or the full-length ymIF2 and initiator tRNA was also assessed by fluorescence measurements. The binding of coumarin-Met-tRNA f to either protein caused a blue shift of the coumarin emission spectrum and an increase in anisotropy. Full-length ymIF2 is functionally competent in forming an initiation complex and supporting formation of the first peptide bond on Escherichia coli ribosomes. The results demonstrate that ymIF2 has the same domain structure and biochemical properties of a typical IF2 species as found in bacteria or mammalian mitochondria—but with enhanced ability to bind unformylated initiator Met-tRNA.