Mammalian Mitochondrial Initiation Factor Research Articles

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.

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.

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.

Mammalian Mitochondrial Initiation Factor 2 Supports Yeast Mitochondrial Translation without Formylated Initiator tRNA

Initiation of protein synthesis in mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl-tRNA (fMet-tRNAfMet) in a process involving initiation factor 2 (IF2). However, yeast strains disrupted at the FMT1 locus, encoding mitochondrial methionyl-tRNA formyltransferase, lack detectable fMet-tRNAfMet but exhibit normal mitochondrial function as evidenced by normal growth on non-fermentable carbon sources. Here we show that mitochondrial translation products in Saccharomyces cerevisiae were synthesized in the absence of formylated initiator tRNA. ifm1 mutants, lacking the mitochondrial initiation factor 2 (mIF2), are unable to respire, indicative of defective mitochondrial protein synthesis, but their respiratory defect could be complemented by plasmid-borne copies of either the yeast IFM1 gene or a cDNA encoding bovine mIF2. Moreover, the bovine mIF2 sustained normal respiration in ifm1 fmt1 double mutants. Bovine mIF2 supported the same pattern of mitochondrial translation products as yeast mIF2, and the pattern did not change in cells lacking formylated Met-tRNAfMet. Mutant yeast lacking any mIF2 retained the ability to synthesize low levels of a subset of mitochondrially encoded proteins. The ifm1 null mutant was used to analyze the domain structure of yeast mIF2. Contrary to a previous report, the C terminus of yeast mIF2 is required for its function in vivo, whereas the N-terminal domain could be deleted. Our results indicate that formylation of initiator methionyl-tRNA is not required for mitochondrial protein synthesis. The ability of bovine mIF2 to support mitochondrial translation in the yeast fmt1 mutant suggests that this phenomenon may extend to mammalian mitochondria as well.