Evidence for an Active Role of IF3_mt in the Initiation of Translation in Mammalian Mitochondria

Abstract

Mitochondrial translational initiation factor 3 (IF3mt) is a 29 kDa protein that has N-and C-terminal domains, homologous to prokaryotic IF3, connected by a linker region. The homology domains are preceded and followed by short extensions. No information is currently available on the specific residues in IF3mt important for its activity. Based on homology models of IF3mt, mutations were designed in the N-terminal, C-terminal, and linker domains to identify the functionally important regions. Mutation of residues 170–171, and 175 in the C-terminal domain to alanine resulted in a nearly complete loss of activity in initiation complex formation and in the dissociation of mitochondrial 55S ribosomes. However, these mutated proteins bind to the small (28S) subunit of the mammalian mitochondrial ribosome with Kd values similar to the wild-type factor. These mutations appear to lead to a factor defective in the ability to displace the large (39S) subunit of the ribosome from the 55S monosomes in an active process. Other mutations in the N-terminal domain, the linker region, and the C-terminal domain had little or no effect on the ability of IF3mt to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 247–248 in the C-terminal extension abolished the ability of IF3mt to reduce the binding of fMet-tRNA to the ribosome in the absence of mRNA. The results from this paper suggest that IF3mt plays an active role in initiation of translation.

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

“…In our current model for initiation in mammalian mitochondria (Fig. 5), IF3 mt first interacts with the 55 S ribosome, forming a transient 28 S:IF3 mt :39 S intermediate complex (step 1) (24). This transient complex dissociates into free 28 S subunits bound to IF3 mt and free 39 S subunits (Fig.…”

“…However, in the mitochondrial system, IF3 mt is known to be stimulatory to initiation complex formation using poly(A,U,G) as the mRNA (24). To test whether IF3 mt has the same effect on initiation complex formation using an mRNA construct derived from a naturally occurring mitochondrially encoded mRNA, initiation complex formation assays were performed using ND2 5Ј-AUG, AUG 91 , and 0 AUG mRNAs with increasing amounts of IF3 mt .…”

Creatine maintains intestinal homeostasis and protects against colitis

“…For example, delta(4)-desaturase, sphingolipid 2 (Degs2) regulates biosynthesis of the lipid ceramide, which is a regulator of whole organism metabolism (26)(27)(28). In addition, mitochondrial translation initiation factor 3 (Mtif3), glycine N-acyltransferase (Glyat), and glycerol-3-phosphate acyltransferase-2 (Gpat2) are localized to the mitochondria and presumably exert their functions there (29)(30)(31).…”

The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly

“…In humans, the mtDNA encodes 13 proteins, all subunits of the oxidative phosphorylation (OXPHOS) system enzymes, two ribosomal RNAs (12S and 16S rRNAs) and 22 tRNAs. Mitoribosomes sediment as 55S particles and consist of a 28S small subunit (mt-SSU), formed by a 12S rRNA and ~29 ribosomal proteins (MRPs), and a 39S large subunit (mt-LSU), formed by a 16S rRNA and ~50 MRPs (Christian and Spremulli, 2009; O’Brien, 1971). The three-dimensional structures of the human 39S large subunit at 3.4 Å resolution (Brown et al 2014), of porcine mt-LSU at 4.9 Å and 3.4 Å resolution (Greber et al, 2014) and the 7-Å structure of the 28S small subunit (Kaushal et al, 2014) were recently determined by cryo-electron (cryo-EM) microscopy.…”