Linda Lucy Spremulli

Mammalian mitochondria synthesize polypeptides crucial for energy generation using ribosomes with a number of unique features. These ribosomes are very protein rich and have very truncated ribosomal RNAs. The bulk of the mammalian mitochondrial ribosome is composed of proteins, only about half of which are homologs of ribosomal proteins found in other translational systems. A number of distinctive features are found in these ribosomes. Among these is a gate‐like structure that allows entrance of the primarily leaderless mRNAs that characterize this system. The exit tunnel of the large subunit is also quite unusual and includes a site in which the nascent peptide is visible to solvent prior to the normal exit site. Further, this region of the mitochondrial ribosome is dominated by ribosomal proteins rather than rRNA and is involved in the interaction of the ribosome with the inner membrane where all of the translation products are ultimately located. The proteins of the mitochondrial ribosome appear to play a number of important roles in the cell in addition to their function in protein biosynthesis, including roles in apoptosis and in cell cycle control.

Mitochondrial translational initiation factor 3 (IF3_mt) 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 IF3_mt important for its activity. On the basis of homology models of IF3_mt, 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 K_d values similar to that of 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 IF3_mt to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 247 and 248 in the C-terminal extension abolished the ability of IF3_mt to reduce the level of binding of fMet-tRNA to the ribosome in the absence of mRNA. Our results suggest that IF3_mt plays an active role in initiation of translation.