The mitochondrial ribosome is responsible for the biosynthesis of protein components crucial to the generation of ATP in the eukaryotic cell. Because the protein:RNA ratio in the mitochondrial ribosome (approximately 69:approximately 31) is the inverse of that of its prokaryotic counterpart (approximately 33:approximately 67), it was thought that the additional and/or larger proteins of the mitochondrial ribosome must compensate for the shortened rRNAs. Here, we present a three-dimensional cryo-electron microscopic map of the mammalian mitochondrial 55S ribosome carrying a tRNA at its P site, and we find that instead, many of the proteins occupy new positions in the ribosome. Furthermore, unlike cytoplasmic ribosomes, the mitochondrial ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided.

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 .Mammalian mitochondria synthesize 13 polypeptides that are essential for oxidative phosphorylation. These 13 proteins are translated from nine monocistronic and two dicistronic mRNAs with overlapping reading frames (1, 2). The proteinsynthesizing system of mammalian mitochondria has a number of interesting features not observed in prokaryotes or the cell cytoplasm (3). The mRNAs in this organelle have an almost complete lack of 5Ј-and 3Ј-untranslated nucleotides. The start codon is generally located within three nucleotides of the 5Ј end of the mRNA (1, 4). Thus, mammalian mitochondrial ribosomes do not recognize the start codon using the Shine/Dalgarno interaction between the mRNA and the 16 S rRNA as observed in prokaryotes. Further, this system does not use a cap-binding and scanning mechanism such as observed in the eukaryotic cytoplasm.Three translational initiation factors, IF1, IF2, and IF3, 1 are required for the initiation of protein synthesis in bacteria (5-7). Prior to the present report, the homolog of only one of these factors, IF2 mt , had been identified, cloned, and characterized in mammalian mitochondria (8 -12). Similar to its prokaryotic counterpart, IF2 mt promotes the binding of fMet-tRNA to the small subunit of mitochondrial ribosomes in response to synthetic polynucleotides such as poly(A,U,G).The current report describes the identification and initial characterization of the mammalian mitochondrial factor equivalent to IF3. In prokaryotes IF3 has a number of roles in the initiation of protein synthesis. IF3 b...

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3 , L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, Mammalian mitochondria are responsible for the synthesis of 13 proteins localized in the inner membrane. These proteins are components of the oligomeric complexes essential for oxidative phosphorylation and, hence, for the synthesis of about 90% of the ATP in eukaryotic organisms. The 55 S mammalian mitochondrial ribosomes consists of small (28 S) and large (39 S) subunits (1). In contrast to bacterial ribosomes which are about 65% RNA, mammalian mitochondrial ribosomes are only 33% RNA. The low percentage of RNA in these ribosomes reflects a reduction in the size of the rRNA and a compensating increase in the number of ribosomal proteins. For example, the small subunit of the mammalian mitochondrial ribosome contains a 12 S rRNA (about 950 nucleotides) and an estimated 29 proteins (2). In contrast, the Escherichia coli 30 S subunit has a 16 S rRNA (1542 nucleotides in length) and 21 proteins (3). The large subunit of the mammalian mitochondrial ribosome contains a 16 S rRNA (about 1560 nucleotides) and about 50 proteins (4, 5).The identification of proteins in mammalian mitochondrial ribosomes has been challenging due to their low abundance. Recently 60 mammalian mitochondrial ribosomal proteins, 31 proteins from the large subunit and 29 proteins from the small subunit, have been characterized by different laboratories (2, 6 -14). The identification of these proteins used two approaches. The traditional approach was to separate the proteins on two-dimensional gels or high performance liquid chromatography followed by sequence analysis using Edman chemistry or mass spectrometry (MS). More recently, proteins present in the 28 S subunit have been characterized by proteolytic digestion of whole subunits. Sequence information on the peptides present in this complex mixture was obtained by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS).1 This strategy allowed the identification of 28 proteins of the small subunit including 14 proteins that had not previously been identified (2). In the present study, we have extended this approach to the 39 S subunit. In addition to direct analysis of 39 S digests by LC/MS, aliquots of the total digest were fractionated prior to reversed-phase LC/MS analysis to maximize the number of peptides sequenced. In the first approach, a portion of the total digest was fractionated by affinity selection ...

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography^tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways. ß