Linda L. Spremulli

Structure of the Mammalian Mitochondrial Ribosome Reveals an Expanded Functional Role for Its Component Proteins

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.

Mechanism of Protein Biosynthesis in Mammalian Mitochondria

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. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

A new face on apoptosis: Death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins

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.

Interaction of Mammalian Mitochondrial Ribosomes with the Inner Membrane

All of the products of mitochondrial protein biosynthesis in animals are hydrophobic proteins that are localized in the inner membrane. Hence, it is possible that the synthesis of these proteins could occur on ribosomes associated with the inner membrane. To examine this possibility, inner membrane and matrix fractions of bovine mitochondria were examined for the presence of ribosomes using probes for the rRNAs. Between 40 and 50% of the ribosomes were found to fractionate with the inner membrane. About half of the ribosomes associated with the inner membrane could be released by high salt treatment, indicating that they interact with the membrane largely through electrostatic forces. No release of the ribosome was observed upon treatment with puromycin, suggesting that the association observed is not due to insertion of a nascent polypeptide chain into the membrane. A fraction of the ribosomes remained with residual portions of the membranes that cannot be solubilized in the presence of Triton X-100. These ribosomes may be associated with large oligomeric complexes in the membrane.

Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs

Human mitochondrial translational initiation factor 3 (IF3mt) 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. IF3mt 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 IF3mt was cloned, and the protein was expressed inEscherichia coli in a His-tagged form. The mature form of IF3mt has short extensions on the N and C termini surrounding a region homologous to bacterial IF3. The region of IF3mt homologous to prokaryotic factors ranges between 21–26% identical to the bacterial proteins. Purified IF3mt promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2mt), [35S]fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA. IF3mtshifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation. In addition, the ability ofE. 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 IF2mt and IF3mt.

A Proteomics Approach to the Identification of Mammalian Mitochondrial Small Subunit Ribosomal Proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coliS10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.

Unconventional decoding of the AUA codon as methionine by mitochondrial tRNAMet with the anticodon f5CAU as revealed with a mitochondrial in vitro translation system

Mitochondrial (mt) tRNAMet has the unusual modified nucleotide 5-formylcytidine (f5C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNAMet carrying f5C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNAMet lacking f5C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNAMetm with the anticodon ac4CAU (ac4C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNAMet (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f5C modification in mt tRNAMet plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f5C-G and f5C-A based on the chemical properties of f5C are presented.

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.

Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver PURIFICATION, CHARACTERIZATION, AND GENE STRUCTURE

The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coliand yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmtformylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria.

Interaction of Mitochondrial Elongation Factor Tu with Aminoacyl-tRNA and Elongation Factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu·GTP·aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu·GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu·Ts). Mammalian mitochondrial EF-Tu (EF-Tumt) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tumt subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K tRNA) and for the dissociation of the EF-Tu·Tsmt complex (K Ts) were investigated. The equilibrium dissociation constant for the ternary complex was 18 ± 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 × 10− 4 s− 1, which is essentially equivalent to that observed for the ternary complex inEscherichia coli. The binding of EF-Tumt to EF-Tsmt is mutually exclusive with the formation of the ternary complex. K Ts was determined by quantifying the effects of increasing concentrations of EF-Tsmt on the amount of ternary complex formed with EF-Tumt. The value obtained for K Ts(5.5 ± 1.3 nm) is comparable to the value ofK tRNA.