The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria.

Mutations in mitochondrial small subunit ribosomal proteins MRPS16 or MRPS22 cause severe, fatal respiratory chain dysfunction due to impaired translation of mitochondrial mRNAs. The loss of either MRPS16 or MRPS22 was accompanied by the loss of most of another small subunit protein MRPS11. However, MRPS2 was reduced only about 2-fold in patient fibroblasts. This observation suggests that the small ribosomal subunit is only partially able to assemble in these patients. Two large subunit ribosomal proteins, MRPL13 and MRPL15, were present in substantial amounts suggesting that the large ribosomal subunit is still present despite a non-functional small subunit.

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.

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt.

Mammalian mitochondrial initiation factor 3 (IF3(mt)) 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 IF3(mt) to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K(d) for the binding of IF3(mt) to 28S subunits is approximately 30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3(mt) has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K(d) = 1.5 muM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K(d) = 0.12-0.25 muM). IF3(mt) does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3(mt) 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 IF3(mt) from the 28S subunits upon 39S subunit joining.

Overexpression and purification of mammalian mitochondrial translational initiation factor 2 and initiation factor 3.

Two mammalian mitochondrial initiation factors have been identified. Initiation factor 2 (IF2(mt)) selects the initiator tRNA (fMet-tRNA) and promotes its binding to the ribosome. Initiation factor 3 (IF3(mt)) promotes the dissociation of the 55S mitochondrial ribosome into subunits and may play additional, less-well-understood, roles in initiation complex formation. Native bovine IF2(mt) was purified from liver a number of years ago. The yield of this factor is very low making biochemical studies difficult. The cDNA for bovine IF2(mt) was expressed in Escherichia coli under the control of the T7 polymerase promoter in a vector that provides a His(6)-tag at the C-terminus of the expressed protein. This factor was expressed in E. coli and purified by chromatography on Ni-NTA resins. The expressed protein has a number of degradation products in partially purified preparations and this factor is then further purified by high-performance liquid chromatography or gravity chromatography on anion exchange resins. IF3(mt) has never been purified from any mammalian system. However, the cDNA for this protein can be identified in the expressed sequence tag (EST) libraries. The portion of the sequence encoding the region of human IF3(mt) predicted to be present in the mitochondrially imported form of this factor was cloned and expressed in E. coli using a vector that provides a C-terminal His(6)-tag. The tagged factor is partially purified on Ni-NTA resins. However, a major proteolytic fragment arising from a defined cleavage of this protein is present in these preparations. This contaminant can be removed by a single step of high-performance liquid chromatography on a cation exchange resin. Alternatively, the mature form of IF3(mt) can be purified by two sequential passes through a gravity S-Sepharose column.

Proteomics and electron microscopic characterization of the unusual mitochondrial ribosome-related 45S complex in Leishmania tarentolae.

A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein-protein (coiled-coil, Rhodanese) or protein-RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania mitochondrial ribosome is associated with an additional protein mass. The biological role of these particles is not known.