Bovine mitochondrial ribosomes: Effect of cations and heterologous dissociation factors on subunit interactions

Spremulli, Linda L.; Kraus, Betsy L.

doi:10.1016/s0006-291x(87)80180-8

Cited by 13 publications

(8 citation statements)

References 9 publications

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“…Effects of IF3 mt on the dissociation of mitochondrial ribosomal subunits. A, mitochondrial 55 S ribosomes (0.2 OD 260 ) were incubated as described under "Materials and Methods" at 2 mM Mg 2ϩ in the absence of added IF3 mt , then the Mg 2ϩ concentration was subsequently raised to 7 mM, and the mixture was analyzed for monosomes and subunits by sucrose density gradient centrifugation as described previously (38). B, mitochondrial ribosomes (0.2 OD 260 , ϳ6 pmol) were incubated at 2 mM Mg 2ϩ in the presence of 1.72 g of IF3 mt .…”

Section: Resultsmentioning

confidence: 99%

“…The reactions were incubated for 15 min at 37°C. The Mg 2ϩ concentration was then adjusted to 7 mM by the addition of 2.5 l of 0.1 M MgCl 2 , and the samples were analyzed for 28, 39, and 55 S particles on 10 -30% (w/v) linear sucrose gradients prepared in the buffer described above containing 7 mM Mg 2ϩ and analyzed as described previously (38).…”

Section: Preparation Of Ribosomes and Initiationmentioning

confidence: 99%

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

Koc

Spremulli

2002

Journal of Biological Chemistry

109

121

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Preparation Of Ribosomes and Initiationmentioning

confidence: 99%

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

Koc

Spremulli

2002

Journal of Biological Chemistry

109

121

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“…Although this process is indeed compromised in the Letm1 knockdowns, it persists when these cells are treated with nigericin. Because mitochondrial ribosomes disassociate in high K ϩ concentration environments (61,62), we propose that the apparent effect on mitochondrial translation represents an epiphenomenon to the accumulation of the cation in the matrix upon Letm1 ablation. Furthermore, the similar growth rates of the Letm1-depleted and uninduced controls in the optimal doses of the ionophores nigericin and monensin suggest that additional processes are not affected besides mt K ϩ /H ϩ antiport.…”

Section: Discussionmentioning

confidence: 98%

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

Hashimi

McDonald

Stříbrná

et al. 2013

Journal of Biological Chemistry

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Background: Letm1 is a mitochondrial protein attributed disparate roles, including cation/proton antiport and translation. Results: Letm1 RNAi silencing in Trypanosoma brucei triggers swelling mitochondria and translation arrest that is ameliorated by chemical potassium/proton exchangers. Conclusion:The ancestral function of Letm1, to maintain mitochondrial potassium homeostasis, shows remarkable conservation. Significance: Results from diverged T. brucei provide a better understanding of Letm1 function throughout eukaryotes.

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“…It also reduces the electrostatic repulsion between the subunits by masking the charges on the rRNA [25]. In both prokaryotes and mitochondria, spermine has been shown to tighten the interaction between the ribosomal subunits [15,26]. The effect of spermine on the ability of IF3 mt to promote subunit dissociation was tested directly using sucrose density gradient centrifugation (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

“…Spermine affects translation in mitochondria by lowering the Mg 2+ requirement for the association of mitochondrial ribosomal subunits into 55S monosomes by about 1 mM [15]. Little is known about other effects that spermine may have on protein synthesis in mitochondria.…”

Section: Introductionmentioning

confidence: 99%

The effect of spermine on the initiation of mitochondrial protein synthesis

Christian¹,

Haque²,

Spremulli³

2010

Biochemical and Biophysical Research Communications

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Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2 mt ) and mitochondrial initiation factor 3 (IF3 mt ). This observation indicates that IF2 mt and IF3 mt , while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3 mt dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2 mt . The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.

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Bovine mitochondrial ribosomes: Effect of cations and heterologous dissociation factors on subunit interactions

Cited by 13 publications

References 9 publications

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

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

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

The effect of spermine on the initiation of mitochondrial protein synthesis

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