Abstract
HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1½–2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a–a 3 and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.
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- Barnett W. E., Brown D. H. Mitochondrial transfer ribonucleic acids. Proc Natl Acad Sci U S A. 1967 Feb;57(2):452–458. doi: 10.1073/pnas.57.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bauer W., Vinograd J. The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye. J Mol Biol. 1968 Apr 14;33(1):141–171. doi: 10.1016/0022-2836(68)90286-6. [DOI] [PubMed] [Google Scholar]
- Bretscher M. S., Marcker K. A. Polypeptidyl-sigma-ribonucleic acid and amino-acyl-sigma-ribonucleic acid binding sites on ribosomes. Nature. 1966 Jul 23;211(5047):380–384. doi: 10.1038/211380a0. [DOI] [PubMed] [Google Scholar]
- Brown J. C., Smith A. E. Initiator codons in eukaryotes. Nature. 1970 May 16;226(5246):610–612. doi: 10.1038/226610a0. [DOI] [PubMed] [Google Scholar]
- Buck C. A., Nass M. M. Studies on mitochondrial tRNA from animal cells. I. A comparison of mitochondrial and cytoplasmic trna and aminoacyl-tRNA synthetases. J Mol Biol. 1969 Apr 14;41(1):67–82. doi: 10.1016/0022-2836(69)90126-0. [DOI] [PubMed] [Google Scholar]
- EAGLE H. Amino acid metabolism in mammalian cell cultures. Science. 1959 Aug 21;130(3373):432–437. doi: 10.1126/science.130.3373.432. [DOI] [PubMed] [Google Scholar]
- Eisenstadt J., Lengyel P. Formylmethionyl-tRNA dependence of amino acid incorporation in extracts of trimethoprim-treated Escherichia coli. Science. 1966 Oct 28;154(3748):524–527. [PubMed] [Google Scholar]
- Firkin F. C., Linnane A. W. Differential effects of chloramphenicol on the growth and respiration of mammalian cells. Biochem Biophys Res Commun. 1968 Aug 13;32(3):398–402. doi: 10.1016/0006-291x(68)90674-8. [DOI] [PubMed] [Google Scholar]
- Galper J. B., Darnell J. E. Mitochondrial protein synthesis in HeLa cells. J Mol Biol. 1971 Apr 28;57(2):363–367. doi: 10.1016/0022-2836(71)90354-8. [DOI] [PubMed] [Google Scholar]
- Galper J. B., Darnell J. E. The presence of N-formyl-methionyl-tRNA in HeLa cell mitochondria. Biochem Biophys Res Commun. 1969 Jan 27;34(2):205–214. doi: 10.1016/0006-291x(69)90633-0. [DOI] [PubMed] [Google Scholar]
- Guerineau M., Grandchamp C., Yotsuyanagi Y., Slonimski P. P. Examen au microscope électronique, du DNA mitochondrial de la levure: molécules circulaires. C R Acad Sci Hebd Seances Acad Sci D. 1968 May 6;266(19):2000–2003. [PubMed] [Google Scholar]
- Housman D., Jacobs-Lorena M., Rajbhandary U. L., Lodish H. F. Initiation of haemoglobin synthesis by methionyl-tRNA. Nature. 1970 Aug 29;227(5261):913–918. doi: 10.1038/227913a0. [DOI] [PubMed] [Google Scholar]
- Hudson B., Vinograd J. Catenated circular DNA molecules in HeLa cell mitochondria. Nature. 1967 Nov 18;216(5116):647–652. doi: 10.1038/216647a0. [DOI] [PubMed] [Google Scholar]
- King M. E., Godman G. C., King D. W. Respiratory enzymes and mitochondrial morphology of HeLa and L cells treated with chloramphenicol and ethidium bromide. J Cell Biol. 1972 Apr;53(1):127–142. doi: 10.1083/jcb.53.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Küntzel H., Noll H. Mitochondrial and cytoplasmic polysomes from Neurospora crassa. Nature. 1967 Sep 23;215(5108):1340–1345. doi: 10.1038/2151340a0. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Lamb A. J., Clark-Walker G. D., Linnane A. W. The biogenesis of mitochondria. 4. The differentiation of mitochondrial and cytoplasmic protein synthesizing systems in vitro by antibiotics. Biochim Biophys Acta. 1968 Jul 23;161(2):415–427. [PubMed] [Google Scholar]
- Leder P., Bursztyn H. Initiation of protein synthesis II. A convenient assay for the ribosome-dependent synthesis of N-formyl-C14-methionylpuromycin. Biochem Biophys Res Commun. 1966 Oct 20;25(2):233–238. doi: 10.1016/0006-291x(66)90586-9. [DOI] [PubMed] [Google Scholar]
- Lederman M., Attardi G. In vitro protein synthesis in a mitochondrial fraction from HeLa cells: sensitivity to antibiotic and ethidium bromide. Biochem Biophys Res Commun. 1970 Sep 30;40(6):1492–1500. doi: 10.1016/0006-291x(70)90037-9. [DOI] [PubMed] [Google Scholar]
- Loeb J. N., Hubby B. G. Amino acid incorporation by isolated mitochondria in the presence of cycloheximide. Biochim Biophys Acta. 1968 Oct 29;166(3):745–748. doi: 10.1016/0005-2787(68)90392-4. [DOI] [PubMed] [Google Scholar]
- Maizel J. V., Jr Acrylamide-gel electrophorograms by mechanical fractionation: radioactive adenovirus proteins. Science. 1966 Feb 25;151(3713):988–990. doi: 10.1126/science.151.3713.988. [DOI] [PubMed] [Google Scholar]
- Montenecourt B. S., Dubin D. T. The ribosomal subunits of hamster cell mitochondria. Biochem Biophys Res Commun. 1970 Oct 23;41(2):458–463. doi: 10.1016/0006-291x(70)90527-9. [DOI] [PubMed] [Google Scholar]
- Perlman S., Abelson H. T., Penman S. Mitochondrial protein synthesis: RNA with the properties of Eukaryotic messenger RNA. Proc Natl Acad Sci U S A. 1973 Feb;70(2):350–353. doi: 10.1073/pnas.70.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Perlman S., Penman S. Mitochondrial protein synthesis: resistance to emetine and response to RNA synthesis inhibitors. Biochem Biophys Res Commun. 1970 Aug 24;40(4):941–948. doi: 10.1016/0006-291x(70)90994-0. [DOI] [PubMed] [Google Scholar]
- Rifkin M. R., Wood D. D., Luck D. J. Ribosomal RNA and ribosomes from mitochondria of Neurospora crassa. Proc Natl Acad Sci U S A. 1967 Sep;58(3):1025–1032. doi: 10.1073/pnas.58.3.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roodyn D. B. Further study of factors affecting amino acid incorporation into protein by isolated mitochondria. Biochem J. 1965 Dec;97(3):782–793. doi: 10.1042/bj0970782. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith A. E., Marcker K. A. Cytoplasmic methionine transfer RNAs from eukaryotes. Nature. 1970 May 16;226(5246):607–610. doi: 10.1038/226607a0. [DOI] [PubMed] [Google Scholar]
- Soslau G., Nass M. M. Effects of ethidium bromide on the cytochrome content and ultrastructure of L cell mitochondria. J Cell Biol. 1971 Nov;51(21):514–524. doi: 10.1083/jcb.51.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strauss J. H., Jr, Burge B. W., Darnell J. E. Sindbis virus infection of chick and hamster cells: synthesis of virus-specific proteins. Virology. 1969 Mar;37(3):367–376. doi: 10.1016/0042-6822(69)90220-7. [DOI] [PubMed] [Google Scholar]
- Vesco C., Penman S. The cytoplasmic RNA of HeLa cells: new discrete species associated with mitochondria. Proc Natl Acad Sci U S A. 1969 Jan;62(1):218–225. doi: 10.1073/pnas.62.1.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zylber E., Vesco C., Penman S. Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide. J Mol Biol. 1969 Aug 28;44(1):195–204. doi: 10.1016/0022-2836(69)90414-8. [DOI] [PubMed] [Google Scholar]