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. 1983;2(12):2161–2168. doi: 10.1002/j.1460-2075.1983.tb01717.x

Import of proteins into mitochondria: a 70 kilodalton outer membrane protein with a large carboxy-terminal deletion is still transported to the outer membrane.

H Riezman, T Hase, A P van Loon, L A Grivell, K Suda, G Schatz
PMCID: PMC555428  PMID: 6321150

Abstract

The yeast mitochondrial outer membrane contains a major 70-kd protein which is coded by a nuclear gene. Two forms of this gene were isolated from a yeast genomic clone bank: the intact gene, and a truncated gene which had lost a large part of its 3' end during the cloning procedure. Upon transformation into yeast, both the intact and the truncated gene are expressed; the truncated gene generates a shortened protein missing 203 amino acids from the carboxy-terminus. This truncated polypeptide reacts with a monoclonal antibody against the authentic 70-kd protein and is transported to the mitochondrial outer membrane. By integrative transformation, we have constructed a yeast mutant which lacks the 70-kd protein and is unable to adapt to growth on a nonfermentable carbon source at 37 degrees C. This phenotypic lesion can be corrected by transforming the mutant with the intact, but not the truncated gene. The carboxy-terminal sequence of 203 amino acids is thus necessary for the function of the protein, but not for its targeting to the mitochondrion.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  3. Chevallier M. R., Bloch J. C., Lacroute F. Transcriptional and translational expression of a chimeric bacterial-yeast plasmid in yeasts. Gene. 1980 Oct;11(1-2):11–19. doi: 10.1016/0378-1119(80)90082-7. [DOI] [PubMed] [Google Scholar]
  4. Cochet M., Perrin F., Gannon F., Krust A., Chambon P., McKnight G. S., Lee D. C., Mayo K. E., Palmiter R. Cloning of an almost full-length chicken conalbumin double-stranded cDNA. Nucleic Acids Res. 1979 Jun 11;6(7):2435–2452. doi: 10.1093/nar/6.7.2435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Douglas M. G., Butow R. A. Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1083–1086. doi: 10.1073/pnas.73.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Freitag H., Janes M., Neupert W. Biosynthesis of mitochondrial porin and insertion into the outer mitochondrial membrane of Neurospora crassa. Eur J Biochem. 1982 Aug;126(1):197–202. doi: 10.1111/j.1432-1033.1982.tb06766.x. [DOI] [PubMed] [Google Scholar]
  7. Gasser S. M., Schatz G. Import of proteins into mitochondria. In vitro studies on the biogenesis of the outer membrane. J Biol Chem. 1983 Mar 25;258(6):3427–3430. [PubMed] [Google Scholar]
  8. Goldbach R. W., Borst P., Bollen-de Boer J. E., van Bruggen E. F. The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST. Biochim Biophys Acta. 1978 Nov 21;521(1):169–186. doi: 10.1016/0005-2787(78)90260-5. [DOI] [PubMed] [Google Scholar]
  9. Hase T., Riezman H., Suda K., Schatz G. Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane. EMBO J. 1983;2(12):2169–2172. doi: 10.1002/j.1460-2075.1983.tb01718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holland M. J., Hager G. L., Rutter W. J. Characterization of purified poly(adenylic acid)-containing messenger ribonucleic acid from Saccharomyces cerevisiae. Biochemistry. 1977 Jan 11;16(1):8–16. doi: 10.1021/bi00620a002. [DOI] [PubMed] [Google Scholar]
  12. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maccecchini M. L., Rudin Y., Blobel G., Schatz G. Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc Natl Acad Sci U S A. 1979 Jan;76(1):343–347. doi: 10.1073/pnas.76.1.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  15. Matsuura S., Arpin M., Hannum C., Margoliash E., Sabatini D. D., Morimoto T. In vitro synthesis and posttranslational uptake of cytochrome c into isolated mitochondria: role of a specific addressing signal in the apocytochrome. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4368–4372. doi: 10.1073/pnas.78.7.4368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  17. Mihara K., Blobel G., Sato R. In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7102–7106. doi: 10.1073/pnas.79.23.7102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Neff N. F., Thomas J. H., Grisafi P., Botstein D. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell. 1983 May;33(1):211–219. doi: 10.1016/0092-8674(83)90350-1. [DOI] [PubMed] [Google Scholar]
  19. Ohashi A., Gibson J., Gregor I., Schatz G. Import of proteins into mitochondria. The precursor of cytochrome c1 is processed in two steps, one of them heme-dependent. J Biol Chem. 1982 Nov 10;257(21):13042–13047. [PubMed] [Google Scholar]
  20. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  22. Riezman H., Hay R., Gasser S., Daum G., Schneider G., Witte C., Schatz G. The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles. EMBO J. 1983;2(7):1105–1111. doi: 10.1002/j.1460-2075.1983.tb01553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Riezman H., Hay R., Witte C., Nelson N., Schatz G. Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins. EMBO J. 1983;2(7):1113–1118. doi: 10.1002/j.1460-2075.1983.tb01554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  25. Schatz G., Butow R. A. How are proteins imported into mitochondria? Cell. 1983 Feb;32(2):316–318. doi: 10.1016/0092-8674(83)90450-6. [DOI] [PubMed] [Google Scholar]
  26. Shortle D., Haber J. E., Botstein D. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science. 1982 Jul 23;217(4557):371–373. doi: 10.1126/science.7046050. [DOI] [PubMed] [Google Scholar]
  27. Strausberg R. L., Butow R. A. Expression of petite mitochondrial DNA in vivo: zygotic gene rescue. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2715–2719. doi: 10.1073/pnas.74.7.2715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. van Loon A. P., de Groot R. J., van Eyk E., van der Horst G. T., Grivell L. A. Isolation and characterization of nuclear genes coding for subunits of the yeast ubiquinol-cytochrome c reductase complex. Gene. 1982 Dec;20(3):323–337. doi: 10.1016/0378-1119(82)90201-3. [DOI] [PubMed] [Google Scholar]

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