Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 May 15;356(Pt 1):207–215. doi: 10.1042/0264-6021:3560207

Self-association and precursor protein binding of Saccharomyces cerevisiae Tom40p, the core component of the protein translocation channel of the mitochondrial outer membrane.

D M Gordon 1, J Wang 1, B Amutha 1, D Pain 1
PMCID: PMC1221829  PMID: 11336653

Abstract

The precursor protein translocase of the mitochondrial outer membrane (Tom) is a multi-subunit complex containing receptors and a general import channel, of which the core component is Tom40p. Nuclear-encoded mitochondrial precursor proteins are first recognized by surface receptors and then pass through the import channel. The Tom complex has been purified; however, the protein-protein interactions that drive its assembly and maintain its stability have been difficult to study. Here we show that Saccharomyces cerevisiae Tom40p expressed in bacteria and purified to homogeneity associates efficiently with itself. The self-association is very strong and can withstand up to 4 M urea or 1 M salt. The tight self-association does not require the N-terminal segment of Tom40p. Furthermore, purified Tom40p preferentially recognizes the targeting sequence of mitochondrial precursor proteins. Although the binding of the targeting sequence to Tom40p is inhibited by urea concentrations in excess of 1 M, it is moderately resistant to 1 M salt. Simultaneous self-assembly and precursor protein binding suggest that Tom40p contains at least two different domains mediating these processes. The experimental approach described here should be useful for analysing protein-protein interactions involving individual or groups of components of the mitochondrial import machinery.

Full Text

The Full Text of this article is available as a PDF (271.1 KB).

Selected References

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

  1. Ahting U., Thun C., Hegerl R., Typke D., Nargang F. E., Neupert W., Nussberger S. The TOM core complex: the general protein import pore of the outer membrane of mitochondria. J Cell Biol. 1999 Nov 29;147(5):959–968. doi: 10.1083/jcb.147.5.959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alconada A., Kübrich M., Moczko M., Hönlinger A., Pfanner N. The mitochondrial receptor complex: the small subunit Mom8b/Isp6 supports association of receptors with the general insertion pore and transfer of preproteins. Mol Cell Biol. 1995 Nov;15(11):6196–6205. doi: 10.1128/mcb.15.11.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolliger L., Junne T., Schatz G., Lithgow T. Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria. EMBO J. 1995 Dec 15;14(24):6318–6326. doi: 10.1002/j.1460-2075.1995.tb00322.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brix J., Dietmeier K., Pfanner N. Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J Biol Chem. 1997 Aug 15;272(33):20730–20735. doi: 10.1074/jbc.272.33.20730. [DOI] [PubMed] [Google Scholar]
  5. Dekker P. J., Martin F., Maarse A. C., Bömer U., Müller H., Guiard B., Meijer M., Rassow J., Pfanner N. The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44. EMBO J. 1997 Sep 1;16(17):5408–5419. doi: 10.1093/emboj/16.17.5408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dekker P. J., Ryan M. T., Brix J., Müller H., Hönlinger A., Pfanner N. Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex. Mol Cell Biol. 1998 Nov;18(11):6515–6524. doi: 10.1128/mcb.18.11.6515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dietmeier K., Hönlinger A., Bömer U., Dekker P. J., Eckerskorn C., Lottspeich F., Kübrich M., Pfanner N. Tom5 functionally links mitochondrial preprotein receptors to the general import pore. Nature. 1997 Jul 10;388(6638):195–200. doi: 10.1038/40663. [DOI] [PubMed] [Google Scholar]
  8. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  9. Gordon D. M., Dancis A., Pain D. Mechanisms of mitochondrial protein import. Essays Biochem. 2000;36:61–73. doi: 10.1042/bse0360061. [DOI] [PubMed] [Google Scholar]
  10. Gordon D. M., Kogan M., Knight S. A., Dancis A., Pain D. Distinct roles for two N-terminal cleaved domains in mitochondrial import of the yeast frataxin homolog, Yfh1p. Hum Mol Genet. 2001 Feb 1;10(3):259–269. doi: 10.1093/hmg/10.3.259. [DOI] [PubMed] [Google Scholar]
  11. Gordon D. M., Roof D. M. The kinesin-related protein Kip1p of Saccharomyces cerevisiae is bipolar. J Biol Chem. 1999 Oct 1;274(40):28779–28786. doi: 10.1074/jbc.274.40.28779. [DOI] [PubMed] [Google Scholar]
  12. Gratzer S., Lithgow T., Bauer R. E., Lamping E., Paltauf F., Kohlwein S. D., Haucke V., Junne T., Schatz G., Horst M. Mas37p, a novel receptor subunit for protein import into mitochondria. J Cell Biol. 1995 Apr;129(1):25–34. doi: 10.1083/jcb.129.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haucke V., Lithgow T., Rospert S., Hahne K., Schatz G. The yeast mitochondrial protein import receptor Mas20p binds precursor proteins through electrostatic interaction with the positively charged presequence. J Biol Chem. 1995 Mar 10;270(10):5565–5570. doi: 10.1074/jbc.270.10.5565. [DOI] [PubMed] [Google Scholar]
  14. Hill K., Model K., Ryan M. T., Dietmeier K., Martin F., Wagner R., Pfanner N. Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment]. Nature. 1998 Oct 1;395(6701):516–521. doi: 10.1038/26780. [DOI] [PubMed] [Google Scholar]
  15. Hönlinger A., Bömer U., Alconada A., Eckerskorn C., Lottspeich F., Dietmeier K., Pfanner N. Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import. EMBO J. 1996 May 1;15(9):2125–2137. [PMC free article] [PubMed] [Google Scholar]
  16. Hönlinger A., Kübrich M., Moczko M., Gärtner F., Mallet L., Bussereau F., Eckerskorn C., Lottspeich F., Dietmeier K., Jacquet M. The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins. Mol Cell Biol. 1995 Jun;15(6):3382–3389. doi: 10.1128/mcb.15.6.3382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kiebler M., Pfaller R., Söllner T., Griffiths G., Horstmann H., Pfanner N., Neupert W. Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature. 1990 Dec 13;348(6302):610–616. doi: 10.1038/348610a0. [DOI] [PubMed] [Google Scholar]
  18. Knight S. A., Kim R., Pain D., Dancis A. The yeast connection to Friedreich ataxia. Am J Hum Genet. 1999 Feb;64(2):365–371. doi: 10.1086/302270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Knight S. A., Sepuri N. B., Pain D., Dancis A. Mt-Hsp70 homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis. J Biol Chem. 1998 Jul 17;273(29):18389–18393. doi: 10.1074/jbc.273.29.18389. [DOI] [PubMed] [Google Scholar]
  20. Komiya T., Rospert S., Koehler C., Looser R., Schatz G., Mihara K. Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis. EMBO J. 1998 Jul 15;17(14):3886–3898. doi: 10.1093/emboj/17.14.3886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Künkele K. P., Heins S., Dembowski M., Nargang F. E., Benz R., Thieffry M., Walz J., Lill R., Nussberger S., Neupert W. The preprotein translocation channel of the outer membrane of mitochondria. Cell. 1998 Jun 12;93(6):1009–1019. doi: 10.1016/s0092-8674(00)81206-4. [DOI] [PubMed] [Google Scholar]
  22. Künkele K. P., Juin P., Pompa C., Nargang F. E., Henry J. P., Neupert W., Lill R., Thieffry M. The isolated complex of the translocase of the outer membrane of mitochondria. Characterization of the cation-selective and voltage-gated preprotein-conducting pore. J Biol Chem. 1998 Nov 20;273(47):31032–31039. doi: 10.1074/jbc.273.47.31032. [DOI] [PubMed] [Google Scholar]
  23. Mannella C. A., Neuwald A. F., Lawrence C. E. Detection of likely transmembrane beta strand regions in sequences of mitochondrial pore proteins using the Gibbs sampler. J Bioenerg Biomembr. 1996 Apr;28(2):163–169. doi: 10.1007/BF02110647. [DOI] [PubMed] [Google Scholar]
  24. Mayer A., Nargang F. E., Neupert W., Lill R. MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19. EMBO J. 1995 Sep 1;14(17):4204–4211. doi: 10.1002/j.1460-2075.1995.tb00094.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mayer A., Neupert W., Lill R. Mitochondrial protein import: reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding. Cell. 1995 Jan 13;80(1):127–137. doi: 10.1016/0092-8674(95)90457-3. [DOI] [PubMed] [Google Scholar]
  26. Neupert W. Protein import into mitochondria. Annu Rev Biochem. 1997;66:863–917. doi: 10.1146/annurev.biochem.66.1.863. [DOI] [PubMed] [Google Scholar]
  27. Rapaport D., Künkele K. P., Dembowski M., Ahting U., Nargang F. E., Neupert W., Lill R. Dynamics of the TOM complex of mitochondria during binding and translocation of preproteins. Mol Cell Biol. 1998 Sep;18(9):5256–5262. doi: 10.1128/mcb.18.9.5256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rapaport D., Mayer A., Neupert W., Lill R. cis and trans sites of the TOM complex of mitochondria in unfolding and initial translocation of preproteins. J Biol Chem. 1998 Apr 10;273(15):8806–8813. doi: 10.1074/jbc.273.15.8806. [DOI] [PubMed] [Google Scholar]
  29. Rapaport D., Neupert W. Biogenesis of Tom40, core component of the TOM complex of mitochondria. J Cell Biol. 1999 Jul 26;146(2):321–331. doi: 10.1083/jcb.146.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rapaport D., Neupert W., Lill R. Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence. J Biol Chem. 1997 Jul 25;272(30):18725–18731. doi: 10.1074/jbc.272.30.18725. [DOI] [PubMed] [Google Scholar]
  31. Scharfe C., Zaccaria P., Hoertnagel K., Jaksch M., Klopstock T., Lill R., Prokisch H., Gerbitz K. D., Mewes H. W., Meitinger T. MITOP: database for mitochondria-related proteins, genes and diseases. Nucleic Acids Res. 1999 Jan 1;27(1):153–155. doi: 10.1093/nar/27.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schatz G. Just follow the acid chain. Nature. 1997 Jul 10;388(6638):121–122. doi: 10.1038/40510. [DOI] [PubMed] [Google Scholar]
  33. Schleiff E., Shore G. C., Goping I. S. Human mitochondrial import receptor, Tom20p. Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins. FEBS Lett. 1997 Mar 10;404(2-3):314–318. doi: 10.1016/s0014-5793(97)00145-2. [DOI] [PubMed] [Google Scholar]
  34. Schlossmann J., Dietmeier K., Pfanner N., Neupert W. Specific recognition of mitochondrial preproteins by the cytosolic domain of the import receptor MOM72. J Biol Chem. 1994 Apr 22;269(16):11893–11901. [PubMed] [Google Scholar]
  35. Sepuri N. B., Gordon D. M., Pain D. A GTP-dependent "push" is generally required for efficient protein translocation across the mitochondrial inner membrane into the matrix. J Biol Chem. 1998 Aug 14;273(33):20941–20950. doi: 10.1074/jbc.273.33.20941. [DOI] [PubMed] [Google Scholar]
  36. Sirrenberg C., Endres M., Becker K., Bauer M. F., Walther E., Neupert W., Brunner M. Functional cooperation and stoichiometry of protein translocases of the outer and inner membranes of mitochondria. J Biol Chem. 1997 Nov 21;272(47):29963–29966. doi: 10.1074/jbc.272.47.29963. [DOI] [PubMed] [Google Scholar]
  37. Stan T., Ahting U., Dembowski M., Künkele K. P., Nussberger S., Neupert W., Rapaport D. Recognition of preproteins by the isolated TOM complex of mitochondria. EMBO J. 2000 Sep 15;19(18):4895–4902. doi: 10.1093/emboj/19.18.4895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vestweber D., Brunner J., Baker A., Schatz G. A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site. Nature. 1989 Sep 21;341(6239):205–209. doi: 10.1038/341205a0. [DOI] [PubMed] [Google Scholar]
  39. Voos W., Martin H., Krimmer T., Pfanner N. Mechanisms of protein translocation into mitochondria. Biochim Biophys Acta. 1999 Nov 16;1422(3):235–254. doi: 10.1016/s0304-4157(99)00007-6. [DOI] [PubMed] [Google Scholar]
  40. Yano M., Hoogenraad N., Terada K., Mori M. Identification and functional analysis of human Tom22 for protein import into mitochondria. Mol Cell Biol. 2000 Oct;20(19):7205–7213. doi: 10.1128/mcb.20.19.7205-7213.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES