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. 1997 May 1;16(9):2217–2226. doi: 10.1093/emboj/16.9.2217

Insertion into the mitochondrial inner membrane of a polytopic protein, the nuclear-encoded Oxa1p.

J M Herrmann 1, W Neupert 1, R A Stuart 1
PMCID: PMC1169824  PMID: 9171337

Abstract

Oxa1p, a nuclear-encoded protein of the mitochondrial inner membrane with five predicted transmembrane (TM) segments is synthesized as a precursor (pOxa1p) with an N-terminal presequence. It becomes imported in a process requiring the membrane potential, matrix ATP, mt-Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the negatively charged N-terminus of Oxa1p (approximately 90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native N(out)-C(in) orientation. This export event is dependent on the membrane potential. Chimeric preproteins containing N-terminal stretches of increasing lengths of Oxa1p fused on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner membrane into the matrix. Thus Oxa1p apparently does not contain a stop transfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion. The sorting pathway of pOxa1p is suggested to combine the pathways of general import into the matrix with a bacterial-type export process. We postulate that at least two different sorting pathways exist in mitochondria for polytopic inner membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p-type pathway.

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Selected References

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  1. Altamura N., Capitanio N., Bonnefoy N., Papa S., Dujardin G. The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin-sensitive ATP synthase. FEBS Lett. 1996 Mar 11;382(1-2):111–115. doi: 10.1016/0014-5793(96)00165-2. [DOI] [PubMed] [Google Scholar]
  2. Andersson H., von Heijne G. Sec dependent and sec independent assembly of E. coli inner membrane proteins: the topological rules depend on chain length. EMBO J. 1993 Feb;12(2):683–691. doi: 10.1002/j.1460-2075.1993.tb05702.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arretz M., Schneider H., Guiard B., Brunner M., Neupert W. Characterization of the mitochondrial processing peptidase of Neurospora crassa. J Biol Chem. 1994 Feb 18;269(7):4959–4967. [PubMed] [Google Scholar]
  4. Bassilana M., Gwizdek C. In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force. EMBO J. 1996 Oct 1;15(19):5202–5208. [PMC free article] [PubMed] [Google Scholar]
  5. Bauer M., Behrens M., Esser K., Michaelis G., Pratje E. PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast. Mol Gen Genet. 1994 Nov 1;245(3):272–278. doi: 10.1007/BF00290106. [DOI] [PubMed] [Google Scholar]
  6. Bonnefoy N., Chalvet F., Hamel P., Slonimski P. P., Dujardin G. OXA1, a Saccharomyces cerevisiae nuclear gene whose sequence is conserved from prokaryotes to eukaryotes controls cytochrome oxidase biogenesis. J Mol Biol. 1994 Jun 3;239(2):201–212. doi: 10.1006/jmbi.1994.1363. [DOI] [PubMed] [Google Scholar]
  7. Bonnefoy N., Kermorgant M., Groudinsky O., Minet M., Slonimski P. P., Dujardin G. Cloning of a human gene involved in cytochrome oxidase assembly by functional complementation of an oxa1- mutation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11978–11982. doi: 10.1073/pnas.91.25.11978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boyd D., Beckwith J. The role of charged amino acids in the localization of secreted and membrane proteins. Cell. 1990 Sep 21;62(6):1031–1033. doi: 10.1016/0092-8674(90)90378-r. [DOI] [PubMed] [Google Scholar]
  9. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  10. Cao G., Cheng S., Whitley P., von Heijne G., Kuhn A., Dalbey R. E. Synergistic insertion of two hydrophobic regions drives Sec-independent membrane protein assembly. J Biol Chem. 1994 Oct 28;269(43):26898–26903. [PubMed] [Google Scholar]
  11. Cao G., Dalbey R. E. Translocation of N-terminal tails across the plasma membrane. EMBO J. 1994 Oct 3;13(19):4662–4669. doi: 10.1002/j.1460-2075.1994.tb06789.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cao G., Kuhn A., Dalbey R. E. The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism. EMBO J. 1995 Mar 1;14(5):866–875. doi: 10.1002/j.1460-2075.1995.tb07068.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dalbey R. E., Kuhn A., von Heijne G. Directionality in protein translocation across membranes: the N-tail phenomenon. Trends Cell Biol. 1995 Oct;5(10):380–383. doi: 10.1016/s0962-8924(00)89079-0. [DOI] [PubMed] [Google Scholar]
  14. Engelman D. M., Steitz T. A. The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell. 1981 Feb;23(2):411–422. doi: 10.1016/0092-8674(81)90136-7. [DOI] [PubMed] [Google Scholar]
  15. Errington J., Appleby L., Daniel R. A., Goodfellow H., Partridge S. R., Yudkin M. D. Structure and function of the spoIIIJ gene of Bacillus subtilis: a vegetatively expressed gene that is essential for sigma G activity at an intermediate stage of sporulation. J Gen Microbiol. 1992 Dec;138(12):2609–2618. doi: 10.1099/00221287-138-12-2609. [DOI] [PubMed] [Google Scholar]
  16. Fölsch H., Guiard B., Neupert W., Stuart R. A. Internal targeting signal of the BCS1 protein: a novel mechanism of import into mitochondria. EMBO J. 1996 Feb 1;15(3):479–487. [PMC free article] [PubMed] [Google Scholar]
  17. Gavel Y., von Heijne G. The distribution of charged amino acids in mitochondrial inner-membrane proteins suggests different modes of membrane integration for nuclearly and mitochondrially encoded proteins. Eur J Biochem. 1992 May 1;205(3):1207–1215. doi: 10.1111/j.1432-1033.1992.tb16892.x. [DOI] [PubMed] [Google Scholar]
  18. Herrmann J. M., Koll H., Cook R. A., Neupert W., Stuart R. A. Topogenesis of cytochrome oxidase subunit II. Mechanisms of protein export from the mitochondrial matrix. J Biol Chem. 1995 Nov 10;270(45):27079–27086. doi: 10.1074/jbc.270.45.27079. [DOI] [PubMed] [Google Scholar]
  19. Herrmann J. M., Stuart R. A., Craig E. A., Neupert W. Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA. J Cell Biol. 1994 Nov;127(4):893–902. doi: 10.1083/jcb.127.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Mahlke K., Pfanner N., Martin J., Horwich A. L., Hartl F. U., Neupert W. Sorting pathways of mitochondrial inner membrane proteins. Eur J Biochem. 1990 Sep 11;192(2):551–555. doi: 10.1111/j.1432-1033.1990.tb19260.x. [DOI] [PubMed] [Google Scholar]
  23. Ogasawara N., Yoshikawa H. Genes and their organization in the replication origin region of the bacterial chromosome. Mol Microbiol. 1992 Mar;6(5):629–634. doi: 10.1111/j.1365-2958.1992.tb01510.x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Pfanner N., Hartl F. U., Guiard B., Neupert W. Mitochondrial precursor proteins are imported through a hydrophilic membrane environment. Eur J Biochem. 1987 Dec 1;169(2):289–293. doi: 10.1111/j.1432-1033.1987.tb13610.x. [DOI] [PubMed] [Google Scholar]
  26. Rojo E. E., Stuart R. A., Neupert W. Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP. EMBO J. 1995 Jul 17;14(14):3445–3451. doi: 10.1002/j.1460-2075.1995.tb07350.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sirrenberg C., Bauer M. F., Guiard B., Neupert W., Brunner M. Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22. Nature. 1996 Dec 12;384(6609):582–585. doi: 10.1038/384582a0. [DOI] [PubMed] [Google Scholar]
  28. Stuart R. A., Gruhler A., van der Klei I., Guiard B., Koll H., Neupert W. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space. Eur J Biochem. 1994 Feb 15;220(1):9–18. doi: 10.1111/j.1432-1033.1994.tb18593.x. [DOI] [PubMed] [Google Scholar]
  29. Stuart R. A., Neupert W. Topogenesis of inner membrane proteins of mitochondria. Trends Biochem Sci. 1996 Jul;21(7):261–267. [PubMed] [Google Scholar]
  30. Teichmann U., van Dyck L., Guiard B., Fischer H., Glockshuber R., Neupert W., Langer T. Substitution of PIM1 protease in mitochondria by Escherichia coli Lon protease. J Biol Chem. 1996 Apr 26;271(17):10137–10142. doi: 10.1074/jbc.271.17.10137. [DOI] [PubMed] [Google Scholar]
  31. Voos W., Gambill B. D., Guiard B., Pfanner N., Craig E. A. Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. J Cell Biol. 1993 Oct;123(1):119–126. doi: 10.1083/jcb.123.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wachter C., Schatz G., Glick B. S. Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator. EMBO J. 1992 Dec;11(13):4787–4794. doi: 10.1002/j.1460-2075.1992.tb05584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Waterston R., Martin C., Craxton M., Huynh C., Coulson A., Hillier L., Durbin R., Green P., Shownkeen R., Halloran N. A survey of expressed genes in Caenorhabditis elegans. Nat Genet. 1992 May;1(2):114–123. doi: 10.1038/ng0592-114. [DOI] [PubMed] [Google Scholar]
  34. Whitley P., Gafvelin G., von Heijne G. SecA-independent translocation of the periplasmic N-terminal tail of an Escherichia coli inner membrane protein. Position-specific effects on translocation of positively charged residues and construction of a protein with a C-terminal translocation signal. J Biol Chem. 1995 Dec 15;270(50):29831–29835. doi: 10.1074/jbc.270.50.29831. [DOI] [PubMed] [Google Scholar]
  35. von Heijne G. Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature. 1989 Oct 5;341(6241):456–458. doi: 10.1038/341456a0. [DOI] [PubMed] [Google Scholar]

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