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Functional analysis of a recently originating, atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast

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Abstract

A gene family of at least five members encodes the tobacco mitochondrial Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichia coli β-glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS proteins were localized by cell fractionation. The aminoterminal 75 and 71 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-terminal sequence of RISP4, which contains an atypical mitochondrial presequence, can translocate the GUS protein specifically into tobacco mitochondria with apparently low efficiency.

Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the tobacco RISP3 and RISP4 presequences can direct import and processing of a GUS fusion protein in yeast mitochondria. Plant presequences, however, direct mitochondrial import in yeast less efficiently than the yeast presequence, indicating subtle differences between the plant and yeast mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an amphipathic secondary structure; however, these structural properties may improve the efficiency of mitochondrial import.

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References

  1. An G, Ebert PR, Mitra A, Ha SB: Binary vectors. In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant Molecular Biology Manual, pp. A3/1-A3/19. Kluwer Academic Publishers, Dordrecht (1988).

    Google Scholar 

  2. Becker DM, Guarente L: High-efficiency transformation of yeast by electroporation. In: Guthrie C, Fink GR (eds) Guide to Yeast Genetics and Molecular Biology, pp. 182–187. Academic Press, San Diego (1991).

    Google Scholar 

  3. Beckmann JD, Ljungdahl PO, Lopez JL, Trumpower BL: Isolation and characterization of the nuclear gene encoding the Rieske iron-sulfur protein (RIP1) from Saccharomyces cerevisiae. J Biol Chem 262: 8901–8909 (1987).

    PubMed  Google Scholar 

  4. Bedwell DM, Strobel SA, Yun K, Jongeward GD, Emr SD: Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis. Mol Cell Biol 9: 1014–1025 (1989).

    PubMed  Google Scholar 

  5. Bibus CR, Lemire BD, Suda K, Schatz G: Mutations restoring import of a yeast mitochondrial protein with a nonfunctional presequence. J Biol Chem 263: 13097–13102 (1988).

    PubMed  Google Scholar 

  6. Boutry M, Nagy F, Poulsen C, Aoyagi K, Chua N-H: Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants. Nature 328: 340–342 (1987).

    PubMed  Google Scholar 

  7. Bowler C, Alliotte T, Van den Bulcke M, Bauw G, Vandekerckhove J, Van Montagu M, Inzé D: A plant manganese superoxide dismutase is efficiently imported and correctly processed by yeast mitochondria. Proc Natl Acad Sci USA 86: 3237–3241 (1989).

    PubMed  Google Scholar 

  8. Brandt U, Yu L, Yu C-A, Trumpower BL: The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc 1 complex in mammals and retained as a subunit in the complex. J Biol Chem 268: 8387–8390 (1993).

    PubMed  Google Scholar 

  9. Braun H-P, Emmermann M, Kruft V, Schmitz UK: The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain. EMBO J 11: 3219–3227 (1992).

    PubMed  Google Scholar 

  10. Chaumont F, Boutry M: Protein import into plant mitochondria. In: Levings CS III, Vasil IK (eds) Molecular Biology of the Mitochondria. Kluwer Academic Publishers. Dordrecht (in press).

  11. Chaumont F, O'Riordan V, Boutry M: Protein transport into mitochondria is conserved between plant and yeast species. J Biol Chem 265: 16856–16862 (1990).

    PubMed  Google Scholar 

  12. Cheng MY, Pollock RA, Hendrick JP, Horwich AL: Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae. Proc Natl Acad Sci USA 84: 4063–4067 (1987).

    PubMed  Google Scholar 

  13. Cigan AM, Donahue TF: Sequence and structural features associated with translational initiator regions in yeast: a review. Gene 59: 1–18 (1987).

    Article  PubMed  Google Scholar 

  14. Daum G, Böhni PC, Schatz G: Import of proteins into mitochondria. Cytochrome b 2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem 257: 13028–13033 (1982).

    PubMed  Google Scholar 

  15. Dircks LK, Poyton RO: Overexpression of a leaderless form of yeast cytochrome c oxidase subunit Va circumvents the requirement for a leader peptide in mitochondrial import. Mol Cell Biol 10: 4984–4986 (1990).

    PubMed  Google Scholar 

  16. Eisenberg D, Schwarz E, Komaromy M, Wall R: Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179: 125–142 (1984).

    Article  PubMed  Google Scholar 

  17. Eisenberg D, Weiss RM, Terwilliger TC: The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature 299: 371–374 (1982).

    PubMed  Google Scholar 

  18. Emmermann M, Clericus M, Braun H-P, Mozo T, Heins L, Kruft V, Schmitz UK: Molecular features, processing and import of the Rieske iron-sulfur protein from potato mitochondria. Plant Mol Biol 25: 271–281 (1994).

    Article  PubMed  Google Scholar 

  19. Emmermann M, Schmitz UK: The cytochrome c reductase integrated processing peptidase from potato mitochondria belongs to a new class of metalloendoproteases. Plant Physiol 103: 615–620 (1993).

    PubMed  Google Scholar 

  20. Eriksson AC, Glaser E: Mitochondrial processing proteinase: a general processing proteinase of spinach leaf mitochondria is a membrane-bound enzyme. Biochim Biophys Acta 1140: 208–214 (1992).

    Google Scholar 

  21. Fu W, Japa S, Beattie DS: Import of the iron-sulfur protein of the cytochrome b.c 1 complex into yeast mitochondria. J Biol Chem 265: 16541–16547 (1990).

    PubMed  Google Scholar 

  22. Gavel Y, Nilsson L, von Heijne G: Mitochondrial targeting sequences. Why ‘non-amphiphilic’ peptides may still be amphiphilic. FEBS Lett 235: 173–177 (1988).

    Article  PubMed  Google Scholar 

  23. Graham LA, Brandt U, Sargent JS, Trumpower BL: Mutational analysis of assembly and function of the ironsulfur protein of the cytochrome bc 1 complex in Saccharomyces cerevisiae. J Bioenerget Biomembr 25: 245–257 (1993).

    Google Scholar 

  24. Graham LA, Trumpower BL: Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc 1 complex of iron-sulfur protein lacking the iron-sulfur cluster. J Biol Chem 266: 22485–22492 (1991).

    PubMed  Google Scholar 

  25. Hartl F-U. Pfanner N, Nicholson DW, Neupert W: Mitochondrial protein import. Biochim Biophys Acta 988: 1–45 (1989).

    PubMed  Google Scholar 

  26. Hartl F-U, Schmidt B, Wachter E, Weiss H, Neupert W: Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase. Cell 47: 939–951 (1986).

    Article  PubMed  Google Scholar 

  27. Hendrick JP, Hodges PE, Rosenberg LE: Survey of amino-terminal proteolytic cleavage sites in mitochondrial precursor proteins: leader peptides cleaved by two matrix proteases share a three-amino acid motif. Proc Natl Acad Sci USA 86: 4056–4060 (1989).

    PubMed  Google Scholar 

  28. Hill JE, Myers AM, Koerner TJ, Tzagoloff A: Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2: 163–167 (1986).

    PubMed  Google Scholar 

  29. Huang J, Hack E, Thornburg RW, Myers AM: A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria. Plant Cell 2: 1249–1260 (1990).

    Article  PubMed  Google Scholar 

  30. Huang J, Struck F, Matzinger DF, Levings CS III: Functional analysis in yeast of cDNA coding for the mitochondrial Rieske iron-sulfur protein of higher plants. Proc Natl Acad Sci USA 88: 10716–10720 (1991).

    PubMed  Google Scholar 

  31. Huang J, Struck F, Matzinger DF, Levings CS III: Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number. Plant Cell 6: 439–448 (1994).

    Article  PubMed  Google Scholar 

  32. Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).

    Google Scholar 

  33. Kimura T, Takeda S, Kyozuka J, Asahi T, Shimamoto K, Nakamura K: The presequence of a precursor to the δ-subunit of sweet potato mitochondrial F1ATPase is not sufficient for the transport of β-glucuronidase (GUS) into mitochondria of tobacco, rice and yeast cells. Plant Cell Physiol 34: 345–355 (1993).

    PubMed  Google Scholar 

  34. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).

    PubMed  Google Scholar 

  35. Leonard K, Wingfield P, Arad T, Weiss H: Three-dimensional structure of ubiquinol:cytochrome c reductase from Neurospora mitochondria determined by electron microscopy of membrane crystals. J Mol Biol 149: 259–274 (1981).

    PubMed  Google Scholar 

  36. Martin T, Wöhner R-V, Hummel S, Willmitzer L, Frommer WB: The GUS reporter system as a tool to study plant gene expression. In: Gallagher SR (ed), GUS protocols: using the GUS Gene as a Reporter of Gene Expression, pp. 23–43. Academic Press, San Diego (1992).

    Google Scholar 

  37. Mersereau M, Pazour GJ, Das A: Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90: 149–151 (1990).

    Article  PubMed  Google Scholar 

  38. Moore AL, Wood CK, Watts FZ: Protein import into plant mitochondria. Annu Rev Plant Physiol Plant Mol Biol 45: 545–575 (1994).

    Article  Google Scholar 

  39. Pfaller R, Pfanner N, Neupert W: Mitochondrial protein import. Bypass of proteinaceous surface receptors can occur with low specificity and efficiency. J Biol Chem 264: 34–39 (1989).

    PubMed  Google Scholar 

  40. Pfanner N, Pfaller R, Neupert W: How finicky is mitochondrial protein import? Trends Biochem Sci 13: 165–167 (1988).

    Article  PubMed  Google Scholar 

  41. Roise D, Schatz G: Mitochondrial presequences. J Biol Chem 263: 4509–4511 (1988).

    PubMed  Google Scholar 

  42. Roise D, Theiler F, Horvath SJ, Tomich JM, Richards JH, Allison DS, Schatz G: Amphiphilicity is essential for mitochondrial presequence function. EMBO J 7: 649–653 (1988).

    PubMed  Google Scholar 

  43. Schiffer M, Edmundson AB: Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J 7: 121–135 (1967).

    PubMed  Google Scholar 

  44. Schmitz UK, Lonsdale DM: A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo. Plant Cell 1: 783–791 (1989).

    Article  PubMed  Google Scholar 

  45. Tamm LK: Membrane insertion and lateral mobility of synthetic amphiphilic signal peptides in lipid model membranes. Biochim Biophys Acta 1071: 123–148 (1991).

    PubMed  Google Scholar 

  46. Trumpower BL: Cytochrome bc 1 complexes of microorganisms. Microbiol Rev 54: 101–129 (1990).

    PubMed  Google Scholar 

  47. Trumpower BL: Function of the iron-sulfur protein of the cytochrome b-c1segment in electron-transfer and energy-conserving reactions of the mitochondrial respiratory chain. Biochim Biophys Acta 639: 129–155 (1981).

    PubMed  Google Scholar 

  48. van Steeg H, Oudshoorn P, van Hell B, Polman JEM, Grivell LA: Targeting efficiency of a mitochondrial presequence is dependent on the passenger protein. EMBO J 5: 3643–3650 (1986).

    PubMed  Google Scholar 

  49. Vassarotti A, Stroud R, Douglas M: Independent mutations at the amino terminus of a protein act as surrogate signals for mitochondrial import. EMBO J 6: 705–711 (1987).

    PubMed  Google Scholar 

  50. von Heijne G: Mitochondrial targeting sequences may form amphiphilic helices. EMBO J 5: 1335–1342 (1986).

    PubMed  Google Scholar 

  51. von Heijne G, Steppuhn J, Herrmann RG: Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).

    PubMed  Google Scholar 

  52. Winning BM, Sarah CJ, Purdue PE, Day CD, Leaver CJ: The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria. Plant J 2: 763–773 (1992).

    Article  PubMed  Google Scholar 

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  1. Department of Genetics, North Carolina State University, Box 7614, 27695-7614, Raleigh, NC, USA

    Jintai Huang & Charles S. Levings III

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  1. Jintai Huang
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Huang, J., Levings, C.S. Functional analysis of a recently originating, atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast. Plant Mol Biol 29, 519–533 (1995). https://doi.org/10.1007/BF00020982

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  • DOI: https://doi.org/10.1007/BF00020982

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