Abstract
Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F1-ATPase β-subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial β-presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.
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Alton NK, Vapnek D: Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature 282: 864–869 (1979).
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: A simple phase-extraction assay for CAT activity. In: Current Protocols in Molecular Biology, pp 9.6.6–9.6.7 John Wiley, New York (1989).
Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).
Boutry M, Nagy F, Poulsen C, Aoyagi K, Chua NH: Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants. Nature 328: 340–342 (1987).
Bring S, Flügge UI, Chaumont F, Boutry M, Emmermann M, Schmitz U, Becker K, Pfanner N: Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria. J Biol Chem 269: 16478–16485 (1994).
Caplan AJ, Cyr DM, Douglas MG: YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism Cell 71: 1143–1155 (1992).
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).
Chaumont F, Silva Filho M de C, Thomas D, Leterme S, Boutry M: Truncated presequences of mitochondrial F1-ATPase β subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco. Plant Mol Biol 24: 631–641 (1994).
Cumsky MG, Trueblood CE, Ko C, Poyton RO: Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxydase subunit V. Mol Cell Biol 7: 3511–3519 (1987).
de Boer AD, Weisbeek PJ: Chloroplast protein topogenesis, import, sorting and assembly. Biochim Biophys Acta 1071: 221–253 (1991).
Deshaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R: A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature 332: 800–805 (1988).
Franzen LG, Rochaix JD, von Heijne G: Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplasts presequences. FEBS Lett 260: 165–168 (1990).
Franzen LG, Falk KG: Nucleotide sequence of cDNA clones encoding the β subunit of mitochondrial ATP synthase from the green alga Chlamydomonas reinhardtii: the precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension Plant Mol Biol 19: 771–780 (1992).
Galanis M, Devenish RJ, Nagley P: Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency. FEBS Lett 282: 425–430 (1991).
Glick B, Schatz G: Import of proteins into mitochondria. Annu Rev Genet 25: 21–44 (1991).
Hachiya N, Alam R, Sakasegawa Y, Sakaguchi M, Mihara K, Omura T: A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins. EMBO J 12: 1579–1586 (1993).
Hachiya N, Komiya T, Alam R, Iwahashi J, Sakaguchi M, Omura T, Mihara K: MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria. EMBO J. 13: 5146–5154 (1994).
Hartl FU, Pfanner N, Nicholson DW, Neupert W: Mitochondrial protein import. Biochim Biophys Acta 988: 1–45 (1989).
Horwich AL, Kalousek F, Mellman I, Rosemberg LE: A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J 4: 1129–1135 (1985).
Huang J, Hack E, Thornburg W, Myers AM: A yeast mitochondrial leader peptide functions in vivo a dual targeting signal for both chloroplasts and mitochondria. Plant Cell 2: 1249–1260 (1990).
Hurt EC, Soltanifar N, Goldschmidt-Clermont M, Rochaix JD, Schatz G: The cleavable pre-sequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria. EMBO J 5: 1343–1350 (1986).
Jefferson RA, Burgess SM, Hirsh D: β-glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83: 8447–8451 (1986).
Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).
Kavanagh TA, Jefferson RA, Bevan MW: Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b binding. Mol Gen Genet 215: 38–45 (1988).
Keegstra K, Olsen LJ, Theg S: Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Physiol Plant Mol Biol 40: 471–501 (1989).
Kimura T, Takeda S, Kyozuka J, Asahi T, Shimamoto K, Nakamura K: Presequence of a precursor for the-subunit of sweet potato mitochondrial F1ATPase is not sufficient for the transport of β-glucuronidase (GUS) into mitochondria in tobacco, rice and yeast cells. Plant Cell Physiol 34: 345–355 (1993).
Kübrick M, Dietmeier K, Pfanner N: Genetic and biochemical dissection of the mitochondrial protein-import machinery. Curr Genet 27: 393–403 (1995).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lithgow T, Hoj PB, Hoogenraad NJ: Do cytosolic factors prevent promiscuity at the membrane surface? FEBS Lett 329: 1–4 (1993).
Lithgow T, Glick BS, Schatz G: The protein import receptor of mitochondria. Trends Biochem Sci 20: 98–101 (1995).
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 267–275 (1951).
Maeshima M, Hattori T, Asahi T: Purification of complexesII and IV from plant mitochondria. Meth Enzymol 148: 491–501 (1987).
Murakami K, Tanase S, Morino Y, Mori M: Presequence binding factor-dependent and-independent import of proteins into mitochondria. J Biol Chem 267: 13119–13122 (1992).
Pfaller R, Pfanner N, Neupert W: Mitochondrial protein import. Bypass of a proteinaceous surface receptors can occur with low specificity and efficiency. J Biol Chem 264: 34–39 (1989).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
Schmidt GW, Bartlett SG, Grossman AR, Cashmore AR, Chua NH: Biosynthetic pathways of two polypeptide subunits of light-harvesting chlorophyll a/b protein complex. J Cell Biol 91: 468–478 (1981).
Schmitz UK, Londsdale DM: A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo. Plant Cell 1: 783–791 (1989).
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 (1979).
Turner SR, Ireland R, Rawsthorne S: Purification and primary amino acid sequence of the L subunit of glycine decarboxylase. J Biol Chem 267: 7745–7550 (1992).
Van den Broeck G, Timko MP, Kausch AP, Cashmore AR, Van Montagu M, Herrera-Estrela L: Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. Nature 313: 358–363 (1985).
Vassarotti A, Stroud R, Douglas M: Independent mutations at the amino terminus of a protein act as a surrogate signals for mitochondrial import. EMBO J 6: 705–711 (1987).
Verner K: Co-translational protein import into mitochondria: an alternative view. Trends Biochem Sci 18: 366–371 (1993).
van Heijne G, Steppuhn J, Herrmann RG: Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).
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Silva Filho, M.d.C., Chaumont, F., Leterme, S. et al. Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles. Plant Mol Biol 30, 769–780 (1996). https://doi.org/10.1007/BF00019010
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DOI: https://doi.org/10.1007/BF00019010