Abstract
Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.
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Cashmore A, Szabo L, Timko M, Kausch A, Van denBroeck G, Schreier P, Bohnert H, Herrera-Estrella L, VanMontagu M, Schell J: Import of polypeptides into chloroplasts. Bio/Technology 3: 803–808 (1985).
Chamberlain J: Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate. Anal Biochem 98: 132–135 (1979).
Cline K, Werner-Washburne M, Andrews J, Keegstra K: Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 75: 675–678 (1984).
Cline K, Werner-Washburne M, Lubben T, Keegstra K: Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J Biol Chem 260: 3691–3696 (1985).
Ellis JR: Chloroplast proteins: synthesis, transport and assembly. Ann Rev Plant Physiol 32: 111–137 (1981).
Emr S, Vassarotti A, Garrett J, Geller B, Takeda M, Douglas M: The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol 102: 523–533 (1986).
Gasser S, Daum G, Schatz G: Import of proteins into mitochondria. Energy dependent uptake of precursors by isolated mitochondria. J Biol Chem 257: 13034–13041 (1982).
Hageman J, Robinson C, Smeekens S, Weisbeek P: A thylakoid processing protease required for complete maturation of the lumen protein plastocyanin. Nature 324: 567–569 (1986).
Hay R, Bohni P, Gasser S: How mitochondria import proteins. Biochim Biophys Acta 779: 65–87 (1984).
Horwich A, Kalousek F, Mellman I, Rosenberg L: A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J 4: 1129–1135 (1985).
Hurt E, Pesold-Hurt B, Suda K, Opligger W, Schatz G: The first twelve amino acids (less than half of the presequence) of an imported mitochondrial protein can direct mouse cytosolic dihydropholate reductase into the yeast mitochondrial matrix. EMBO J 4: 2061–2068 (1985).
Hurt E, Soltanifar N, Goldschmidt-Clermont M, Rochaix J-D, Schatz G: The cleavable presequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria. EMBO J 5: 1343–1350 (1986).
Keng T, Alani E, Guarente L: The nine amino-terminal residues of delta-aminolevulinate synthase direct betagalactosidase into the mitochondrial matrix. Mol Cell Biol 6: 355–364 (1986).
Laemmli U: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680–685 (1970).
Laskey R, Mills A: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56: 335–341 (1975).
Lubben T, Keegstra K: Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts. Proc Natl Acad Sci USA 83: 5502–5506 (1986).
Maniatis T, Fritsch E, Sambrook J: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1982).
Marres C, VanLoon A, Oudshoorn P, VanSteeg H, Grivell L, Slater E: Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulphur protein. Eur J Biochem 147: 153–161 (1985).
Melton D, Krieg P, Rebagliati M, Maniatis T, Zinn K, Green M: Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl Acids Res 12: 7035–7056 (1984).
Pelham H, Jackson R: An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem 67: 247–256 (1976).
Schmidt GW, Mishkind M: The transport of proteins into chloroplasts. Ann Rev Biochem 55: 879–912 (1986).
Schreier PH, Seftor EA, Schell J, Bohnert HJ: The use of nuclear encoded sequences to direct the light regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J 4: 25–32 (1985).
Smeekens S, VanBinsbergen J, Weisbeek P: The plant ferredoxin precursor: nucleotide sequence of a full length cDNA clone. Nucl Acids Res 13: 3179–3194 (1985).
Smeekens S, DeGroot M, VanBinsbergen J, Weisbeek P: Sequence of the precursor of the chloroplast lumen protein plastocyanin. Nature 317: 456–458 (1985).
Smeekens S, Bauerle C, Hageman J, Keegstra K, Weisbeek P: The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell 46: 365–375 (1986).
Steuber D, Ibrahimi I, Cutler D, Dobberstein B, Bujard H: A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences. EMBO J 3: 3143–3148 (1984).
Van denBroeck G, Timko MP, Kausch AP, Cashmore AR, VanMontagu M, Herrera-Estrella L: Targeting of foreign proteins to chloroplasts by fusion to the transit peptide from the small subunit of ribulose-1,5-bisphosphate carboxylase. Nature 313: 358–363 (1985).
VanLoon A, Brandli A, Schatz G: The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting. Cell 44: 801–812 (1986).
VanSteeg H, Oudshoorn P, VanHell B, Polman J, Grivell LA: Targeting efficiency of a mitochondrial pre-sequence is dependent on the passenger protein. EMBO J 5: 3643–3650 (1986).
Wasmann C, Reis B, Bartlett S, Bohnert H: The importance of the transit peptide and the transported protein for protein import into chloroplasts. Mol Gen Genet 205: 446–453 (1986).
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Smeekens, S., van Steeg, H., Bauerle, C. et al. Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides. Plant Mol Biol 9, 377–388 (1987). https://doi.org/10.1007/BF00014912
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DOI: https://doi.org/10.1007/BF00014912