Abstract
Protein import into chloroplasts requires the movement of a precursor protein across the envelope membranes. The conformation of a precursor as it passes from the aqueous medium across the hydrophobic membranes is not known in detail. To address this problem we examined precursor conformation during translocation using the chimeric precursor PCDHFR, which contains the plastocyanin (PC) transit peptide in front of mouse cytosolic dihydrofolate reductase (DHFR). The chimeric protein is targeted to chloroplasts and is competent for import. The conformation of PCDHFR can be stabilized by complexing with methotrexate, an analogue of the substrate of DHFR. Methotrexate strongly inhibits DHFR import into yeast mitochondria (M. Eilers and G. Schatz, Nature 322 (1986) 228–232), presumably because the precursor must unfold to cross the membrane and it cannot do so when complexed with methotrexate. We show here that methotrexate does not block PCDHFR import into chloroplasts. Methotrexate does slow the rate of import, and protects DHFR from degradation once inside chloroplasts. The processed protein is localized in the stroma, indicating that import into thylakoids is impeded. Protease sensitivity assays indicate that the complex of precursor protein with methotrexate changes in conformation during the translocation across the envelope.
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References
Beckmann RP, Mizzen LA, Welch WJ: Interaction of Hsp 70 with newly synthesized proteins: Implications for protein folding and assembly. Science 248: 850–854 (1990).
Chen W-J, Douglas MG: The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation. J Biol Chem 262: 15605–15609 (1987).
Chirico WJ, Waters MG, Blobel G, 70kD heat shock related proteins stimulate protein translocation into microsomes. Nature 332: 805–810 (1988).
deBoer AD, Weisbeek PJ: Chloroplast topogenesis: protein import, sorting, and assembly. Biochim Biophys Acta 1071: 221–253 (1991).
Della-Cioppa G, Kishore GM: Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate. EMBO J 7: 1299–1305 (1988).
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).
Deutsch JC, Ellwood PC, Portillo RM, Macey MG, Kolhouse JF: Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells. Arch Biochem Biophys 274: 327–337 (1989).
Eilers M, Hwang S, Schatz G: Unfolding and refolding of a purified precursor protein during import into isolated mitochondria. EMBO J 7: 1139–1145 (1988).
Eilers M, Schatz G: Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature 322: 228–232 (1986).
Endo T, Eilers M, Schatz G: Binding of a tightly folded artificial mitochondrial precursor protein to the mitochondrial outer membrane involves a lipid-mediated conformational change. J Biol Chem 264: 2951–2956 (1989).
Endo T, Schatz G: Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding. EMBO J 7: 1153–1158 (1988).
Guéra A, America T, vanWaas M, Weisbeek PJ: A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes. Plant Mol Biol 23: 309–324 (1993).
Haber DA, Beverly SM, Kiely ML, Schimke RT: Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. J Biol Chem 256: 9501–9510 (1981).
Hageman J, Baecke C, Ebskamp M, Pilon R, Smeekens S, Weisbeek P: Protein import into and sorting inside the chloroplast are independent processes. Plant Cell 2: 479–494 (1990).
Hageman J, Robinson C, Smeekens S, Weisbeek P: A thylakoid processing protease is required for complete maturation of the lumen protein plastocyanin. Nature 324: 567–569 (1986).
Heldt HW: Measurement of metabolite movement across the envelope and of the pH in the stroma and the thylakoid space in intact chloroplasts. Meth Enzymol 69: 604–613 (1980).
Keegstra K, Olsen LJ, Theg SM: Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Physiol Plant Mol Biol 40: 471–502 (1989).
Ko K, Bornemisza O, Kourtz L, Ko ZW, Plaxton WC, Cashmore AR: Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope. J Biol Chem 267: 2986–2993 (1992).
Lubben TH, Keegstra K: Efficient in-vitro import of a cytosolic heat shock protein into pea chloroplasts. Proc Natl Acad Sci USA 83: 5502–5506 (1986).
Marshall J, DeRocher AE, Keegstra K, Vierling E: Identification of heat shock hsp 70 homologues in chloroplasts. Proc Natl Acad Sci USA 87: 374–378 (1990).
Muller G, Zimmermann R: Import of honeybee prepromellitin into the endoplasmic reticulum: energy requirements for membrane insertion. EMBO J 7: 639–648 (1988).
Murakami H, Pain D, Blobel G: 70-kD Heat shockrelated protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J Cell Biol 107: 2051–2057 (1988).
Ono H, Tuboi S: Presence of a cytosolic factor stimulating the import of precursor of mitochondrial proteins in rabbit reticulocytes and rat liver cells. Arch Biochem Biophys 277: 368–373 (1990).
Pfanner N, Tropschug M, Neupert W: Mitochondrial protein import: nucleoside triphosphates are involved in conferring import-competence to precursors. Cell 49: 815–823 (1987).
Price EM, Ratnam M, Rodeman KM, Freisheim JH: Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein. Biochemistry 27: 7853–7858 (1988).
Randall LL, Hardy SJS: Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell 46: 921–928 (1986).
Rassow J, Guiard B, Wienhues U, Herzog V, Hartl F-U, Neupert W: Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites. J Cell Biol 109: 1421–1428 (1989).
Rothman JE: Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell 59: 591–601 (1989).
Sheffield WP, Shore GC, Randall SK: Mitochondrial precursor protein: Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation and import competence. J Biol Chem 265: 11069–11076 (1990).
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: 365–376 (1986).
Smeekens S, deGroot M, vanBinsbergen J, Weisbeek P: Sequence of the precursor of the chloroplast thylakoid lumen protein plastocyanin. Nature 317: 456–458 (1985).
Van'tHoff R, Demel RA, Keegstra K, deKruijff B: Lipid-peptide interactions between fragments of the transit peptide of ribulose-1,5,-biphosphate carboxylase/oxygenase and chloroplast membrane lipids. FEBS Lett 291: 350–354 (1991).
Van'tHoff R, vanKlompenburg W, Pilon M, Kozubek A, deKorte-Kool G, Demel RA, Weisbeek PJ, deKruijff B: The transit sequence mediates the specific interaction of the precursor protein of ferredoxin with chloroplast membrane lipids. J Biol Chem 268: 4037–4042 (1993).
Vestweber D, Schatz G: A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites. J Cell Biol 107: 2037–2043 (1988).
Waegemann K, Paulsen H, Soll J: Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence. FEBS Lett 261: 89–92 (1990).
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America, T., Hageman, J., Guéra, A. et al. Methotrexate does not block import of a DHFR fusion protein into chloroplasts. Plant Mol Biol 24, 283–294 (1994). https://doi.org/10.1007/BF00020168
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DOI: https://doi.org/10.1007/BF00020168