title: Methotrexate does not block import of a DHFR fusion protein into chloroplasts.
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.
authors.affiliation: Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
authors.forename: T
authors.initials: T
authors.lastname: America
authors.forename: J
authors.initials: J
authors.lastname: Hageman
authors.forename: A
authors.initials: A
authors.lastname: Guéra
authors.forename: F
authors.initials: F
authors.lastname: Rook
authors.forename: K
authors.initials: K
authors.lastname: Archer
authors.forename: K
authors.initials: K
authors.lastname: Keegstra
authors.forename: P
authors.initials: P
authors.lastname: Weisbeek
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D002736:Chloroplasts / Q000378:metabolism; D004247:DNA; D008727:Methotrexate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010970:Plastocyanin / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D004247:DNA; D010970:Plastocyanin; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate