METHOTREXATE DOES NOT BLOCK IMPORT OF A DHFR FUSION PROTEIN INTO CHLOROPLASTS

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 mou se cytosolic dihydrofolate reductase (DHFR). The chimeric protein is t argeted to chloroplasts and is competent for import. The conformation of PCDHFR can be stabilized by complexing with methotrexate, an analog ue of the substrate of DHFR. Methotrexate strongly inhibits DHFR impor t into yeast mitochondria (M. filers and G. Schatz, Nature 322 (1986) 228-232), presumably because the precursor must unfold to cross the me mbrane and it cannot do so when complexed with methotrexate. We show h ere that methotrexate does not block PCDHFR import into chloroplasts. Methotrexate does slow the rate of import, and protects DHFR from degr adation once inside chloroplasts. The processed protein is localized i n the stroma, indicating that import into thylakoids is impeded. Prote ase sensitivity assays indicate that the complex of precursor protein with methotrexate changes in conformation during the translocation acr oss the envelope.