REGULATION OF CHLOROPLAST PROTEIN IMPORT THROUGH A PROTOCHLOROPHYLLIDE-RESPONSIVE TRANSIT PEPTIDE

NADPH:protochlorophyllide (Pchlide) oxidoreductase (FOR) is the key en zyme of chlorophyll biosynthesis in angiosperms, In barley, two POR en zymes, termed PORA and PORB, exist. Both are nucleus-encoded plastid p roteins that must he imported posttranslationally from the cytosol. Wh ereas the import of the precursor of PORI, pPORA, previously has been shown to depend on Pchlide, the import of pPORB occurred constitutivel y. To study this striking difference, chimeric precursor proteins were constructed in which the transit sequences of the pPORA and pPORB wer e exchanged and fused to either their cognate polypeptides or to a cyt osolic dihydrofolate reductase (DHFR) reporter protein of mouse, As sh own here, the transit peptide of the pPORA (transA) conferred the Pchl ide requirement of import unto both the mature PORTS and the DHFR. By contrast, the transit peptide of the pPORB directed the reporter prote in into both chloroplasts that contained or lacked translocation-activ e Pchlide, bz vitro binding studies further demonstrated that the tran sit peptide of the pPORA, but not of the pPORB, is able to bind Pchlid e. Wr: conclude that the import of the authentic pPORA and that of the transA-PORB and transA-DHFR fusion proteins is regulated by a direct transit peptide-Pchlide interaction, which is likely to occur in the p lastid envelope, a major site of porphyrin biosynthesis.