A FOLDED PROTEIN CAN BE TRANSPORTED ACROSS THE CHLOROPLAST ENVELOPE AND THYLAKOID MEMBRANES

Many thylakoid lumenal proteins are nuclear encoded, cytosolically syn thesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these t ransmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-t erminal cysteine. This allowed us to chemically link a terminal 6.5-kD a bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to creat e the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to m anipulate the structure of BPTI. Import of prOE17-BPTI into isolated c hloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the D elta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport procee ded even when the BPTI moiety was internally cross-linked into a prote ase-resistant form. These data indicate that unfolding is not a ubiqui tous requirement for protein translocation and that at least some doma ins of targeted proteins can maintain a nonlinear structure during the ir translocation into and within chloroplasts.