The thylakoid ΔpH-dependent pathway machinery facilitates RR-independent N-tail protein integration

The thylakoidal Delta pH-dependent and bacterial twin arginine transport systems are structurally and functionally related protein export machineries. These recently discovered systems have been shown to transport folded proteins brit are not known to assemble integral membrane proteins. We determined the translocation pathway of a thylakoidal FtsH homologue, plastid fusion/protein translocation factor, which is synthesized with a chloroplast-targeting peptide, a hydrophobic signal peptide, and a hydrophobic membrane anchor. The twin arginine motif in its signal peptide and its sole integration requirement of a Delta pH suggested that plastid fusion/protein translocation factor employs the Delta pH pathway. Surprisingly, changing the twin arginine to twin lysine or deleting the signal peptide did not abrogate integration capability or characteristics. Nevertheless, three criteria argue that all three forms require the Delta pH pathway for integration. First, integration was competed by an authentic Delta pH pathway precursor. Second, antibodies to Delta pH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the hcf106 null mutant were unable to integrate Pftf into their thylakoids. Thus, Delta pH pathway machinery facilitates both signal peptide-directed and N-tail-mediated membrane integration and does not strictly require the twin arginine motif.