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

Abstract

The thylakoidal ΔpH-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 but 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 ΔpH suggested that plastid fusion/protein translocation factor employs the ΔpH 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 ΔpH pathway for integration. First, integration was competed by an authentic ΔpH pathway precursor. Second, antibodies to ΔpH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the hcf106 null mutant were unable to integrate Pftf into their thylakoids. Thus, ΔpH pathway machinery facilitates both signal peptide-directed and N-tail-mediated membrane integration and does not strictly require the twin arginine motif.