Targeting of thylakoid proteins by the ΔpH-driven twin-arginine translocation pathway requires a specific signal in the hydrophobic domain in conjunction with the twin-arginine motif

Superficially similar cleavable targeting signals specify whether lumenal proteins are transported across the thylakoid membrane by a Sec- or Delta pH-dependent pathway, A twin-arginine motif is essential but not sufficient to direct Delta pH-dependent targeting; here we show that a second determinant is located in the hydrophohic region. A highly hydrophobic amino acid is found either two or three residues C-terminal to the twin-arginine in all known transfer peptides for the Delta pH-dependent system, and substitution of this residue in the 23-kDa (23K) peptide markedly inhibits translocation, Further, whereas the insertion of twin-arginine in a Sec-dependent precursor does not permit efficient Delta pH-dependent targeting, the simultaneous presence of a leucine at the +3 position (relative to the RR) enables the peptide to function as efficiently as an authentic transfer peptide. RRNVL, RRAAL and RRALA within a Sec targeting signal all support efficient Delta pH-dependent targeting, RRNVA is less effective and RRNAA/RRNAG are totally ineffective, We conclude that the core signal for this pathway is a twin-arginine together with an adjacent hydrophobic determinant, (C) 1998 Federation of European Biochemical Societies.