Pathway specificity for a ΔpH‐dependent precursor thylakoid lumen protein is governed by a 'sec‐avoidance’ motif in the transfer peptide and a 'sec‐incompatible’ mature protein

Cleavable N‐terminal targeting signals direct the translocation of lumenal proteins across the chloroplast thylakoid membrane by either a Sec‐type or ΔpH‐driven protein translocase. The targeting signals specify choice of translocation pathway, yet all resemble typical bacterial 'signal’ peptides in possessing a charged N‐terminus (N‐domain), hydrophobic core region (H‐domain) and more polar C‐terminal region (C‐domain). We have previously shown that a twin‐arginine motif in the N‐domain is essential for targeting by the ΔpH‐dependent pathway, but it has remained unclear why targeting signals for this system (transfer peptides) are not recognized by the Sec apparatus. We show here that the conserved charge distribution around the H‐domain in the 23K transfer peptide (twin‐Arg in the N‐domain, Lys in the C‐domain) constitutes a 'sec‐avoidance’ signal. The C‐domain Lys, while not important for ΔpH‐dependent targeting, is the only barrier to Sec‐dependent translocation; its removal generates an apparently perfect signal peptide. Conversely, insertion of twin‐Arg into the N‐domain of a Sec substrate has little effect, as has insertion of a C‐domain Lys, but the combined substitutions almost totally block transport. We also show that the 23K mature protein is incapable of being targeted by the Sec pathway, and it is proposed that the role of the Sec‐avoidance motif in the transfer peptide is to prevent futile interactions with the Sec apparatus.