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

Abstract

Cleavable N-terminal targeting signals direct the translocation of lumenal proteins across the chloroplast thylakoid membrane by either a Sec-type or delta pH-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 delta pH-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 delta pH-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.

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

“…Earlier studies had indicated that the presence of a positive charge at the C terminus of Tat leader peptides can serve as a ''Sec-avoidance'' signal (5,43). Although ssDmsA, ssSufI, and ssYacK all possess a charged amino acid in this region it appears that the C-domain-positive charge alone is insufficient for preventing promiscuous export by the Sec pathway, at least when these leaders are fused to AP.…”

Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene

“…Feltham First) seedlings using differential centrifugation followed by Percoll gradient centrifugation as described by Bogsch et al (1997). Chloroplasts were washed twice in import buffer, resuspended at 1 mg chlorophyll/mL, and used immediately.…”

Signal peptide–chaperone interactions on the twin-arginine protein transport pathway

“…The twin-arginine motif is always located at the boundary between the n-and h-regions, with the arginine pair being critical for successful translocation (1). Definitive physiological functions have not yet been assigned to the n-regions of any Tat signal peptides, although c-regions sometimes contain a positively charged Sec-avoidance motif thought to prevent mistargeting of some Tat substrates (17).…”