Abstract
Tat (twin arginine translocation) systems transport folded proteins across the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria. Tat precursors are targeted by hydrophobic cleavable signal peptides with twin arginine (RR) motifs. Bacterial precursors possess an extended consensus, (S/T)RRXFLK, of which the two arginines and the phenylalanine are essential for efficient transport. Thylakoid Tat precursors possess twin arginines but lack the consensus phenylalanine. Here, we have characterized two stages of precursor binding to the thylakoid Tat signal peptide receptor, the 700-kDa cpTatC-Hcf106 complex. The OE17 precursor tOE17 binds to the receptor by RR-dependant electrostatic interactions and partially dissociates during blue native gel electrophoresis. In addition, the signal peptide of thylakoid-bound tOE17 is highly exposed to the membrane surface, as judged by accessibility to factor Xa of cleavage sites engineered into signal peptide flanking regions. By contrast, tOE17 containing a consensus phenylalanine in place of Val(-20) (V - 20F) binds the receptor more strongly and is completely stable during blue native gel electrophoresis. Thylakoid bound V - 20F is also completely protected from factor Xa at the identical sites. This suggests that the signal peptide is buried deeply in the cpTatC-Hcf106 binding site. We further provide evidence that the proton gradient, which is required for translocation, induces a tighter interaction between tOE17 and the cpTat machinery, similar to that exhibited by V - 20F. This implies that translocation involves a very intimate association of the signal peptide with the receptor complex binding site.
Highlights
Are distinctive in that they transport proteins in a folded conformation, and they employ only the protonmotive force as energy source
The thylakoid Tat system has been experimentally staged into several steps [8, 9]; the precursor protein binds to a cpTatC-Hcf106 receptor complex, a Tha4 oligomer assembles with the precursor-receptor complex to form the putative translocase [10], and the precursor is transported into the lumen
A Position-specific Phenylalanine Substitution in the Signal Peptide Results in tOE17 That Forms a Tighter and More Stable Association with the cpTat Receptor Complex—As reported previously [13] and designated in Fig. 1A, (Tmd)Phe substitutions of certain residues in the tOE17 signal peptide resulted in a more stable association of the precursor with the 700-kDa receptor complex, cpTatC-Hcf106. One possibility for this effect was that the phenylalanine moiety of (Tmd)Phe increased the overall hydrophobicity of the signal peptide
Summary
Are distinctive in that they transport proteins in a folded conformation, and they employ only the protonmotive force as energy source (for the most recent reviews, see Refs. 1– 4). These results support a model in which, at the moment of transport, the signal peptide of Tat precursors becomes more tightly associated with and deeply buried in the binding site of the cpTatC-Hcf106 receptor complex.
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