The thylakoid proton gradient promotes an advanced stage of signal peptide binding deep within the Tat pathway receptor complex.
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.
Gérard
Fabien
F
Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611, USA.
Cline
Kenneth
K
D020816:Amino Acid Motifs; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005174:Factor XII / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D018532:Pisum sativum / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000502:physiology; D018892:Proton-Motive Force / Q000502:physiology*; D036226:Seedlings / Q000737:chemistry / Q000378:metabolism; D020524:Thylakoids / Q000737:chemistry / Q000378:metabolism*
C109236:Hcf106 protein, plant; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010940:Plant Proteins; D021382:Protein Sorting Signals; C432107:TatC protein, plant; D005174:Factor XII