Abstract
The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of Escherichia coli and plant plastids, a third component – TatB – is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells.
Highlights
The twin-arginine translocation (Tat) system is a general translocation system that serves to translocate folded proteins with N-terminal signal peptides across energized membranes in prokaryotes and plant plastids [1]
We addressed the substrate-translocon interactions of the Tat system by a site-directed in vivo cross-linking method that has been developed by the group of Peter G
A specific amber stop codon suppressor tRNA is loaded exclusively with the artificial amino acid p-benzoyl-L-phenylalanine, which is efficiently incorporated into engineered amber stop codons of recombinant proteins
Summary
The twin-arginine translocation (Tat) system is a general translocation system that serves to translocate folded proteins with N-terminal signal peptides across energized membranes in prokaryotes and plant plastids [1]. Tat signal peptides contain a characteristic amino acid pattern that typically includes the two eponymous arginines [2]. Tat systems minimally consist of the two components TatA and TatC [3]. Often a third component that is sequence-related to TatA is found that has a higher affinity to TatC and that is termed TatB [2]. In E. coli as well as in plant plastids, TatBC-containing complexes recognize the twin-arginine motif and thereby bind the Tat substrates [4,5,6,7]. Some TatA associates with TatBC complexes already before substrate-binding and can be important for high-affinity binding of substrates at physiological
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