Differential Interactions between a Twin-Arginine Signal Peptide and Its Translocase in Escherichia coli

Abstract

The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

“…Tat-signal peptides are recognized at the membrane by a TatBC receptor complex (18)(19)(20)(21)(22)(23). Substrate protein binding to TatBC causes a PMF-dependent recruitment of TatA (and presumably TatE) to assemble the active translocation site (18,24).…”

The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter

“…Projection maps of Tat complexes obtained by negative stain electron microscopy show that each type of complex forms particles of Ϸ90-160 Å (1 Å ϭ 0.1 nm) in diameter (15,17). TatBC has been shown to act as the receptor element of the translocation pathway (13,16,18), and TatC has been shown to contain the primary binding site for the signal peptide (18). The function of TatA is less clear.…”

“…The function of TatA is less clear. It is known that TatA is required subsequent to substrate recognition by the TatBC complex (16,18,19), and this has led to the suggestion that TatA constitutes the protein-conducting channel of the Tat system. Recent chemical crosslinking studies suggest that TatA transiently associates with the TatBC-substrate complex during active protein translocation (18,19).…”

Dual Topology of the Escherichia coli TatA Protein

“…A large complex of ϳ650 kDa containing TatABC has been purified from the detergent-solubilized E. coli membrane (9,10) and shown to be capable of binding a Tat signal peptide (10). Recently, Alami et al (11) have reported a hierarchy in targeting of a Tat substrate to the TatBC complex. For the primary interaction TatC is both necessary and sufficient, whereas a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore (11).…”

“…Recently, Alami et al (11) have reported a hierarchy in targeting of a Tat substrate to the TatBC complex. For the primary interaction TatC is both necessary and sufficient, whereas a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore (11). Apparently, TatB and TatC are present in a constant 1:1 stoichiometry in the E. coli TatABC complex, whereas the vast majority of the TatA protein does not co-purify with the TatBC core complex (9).…”