Separate Analysis of Twin-arginine Translocation (Tat)-specific Membrane Binding and Translocation in Escherichia coli

Abstract

The twin-arginine translocation (Tat) pathway exports those precursor proteins to the periplasmic space of bacteria that harbor a twin-arginine (RR) consensus motif in their signal sequences. We have reproduced translocation of several Tat substrates into inside-out plasma membrane vesicles from Escherichia coli. Translocation proceeding at an efficiency of up to 20% occurs specifically via the Tat pathway as indicated by (i) its requirement for elevated levels of the TatABC proteins in the membrane vesicles, (ii) competition by an intact twin-arginine signal peptide, and (iii) susceptibility toward dissipation of the transmembrane H ؉ gradient. The latter treatment, while blocking translocation, still allows for functional membrane association of Tat precursors. This is shown by the finding that translocation of isolated membrane-bound Tat precursor is restored upon re-energization of the vesicles.

Early Contacts between Substrate Proteins and TatA Translocase Component in Twin-arginine Translocation

“…In complete agreement with an obligate identification of the RR consensus motif prior to establishing contacts with TatA, KK mutant precursors failed to cross-link to TatA, whereas they have repeatedly been found to still physically associate with the TatBC complex (13,36,60). In contrast to the primary membrane targeting of RR precursors at TatB and TatC (51,61), contacts with TatA turned out to be sensitive toward dissipation of the PMF. This means that the observed interaction of RR precursors with TatA does not directly coincide with their binding to TatBC but rather reflects a subsequent targeting event that is distinguished by its requirement for the PMF.…”

“…Of this solution, 5 l were used for SDS-PAGE and Western transfer, when blots were decorated with anti-TatA antibodies, and 20 l were used when using anti-TatB and antiTatC antibodies. Polyclonal antibodies against TatA, TatB, and TatC were raised in rabbits as described (19,51). The second antibody was goat anti-rabbit IgG coupled to alkaline phosphatase (Sigma).…”

“…1A) contained in the tatABCD operon of plasmid p8737. This plasmid allows T7 promoter-dependent high expression of TatABC, which is required to obtain inner membrane vesicles displaying sufficient Tat-specific translocation activity (51,55). These amber stop codon variants of tatA were transformed into E. coli BL21(DE3) cells expressing an amber suppressor tRNA and a cognate Bpa-tRNA synthetase.…”

Cysteine-scanning Mutagenesis and Disulfide Mapping Studies of the Conserved Domain of the Twin-arginine Translocase TatB Component

“…The spheroplasts were resuspended in ice-cold 33 mM Tris-HCl, 40% (w/v) sucrose. Inverted inner membrane vesicles were prepared as described (37).…”

Dual Topology of the Escherichia coli TatA Protein

“…Expression studies suggest that tatE may be a cryptic gene duplication of tatA (6). Functional Tat translocase has been reconstituted in vitro using membrane vesicles derived from cells overproducing TatA, TatB, and TatC proteins (7,8). 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).…”