The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Abstract

Background:The Tat translocon transports folded proteins across energized prokaryotic cytoplasmic membranes. Results: The N-terminal transmembrane domain of TatA interacts with the membrane-stabilizing Psp machinery. Conclusion: Membrane-stabilization takes place where folded proteins are Tat-dependently translocated. Significance: Membrane stress is directly related to Tat transport.

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

“…All constructs were stably integrated into the membrane ( Figure 5E). In the acridine orange assay, full-length TatA had only a slight effect on membrane stability ( Figure 5A), which has been reported before (23). Quenching efficiency was determined as percentage of quenching relative to the fluorescence level after CCCP-induced fluorescence recovery.…”

“…As the extremely short membrane-spanning region of TatA has been suggested to have the membrane-weakening effect, we tested whether production of the TatA membrane anchor (= TatA-NT, residues 1 to 21) affects membrane stability. In earlier studies we had already fused TatA-NT to a small, extremely stable soluble protein (the mature domain of the Tat substrate HiPIP from Allochromatium vinosum, 22), which stabilizes the peptide in vivo and renders it readily detectable by SDS-PAGE/Western blotting (23). In that earlier study we had recognized that the TatA-NT-Hip construct induces the phage shock protein membrane stress response, indirectly indicating a membrane-destabilization (23).…”

“…In earlier studies we had already fused TatA-NT to a small, extremely stable soluble protein (the mature domain of the Tat substrate HiPIP from Allochromatium vinosum, 22), which stabilizes the peptide in vivo and renders it readily detectable by SDS-PAGE/Western blotting (23). In that earlier study we had recognized that the TatA-NT-Hip construct induces the phage shock protein membrane stress response, indirectly indicating a membrane-destabilization (23). In continuation of that study, we systematically analyzed possible membrane-destabilizing effects of the TatA membrane anchor and included TatA-NT extensions to Gly-33 (= TatA-33), Ala-42 (= TatA-42), Lys-49 (= TatA-49), and full-length TatA to assess a possible influence of the adjacent amphipathic helix and further C-terminal regions ( Figure 1A).…”

“…Accessibility of this segment in any orientation to the aqueous surface in the course of substrateinduced conformational changes should thus become detectable. Dyes at positions 22,23,26,27,28,32,33,34,35,42, and 45 cover the region from the hinge to the APH and an adjacent conserved negatively charged stretch. Position 51 served as positive control for quenching, as it is already positioned in an exposed region that is not anymore required for functionality (25).…”

“…In case of intact membranes, acridine orange that is present in the assay becomes protonated inside the acidified vesicles, which causes a fluorescence quenching. When the membranes show proton leakage, the proton gradient is less pronounced, resulting in a reduced acridine orange fluorescence quenching (23). First, we used TatBC-containing vesicles in which TatA was functionally inserted by in vitro translation.…”