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

Abstract

The twin-arginine translocation (Tat) system that comprises the TatA, TatB, and TatC components transports folded proteins across energized membranes of prokaryotes and plant plastids. It is not known, however, how the transport of this protein cargo is achieved. Favored models suggest that the TatA component supports transport by weakening the membrane upon full translocon assembly. Using Escherichia coli as model organism, we now demonstrate in vivo that the N-terminus of TatA can indeed destabilize the membrane, resulting in a lowered membrane energization in growing cells. We found that in full-length TatA, this effect is counterbalanced by its amphipathic helix. Consistent with these observations, the TatA N-terminus induced proton leakage in vitro, indicating membrane destabilization. Fluorescence quenching data revealed that substrate binding causes the TatA hinge region and the N-terminal part of the TatA amphipathic helix to move toward the membrane surface. In the presence of TatBC, substrate binding also reduced the exposure of a specific region in the amphipathic helix, indicating a participation of TatBC. Of note, the substrate-induced reorientation of the TatA amphipathic helix correlated with detectable membrane weakening. We therefore propose a two-state model in which membranedestabilizing effects of the short TatA membrane anchor are compensated by the membraneimmersed N-terminal part of the amphipathic helix in a resting state. We conclude that substrate binding to TatABC complexes switches the position of the amphipathic helix, which locally weakens the membrane on demand to allow substrate translocation across the membrane.The Tat system serves to transport folded proteins in bacteria, archaea, plant plastids, and possibly plant mitochondria (1-4). In Escherichia coli, the Tat system consists of TatA, TatB, and TatC components. TatA and TatB are similar, and two-component "minimal" Tat systems exist in which TatA exerts functions of TatA and TatB (5). TatA/B components are N-terminally membraneanchored by a very short hydrophobic transmembrane domain (TMD), which is followed by a short hinge region, an amphipathic helix (APH), and a variable C-terminal domain (6). TatC is a polytopic membrane protein with six TMDs (7,8). TatB tightly interacts with TatC (9, 10). TatA associates with these TatBC complexes and is thought to permeabilize the membrane for protein transport (11)(12)(13)(14). TatBC complexes recognize and tightly bind the signal peptides of the cargo proteins throughout the translocation process (15,16).The mechanism by which this translocation is achieved must be unusual and is not understood (17). A currently favored model suggests that the N-termini of multiple TatA molecules at the translocon site weaken the membrane upon substrate-binding, thereby permitting a TatCmediated pulling of the substrate through the Control of membrane-weakening by TatA 2 destabilized membrane ("membrane-weakening and pulling mechanism", 18). Molecular dynamics simulations support this view ...

Evidence for a second regulatory binding site on PspF that is occupied by the C-terminal domain of PspA

“…For comparison, we included also full-length PspA and PspA(1–144) that both are known to regulate PspF ( Fig 1 ). To stabilize PspA(145–222) to a detectable level, we fused it to the C-terminus of HiPIP, an unrelated small globular protein that is used to stabilize fused protein fragments [ 23 , 25 ]. The resulting fusion protein, termed Hip-PspA(145–222), was stable and completely soluble ( Fig 1B ).…”

Rapid diffusion of large TatA complexes detected using single particle tracking microscopy

“…E. coli TatA is generally considered to be a genuine trans-membrane protein (De Leeuw et al, 2001). Some more recent data however, showed that E. coli TatA fused to a Strep-tag can be partially extracted from the inner membrane, and that the extractable fraction depends on the growth phase of the cells (Hou et al, 2018, Taubert et al, 2015. Also in other bacteria, such as Bacillus subtilis (Pop, Westermann et al, 2003) and Streptomyces lividans (De Keersmaeker, Van Mellaert et al, 2005a, De Keersmaeker, Van Mellaert et al, 2005b, a substantial portion of TatA is found outside the membrane.…”

“…E. coli TatA is generally considered to be a genuine trans-membrane protein (De Leeuw et al, 2001). Recently however, E. coli TatA fused to a Strep-tag was shown to be partially extracted from the inner membrane (Hou, Heidrich et al, 2018, Taubert, Hou et al, 2015. In order to find out if rapid diffusion of large TatA complexes can be explained by them not being properly incorporated in the membrane, we isolated E. coli membranes and washed them with sodium carbonate.…”

The Twin-Arginine Pathway for Protein Secretion

“…The 'translocation pore model' theorises that TatA forms a channel/pore for protein passage (109). More recent data favor the 'membrane-destabilisation model' (110,116).…”