The Twin-arginine Leader-binding Protein, DmsD, Interacts with the TatB and TatC Subunits of the Escherichia coli Twin-arginine Translocase

Abstract

The twin-arginine translocase (Tat) pathway is involved in the targeting and translocation of fully folded proteins to the inner membrane and periplasm of bacteria. Proteins that use this pathway contain a characteristic twin-arginine signal sequence, which interacts with the receptor complex formed by the TatBC subunits. Recently, the DmsD protein was discovered, which binds to the twin-arginine signal sequences of the anaerobic respiratory enzymes dimethylsulfoxide reductase (DmsABC) and trimethylamine N-oxide (TMAO) reductase. In this work, the targeting of DmsD within Escherichia coli was investigated. Using cell fractionation and Western blot analysis, DmsD is found to be associated with the inner membrane of wild-type E. coli and a dmsABC mutant E. coli under anaerobic conditions. In contrast, DmsD is predominantly found in the cytoplasmic fraction of a ⌬tatABCDE strain, which suggests that DmsD interacts with the membrane-associated Tat complex. Under aerobic conditions DmsD was also found primarily in the cytoplasmic fraction of wildtype E. coli, suggesting that physiological conditions have a significant effect upon the targeting of DmsD to the inner membrane. Size exclusion chromatography data and membrane washing studies indicate that DmsD is interacting tightly with an integral membrane protein and not with the lipid component of the E. coli inner membrane. Additional investigation into the nature of this interaction revealed that the TatB and TatC subunits of the translocase are important for the interaction of DmsD with the E. coli inner membrane.

Signal peptide–chaperone interactions on the twin-arginine protein transport pathway

“…(TorA-SP [23][24][25][26][27][28][29][30][31][32][33][34][35][36] ) was prepared that covered the C-terminal stretch of the TorA signal missing from the TorA-SP 2-22 peptide ( Table 1). Binding of the shorter TorA-SP [23][24][25][26][27][28][29][30][31][32][33][34][35][36] peptide was poor and under increased ionic strength titration of TorD with the TorA-SP [23][24][25][26][27][28][29][30][31][32][33][34][35][36] peptide elicited only a weak response that did not reach saturation and could not be fitted convincingly to any model (Table 1). Complete omission of the SRRRF stretch from the N terminus of the synthetic signal peptide (peptide TorA-SP 15-36 ) did not prevent peptide binding by TorD but did reduce the binding affinity 5-to 9-fold (Table 1).…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…Undoubtedly, DmsA can interact with the Tat translocon independent of DmsD, further supporting the existence of a handover or dissociation step (30). Moreover, DmsD association with the cytoplasmic membrane occurs only in the presence of TatB or TatC (51,52).…”

“…Figure 1 demonstrates the possible processes of these non-substrate proteins that were identified in the DmsD interactome from several studies (30,51,52). These interactions implicate DmsD as a hub of the DMSO reductase maturation pathway for connecting the nascent DmsA polypeptide to proteins that would assist in active holoenzyme maturation.…”

“…Specifically, the RR-leader peptide is recognized at the membrane by a receptor complex formed from copies of TatB and TatC (80,81). It has been established by both in vivo and in vitro experiments that DmsD interacts with both TatB and TatC (34,51,52). It is likely that the associated REMP docks at TatBC to donate the substrate to the TatBC receptor, with unfolded proteins rejected by the TatBC receptor complex through an undefined quality control mechanism (83).…”

Structural Analysis of a Monomeric Form of the Twin-Arginine Leader Peptide Binding Chaperone Escherichia coli DmsD

“…2 The translocation machinery is composed of three proteins: TatA, which is thought to polymerize to form the pore; TatB, a regulatory component; and TatC, which binds the substrate proteins. 3,4 The folding and assembly of Tat substrates with their cofactors must be complete prior to their targeting and translocation. A family of chaperone proteins has been identified in the targeting of Tat substrates to the membrane.…”