of DmsA, and both contain a proline residue and at least one arginine. The N-region differs the most, with that of TorA being shorter and more polar. One might thus expect that the N-region, which contains the twin-arginine motif, would be important for conveying the REMP binding specificity. However, a more detailed analysis of the signal sequences shows that the H- and C-regions of DmsD substrates are rather well conserved, whereas they differ significantly in the N-region (Figure 4). This points to the combined H- and C-regions being the recognition site for DmsD. Indeed, the experiments with chimeric signal sequences (Figure 5) show that the combined H-/C-regions are essential for binding, whereas the N-region has little, if any, effect. These results are in agreement with previous work in which ITC was used to measure binding of TorD to short peptides corresponding to various parts of the TorA signal peptide [26,32]. Interestingly, Chan et al. recently identified a binding pocket on the surface of E. coli DmsD that mainly consists of hydrophobic and aromatic residues [34]. Furthermore, our observations that the conserved arginine pair, which is essential for transport, is not crucial for REMP binding (Figures 2 and 3, Table 2) are also consistent with a central role for the H- and C-regions in REMP binding. Nevertheless, the part of the N-region in which the twin-arginine motif is located is involved in the interaction with REMPs, probably via a charge-interaction, because the mutant ssTor- A(GHP)-GFP does not bind to TorD at all. The N-region is however not used to discriminate between TorA and DmsA. Binding of the H-region to a REMP must be primarily governed by the hydrophobic effect. The entropic hydrophobic effect is driven by the exclusion of water, and on it own as such is not expected to provide specificity of the REMP towards a certain signal peptide. Some data suggest that the signal peptide binds in a particular conformation to the REMP. While being mostly unstructured in solution, signal peptides have a strong tendency to adopt an a- helical conformation in a hydrophobic membrane-mimicking environment [35], and probably also upon binding with a hydrophobic binding pocket on a REMP. Recently, Zakian et al. showed that the REMP NarJ binds to its cognate signal peptide ssNarG in an a-helical conformation, and that the affinity is mostly determined by the hydrophobic effect [36]. The same might be true for ssTorA binding to TorD and ssDmsA binding to DmsD. The second question of this study was how REMP binding relates to the twin-arginine translocation mechanism. The path starts with synthesis of a twin-arginine-containing polypeptide on a ribosome in the cytoplasm, and ends with the translocation of a folded, often cofactor-containing protein to the periplasm. It was earlier shown that membrane binding of Tat pre-proteins is the last step en route to transport that precedes binding to the Tat translocon [11,12]. Here, the possibility is assessed that REMP binding and subsequent release are the steps directly preceding membrane binding. Figure 6A shows that DmsD reduces membrane binding of ssDmsA-GFP in vitro. Hence, the presence of sufficient DmsD in the cytoplasm could reduce membrane binding of ssDmsA-GFP, and thereby retard transport of the pre- References 1. Weiner JH, Bilous PT, Shaw GM, Lubitz SP, Frost L, et al. (1998) A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell 93: 93–101. 2. Sargent F, Bogsch EG, Stanley NR, Wexler M, Robinson C, et al. (1998) Overlapping functions of components of a bacterial Sec-independent protein export pathway. EMBO J 17: 3640–3650. 3. Fisher AC, DeLisa MP (2004) A little help from my friends: quality control of presecretory proteins in bacteria. J Bacteriol 186: 7467–7473. 4. Graubner W, Schierhorn A, Bruser T (2007) DnaK plays a pivotal role in Tat targeting of CueO and functions beside SlyD as a general Tat signal binding chaperone. J Biol Chem 282: 7116–7124. Binding of Tat Signal Sequences to REMPs protein. This might be the simplest way in which REMPs perform their so-called proofreading function. If true, the twin-arginine translocation mechanism would look as follows. After synthesis of the twin-arginine-containing polypeptide on the ribosome, REMPs rapidly bind to the signal peptide of the cognate substrate with a relatively high affinity, and hence prevent premature binding of the substrate to the membrane and subsequently to the TatBC complex. While the REMP is bound to the pre-protein, other maturation enzymes can fold the protein and incorporate the appropriate cofactor. After spontaneous dissociation the REMP will usually rebind rapidly, until occasionally the released signal peptide binds to the membrane. Once membrane bound, the signal peptide is no longer fully available to the REMP, as our data show for DmsD and ssDmsA-GFP (Figure 6B). The REMP cannot recover the pre-protein out of the membrane, and the substrate will proceed to the translocation machinery. In this way, REMPs provide a time window during which the essential redox cofactors can be incorporated. The length of this time window would then be a function of the relative affinity of the signal peptide affinities for the REMP and the target membrane. The model described above is speculative and requires further experimental investigation. Nevertheless, the model is consistent with the available information. Nucleotide hydrolysis might provide sufficient energy to release a tightly bound substrate. There is indeed some evidence that TorD binds guanosine triphosphate (GTP) [26]. However, GTPase activity was recently shown to be specific for a domain-swapped TorD homodimer [37], of which the physiological role is unclear. We do not know and cannot control the oligomeric state of TorD on the SPR chip, although the observed 1:1 binding stoichiometry suggests that TorD is mainly monomeric on the surface (Figure 2). Nevertheless, when we measured release of ssTorA-GFP from TorD in the presence of GTP, we did not observe any effect (data not shown). We thus conclude that TorD binds ssTorA with a micromolar dissociation constant, and that there is no evidence that nucleotide hydrolysis promotes release of an intact pre-protein. In conclusion, REMP binding to signal peptides serves at least two purposes. The first purpose, already well established, is that this interaction prevents premature cleavage of signal peptides by peptidases [19]. The second purpose is to prevent premature interactions of pre-proteins with the translocation machinery. We propose that binding of a REMP to a signal sequence delays binding of the pre-protein to the membrane and subsequently to the Tat translocase. This might provide a time frame in which the mature domain of the pre-protein can fold and the required cofactors can be incorporated. Author Contributions Conceived and designed the experiments: AS HL YB. Performed the experiments: AS AB PV JG YB. Analyzed the data: AS AB JG HL YB. Contributed reagents/materials/analysis tools: AS AB PV. Wrote the paper: AS AB HL YB. 5. Perez-Rodriguez R, Fisher AC, Perlmutter JD, Hicks MG, Chanal A, et al. (2007) An Essential Role for the DnaK Molecular Chaperone in Stabilizing Over-expressed Substrate Proteins of the Bacterial Twin-arginine Translocation Pathway. J Mol Biol 367: 715–730. 6. Sargent F (2007) The twin-arginine transport system: moving folded proteins across membranes. Biochem Soc Trans 35: 835–847. 7. Berks BC (1996) A common export pathway for proteins binding complex redox cofactors? Mol Microbiol 22: 393–404. 8. Bogsch E, Brink S, Robinson C (1997) Pathway specificity for a delta pH- dependent precursor thylakoid lumen protein is governed by a ‘Sec-avoidance’ PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e34159