Tat substrate signal sequences are involved in at least three important steps along the translocation path. First, the signal peptide is involved in the recognition of Tat substrates by REMPs. For example DmsD has been shown to directly bind to the signal peptide of DmsA [9]. Recently, it was shown that many (but not all) REMPs bind to the signal peptides of the binding partner [10]. Second, signal peptides bind to phospholipid bilayers and biological membranes, possible corresponding to the step that directly precedes TatC binding [11,12]. Third, signal peptides are recognized and bound by TatC, which is presumably a trigger for translocation [13]. REMPs are considered bifunctional chaperones, with roles in both the maturation and transport of substrates. The function in maturation is to keep the substrate in a conformation in which it can bind its redox cofactor [14,15]. The role in transport is less clear. Initially, it was thought that DmsD directs DmsA to the Tat translocon [16]. However, other data showed that DmsD does not function as a targeting factor [17]. REMPs were also shown to protect the signal peptide from degradation by peptidases [18,19]. It has been proposed that REMPs prevent the premature interaction of immature substrates with the Tat system which only transports fully folded proteins [20,21]. Unfolded polypep- tides can bind to the TatBC receptor complex, but cannot be transported [22]. Such binding of an unfolded polypeptide to TatBC blocks the Tat system and leads to severe growth defects [22]. REMPs are supposed to prevent such premature interac- tions, a process sometimes referred to as ‘proofreading’ [23]. However, there is little experimental evidence to directly support this hypothesis. An open question is: what is the function of the two arginine residues (RR) in the signal sequence? They are known to be essential for translocation. Even the most conservative mutation to two lysine residues blocks translocation entirely [24,25]. For the Tat substrate SufI it has been shown that the two arginines are essential for binding to TatC, but not for binding to the membrane [12]. Whether the two arginines are important for binding to REMPs is not clear. On one hand, Hatzixanthis et al. have analyzed the binding of the TorA signal peptide to TorD by isothermal titration calorimetry (ITC). They found that the twin- arginine motif is probably not required for TorD binding [26]. On the other hand, Stevens et al. have analyzed the interaction between DmsD and the DmsA signal peptide using docking and molecular dynamics simulations, and concluded that the twin- arginine motif probably interacts with conserved regions on the surface of DmsD [27]. In summary, the interactions between REMPs and their substrates, and the actual function of this interaction in the twin-arginine transport pathway are still not well understood. The work presented here aims to provide more insight into the nature of the interactions between Tat signal sequences and REMPs, and to better understand the function of REMP binding in the Tat pathway. In particular, we focus on two questions: (i) how do REMPs discriminate between their cognate substrate(s) and other Tat substrates, which often have many similar features; and (ii) how does REMP binding affect signal-peptide binding to the membrane relating to the next step in the Tat translocation pathway. Chimeric substrates were created based on green fluorescent protein (GFP) fused to different signal sequences. This approach has three important features: (i) GFP reports on the folding state of the pre-protein: it is only fluorescent when fully folded [28]; (ii) the same mature domain is used in all experiments, which ensures that observed differences in REMP binding are due to the signal sequence, and not to the mature domain; and (iii) such chimeric pre-proteins are functional Tat substrates, since Binding of Tat Signal Sequences to REMPs their expression in E. coli results in periplasmic accumulation of GFP [11,29]. The signal peptides thus behave naturally when fused to GFP. Using surface plasmon resonance (SPR) it is shown that the signal sequences of TorA and DmsA target the cargo GFP-fusion protein to the corresponding REMP. The targeting information resides in the combined H- and C-regions, which for TorA and DmsA are substantially hydrophobic and rather similar in overall composition. The N-region, which includes the twin- arginine motif but otherwise differs considerably between TorA and DmsA, is not involved in providing specificity to the interaction with the REMP. Interestingly, the presence of DmsD reduces membrane binding of ssDmsA-GFP, which might be important in preventing the premature interaction of the pre- protein with the Tat system. Materials and Methods Plasmids and bacterial strains Wild-type signal sequences were amplified from the torA and dmsA genes, which had previously been amplified from the E. coli genome and cloned into vector pBSK. Hybrid signal sequences combining N-, H-, and C-regions from torA and dmsA were created entirely by oligonucleotide synthesis and cloned into pUC57 (Eurogentec, Maastricht, The Netherlands). Subsequently, signal sequences were amplified from pBSK or pUC57 by PCR, and at the same time given a 39-extension that corresponds to the first 15 basepairs of the GFP insert. In a second PCR reaction the signal sequences were fused to the GFP insert and cloned into pTYB11 (New England Biolabs, Ipswich, MA, USA) such that a single fusion protein is encoded consisting of an intein domain, a signal sequence and GFP. Mutations in the twin-arginine regions of the signal sequences were subsequently created by site-directed mutagenesis. pGFP6A, encoding Strep-GFP, was made as follows. First, the internal NdeI restriction site in the GFP gene was removed by site-directed mutagenesis. Subsequently, the mutant GFP gene was amplified by pcr and cloned into the NdeI/BamHI restriction sites of pET3a (New England Biolabs), resulting in pGFP4. Finally, pGFP4 was restricted by NdeI and ligated with two oligonucleotides such that a StrepII-tag is encoded upstream of the GFP-gene. An overview of the expression plasmids used is given in Table 1. To express TorD and DmsD, E. coli torD and dmsD genes were amplified from the genome and cloned into vector pET16b (Invitrogen), which encodes an N-terminal His-tag. E. coli strains used in this study were MC4100 (F2 araD139 D(argF-lac)U169 2 rpsL150 relA1 deoC1 rbsR fthD5301 fruA25 l ), DADE (MC4100 DtatABCD, DtatE, [30]), and BL21(DE3)*. Protein expression and purification For large-scale preparation, the intein-fused signal GFPs were expressed in E. coli BL21(DE3)* cells and column purified as described in previous work [11]. In short, the fusion proteins were bound to a chitin affinity column (New England Biolabs) via the chitin-binding domain that is engineered into the intein domain. Subsequently, self-cleavage of the intein domain is induced by reduction on the column, and GFP is released with an intact and unmodified signal sequence at its N-terminus. StrepII-tagged GFP was expressed from pGFP6A in BL21(DE3)* cells and purified using Strep-Tactin Superflow resin (IBA, Go¨ ttingen, Germany) as recommended by the manufacturer. His-tagged TorD and DmsD were also expressed in E. coli BL21(DE3)* cells and column purified as recommended in the manual, ‘‘The Qiaexpressionist’’ (Qiagen). PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34159