, 2002; Shi et al., 2006; Gimenez et al., 2007; Kwan et al., 2008). To test whether single R to K substitutions affected translocation, we individually replaced the arginine residues at positions 14 and 15 of the AmyH signal peptide (Fig. 2a) with lysine residues. The secretion of these variants (preAmyH-KR and preAmyH-RK) was compared with wild-type AmyH (preAmyH-RR) and the earlier constructed mutant containing two lysines (preAmyH-KK). As shown in

Fig. 2 with starch-plate assays and Western blotting, neither preAmyH-KR nor preAmyH-RK was secreted, indicating that both arginine residues of the Tat motif are critical to translocation. Western blotting indicated a small amount of AmyH in the supernatant fractions Obeticholic Acid chemical structure of the KK and KR mutants,

but, as the precursor and mature forms of AmyH run very close together on SDS-PAGE, we could not determine whether those corresponded to precursor (the result of cellular lysis) or mature AmyH (the result of secretion). To find more investigate the importance of the other residues in the twin-arginine motif, the residues at positions 13, 16, 17, 18, and 19 in preAmyH were all changed to alanine residues. As used in many other studies, alanine was chosen as it removes most of the side chain without affecting the backbone of the peptide chain. As shown in Fig. 3, the secretion was again tested using the starch-plate assays and Western blotting. Ser13, Thr16, and Lys19 were not critical, as Ala residues on those positions did not affect translocation. This was not entirely surprising because residues Protirelin in the same positions in the E. coli Tat substrate SufI also had no significant effect on translocation (Stanley et al., 2000). Two residues that were shown to be important were Val17 and Leu18. When Val17 was substituted by Ala, no amylase activity was detected in the supernatant (Fig. 3a). This was confirmed by Western blotting (Fig. 3b), which showed a complete absence of AmyH

in the medium fraction of the V17A substitution. Our finding that this residue is critical to translocation is similar to what was found for E. coli SufI (which has a Phe in this position; Stanley et al., 2000). At this position, a strongly hydrophobic residue is important and the most common residues found here are Phe, Val, and Leu. It is interesting to note that a number of haloarchaeal Tat substrates, nine out of a total of 209 proteins in our datasets (see Table S1), do contain an Ala in that position. None of these nine proteins have been characterized, but homology searches indicate that at least some of them appear to be genuinely extracytoplasmic proteins (data not shown).