, 2002). The residues surrounding the two arginine residues are present at a high frequency, but can nevertheless still vary. However, only the phenylalanine (the second residue after the arginines) appears to be critical; the functionality of the E. Selleckchem Everolimus coli Tat substrate SufI was only retained when Phe was replaced with another strongly hydrophobic residue such as Leu (Stanley et al., 2000). Surprisingly, replacing the other residues surrounding the two arginines in SufI or YacK (a SufI homologue) only led to minor effects, if at all (Stanley et al., 2000). As mentioned before, in most prokaryotes, the Sec system is the dominant export route. In contrast,

however, in halophilic archaea (haloarchaea), it is the Tat system that is predicted to be the dominant export route (Bolhuis, 2002; Rose et al., 2002). It has been speculated that this is an adaptation to the highly saline conditions in which these organisms thrive (Bolhuis, 2002; Rose et al., 2002). Haloarchaea contain high concentrations of KCl intracellularly, and it may be that secretory proteins fold very rapidly, which in turn leads to a necessity of the Tat system. As a consequence, the haloarchaeal Tat system is essential for viability (Dilks et al., 2005; Thomas & Bolhuis, 2006), corroborating the dominant role of this transport route. The haloarchaeal Tat system is different from the Tat system of nonhalophilic organisms in a number BGB324 supplier of ways. Firstly, as mentioned before, most proteins in haloarchaea

are secreted in a Tat-dependent manner. Secondly, the composition and topology of Tat translocase components in haloarchaea are different. There are one or two TatA proteins, and always two TatC proteins, with one of these TatC proteins being Buspirone HCl a translational fusion between two TatC domains (Bolhuis, 2002); the latter seems unique to haloarchaea. Thirdly, we have shown that transport of the Tat-dependent substrate AmyH, an amylase from the haloarchaeon Haloarcula hispanica, depends on the sodium motive force (Kwan et al., 2008). This is in contrast to bacterial

or chloroplast Tat systems, which depend on the proton motive force. For all of those reasons, it is also conceivable that the nature of signal peptides of haloarchaeal Tat substrates is different from those of nonhalophilic Tat substrates. Thus, it was important to investigate the Tat motif of Tat substrates, as any major differences would have an impact on for instance the prediction of the transport routes used by proteins found through genomic sequencing projects. Here, in this study, we analysed the importance of residues in the Tat motif of the aforementioned AmyH to provide. Unless noted, all chemicals were from Sigma-Aldrich (Dorset, UK) or Fisher Scientific (Loughborough, UK). Haloferax volcanii H26 has been described before (Allers et al., 2004) and was routinely grown at 45 °C in a rich medium (YPC) containing 0.5% yeast extract (Difco, Becton Dickinson, Oxford, UK), 0.1% peptone (Oxoid, Basingstoke, UK), 0.