E PKA target trehalase within the wild-type strain just after addition of
E PKA target trehalase within the wild-type strain immediately after addition of five mM L-citrulline (), IDO MedChemExpress L-histidine (), L-lysine () or L-tryptophan () to nitrogen-starved cells. B. Gap1-dependent uptake. CysLT1 MedChemExpress Transport of five mM L-citrulline, L-histidine, L-lysine or L-tryptophan in wild-type (black bars) and gap1 (white bars) strains. C. The 3 non-signalling amino acids are very poor nitrogen sources. Development on 5 mM L-citrulline (, ), L-histidine (, ), L-lysine (, ), L-tryptophan (, ) or L-asparagine (, ) in wild-type (closed symbols) and gap1 (open symbols) strains. D. L-histidine, L-lysine and L-tryptophan act as inhibitors of Gap1 transport. Transport of 1 mM L-citrulline measured in the presence of different concentrations L-histidine, L-lysine and L-tryptophan (0, 0.5, 1, five and ten mM, white bars to black bars). E. L-histidine, L-lysine and L-tryptophan act as partially or largely competitive inhibitors of Gap1 transport. Transport of five concentrations (0.5, 1, 2.five, five and 10 mM, white bars to black bars) of L-citrulline measured without inhibitor or inside the presence of 0.125 mM L-histidine, 0.5 mM L-lysine or 0.125 mM L-tryptophan. These values are also shown as a Lineweaver-Burk plot (inset): no inhibitor (), or 0.125 mM L-histidine (), 0.5 mM L-lysine (), or 0.125 mM L-tryptophan (). F. Transport in the non-signalling amino acids is reduced by mutagenesis of Ser388 or Val389 to cysteine. Transport of five mM L-citrulline, L-histidine, L-lysine or L-tryptophan by a wild-type (1), gap1S388C (2, 3) in addition to a gap1V389C (4, five) strain, with no (two, 4) or with (three, five) pre-addition of ten mM MTSEA. Error bars in (A) to (F) represent typical deviation (s.d.) between biological repeats.2014 The Authors. Molecular Microbiology published by John Wiley Sons Ltd., Molecular Microbiology, 93, 213216 G. Van Zeebroeck, M. Rubio-Texeira, J. Schothorst and J. M. TheveleinNon-signalling and signalling amino acids look to bind via distinct interactions inside a promiscuous binding pocket The 3 non-signalling amino acids, L-histidine, L-lysine and L-tryptophan acted as inhibitors of L-citrulline uptake (Fig. 1D). In the case of L-lysine or L-histidine the inhibition was purely or largely competitive, respectively, though for L-tryptophan there was a clear non-competitive element (Fig. 1E). According to Fig. 1E, the inhibition constants have been determined as Ki(His) = 0.0025 mM, Ki(Lys) = 0.0095 mM and Ki(Trp) = 0.0033 mM. As described above, tryptophan addition also resulted in an intermediate phenotype in terms of its ability to help growth (Fig. 1C). This indicates that these non-signalling amino acids apparently bind in to the identical binding pocket of Gap1 as the signalling amino acid, L-citrulline, but in a distinct way from the signalling substrate. To obtain further evidence for this conclusion, we’ve created use of two residues, Ser388 and Val389, which were previously located by Substituted Cysteine Accessibility Method (SCAM), and whose side-chains are exposed in to the amino acid binding pocket of Gap1 (Van Zeebroeck et al., 2009). Covalent modification from the Gap1S388C or Gap1V389C proteins together with the sulphydryl-reactive reagent MTSEA (2-aminoethyl methanethiosulphonate hydrobromide) blocked signalling by both transported and nontransported signalling agonists (Van Zeebroeck et al., 2009; Rubio-Texeira et al., 2012). Here we show that, in contrast to the signalling amino acids, transport in the non-signalling amino acids was already decreased in strains expressing the gap.