Figure 2. Development of AmTrac.
(A) Growth of the yeast Δmep1,2,3 mutant transformed with fusion variants on solid media containing 2 mM NH4Cl or 1 mM arginine (growth control) as the sole nitrogen source for 3 days. Composition of linkers connecting AMT1;3 and mcpGFP are indicated. Linkers at the N- and C-termini of mcpGFP are indicated in letter code and separated by a slash. In the cases of variants 1–6, no linkers were inserted between the C-terminal sequence of mcpGFP and the second part of AMT1;3. (B) Screen of 24 linker variants for fluorescence intensity before addition of ammonium and fluorescence intensity change after addition of 1 mM NH4Cl (mean ± SE; n = 3). Variant 16 (in red), carrying LE/FN as linkers, named AmTrac, showed the highest change in fluorescence intensity. (C) Schematic representation of AmTrac. Linkers between AMT1;3 and mcpGFP are indicated in yellow. (D) Protein sequence of AmTrac. Residues in ‘yellow’ constitute synthetic linker segments. Residues in ‘green’ correspond to the mcpGFP moiety. Numbers indicate amino acid position in AMT1;3.