FIGURE 1: The effect of excess histidine on yeast cell growth and copper uptake.

(A) Growth phenotypes of S. cerevisiae Σ1278b wild-type strain L5487 (complemented with pRS415 (LEU2)) carrying an empty vector pYES2 (URA3) or pYES2-CAN1 (URA3). After overnight cultivation in SC-Leu-Ura liquid medium, approximately 10⁶ cells of each strain, and serial dilutions of 10^-1 to 10^-4 (from left to right) were spotted and incubated onto SCGal-Leu-Ura-Lys-Arg-His agar medium in the absence (Control) and presence of excess basic amino acids (25 mM lysine (+ Lys), 25 mM arginine (+ Arg), or 5 mM histidine (+ His)).

(B) Intracellular contents of basic amino acids in strain L5487 (complemented with pRS415) carrying an empty vector pYES2 (white) or pYES2-CAN1 (gray). The values are the means of two independent experiments that produced similar results.

(C) Growth phenotypes of strain L5487 (complemented with pRS415) carrying an empty vector pYES2, pYES2-CTR1, pYES2-CTR3, and pYES2-FRE1. After overnight cultivation in SC-Leu-Ura liquid medium, approximately 10⁶ cells of each strain, and serial dilutions of 10^-1 to 10^-4 (from left to right) were spotted and incubated onto SCGal-Leu-Ura-Lys-Arg-His agar medium in the absence (Control) and presence of excess levels of basic amino acids (25 mM lysine (+ Lys) or 5 mM histidine (+ His)).

(D) Growth phenotypes of strain L5487 (complemented with pRS415 and pRS416 (URA3)) under various concentrations of copper. After overnight cultivation in SD liquid medium, which contains 0.25 µM CuSO₄, approximately 10⁶ cells of each strain, and serial dilutions of 10^-1 to 10^-4 (from left to right) were spotted and incubated onto SD-Cu agar medium in the presence of 0.0025, 0.025, 0.25, 2.5, or 25 µM CuSO₄ and in the absence (Control) and presence of 5 mM histidine (+ His).

(E) Intracellular levels of copper ions of strain L5487 (complemented with pRS415 and pRS416) in the absence (0 mM His) and presence of excess histidine (5 or 10 mM His). The values are the means and standard deviations of three independent experiments. Asterisks indicate a significant increase in copper levels compared to the control sample (0 mM His) (p < 0.05).

(F) Growth phenotypes of strain L5487 (complemented with pRS415 and pRS416) (shown as ρ⁺) and its spontaneous ρ^– mutant. After overnight cultivation in SD liquid medium, approximately 10⁶ cells of each strain, and serial dilutions of 10^-1 to 10^-4 (from left to right) were spotted and incubated onto SD agar medium in the absence (Control) and presence of 5 mM histidine (+ His) under aerobic or anaerobic conditions.