Fig. 4.

Elucidation of putative TA transporter substrates and transport mechanisms. Growth curve data showing (A and B) the effect of AbPUP1 and AbLP1 on alkaloid tolerance, (C) H⁺ dependence of TA transport by AbPUP1 and AbLP1, and (D) the effect of monovalent cations on TA transport by AbPUP1 and AbLP1 in yeast. Transporter substrate specificity (θ_HM) quantified as the ratio of time required for half-of-maximum OD₆₀₀ increase (t_HM), with maximum alkaloid concentration relative to without alkaloid supplementation (θ_HM = t_{HM,max_alk}/t_{HM,no_alk}), is shown as a heatmap in B; heatmap values were extracted from mean growth curves in A. For A through D, the yeast strain AD1-8 expressing BFP (control), AbPUP1, or AbLP1 from low-copy plasmids was cultured in selective media supplemented with (A and B) 0 to 20 mM alkaloids, (C) 10 mM hyoscyamine, and 100 mM potassium phosphate buffer, or (D) 20 mM hyoscyamine and 0 to 100 mM NaCl or KCl. Note that for A and B, tropine was supplemented in media at only 1 mM, as higher tested concentrations severely retarded the growth of AD1-8, such that growth measurements were not feasible. For D, “–” indicates no salt supplementation in excess of that already present in the yeast nitrogen base. For A, C, and D, solid lines and shaded bars, respectively, indicate the mean and SD of n = 3 biologically independent samples.