Figure 1

Gdt1p mediates calcium influx in L. lactis cells and this is strongly dependent on the external Ca²⁺ concentration and pH.

(A) Wild type and evolved DML1 L. lactis cells expressing 10His-Strep-TEV-^Δ23GDT1 were grown to an OD₆₀₀ of 0.4–0.5 and Gdt1p expression was induced with nisin (2.5 μg/L). After 3 h of induction, the total membrane fraction was prepared and Gdt1p expression analyzed by SDS-PAGE followed by Western blotting with anti-Gdt1p antibodies. The negative control (C) consisted of the total membrane fraction from cells containing the empty pNZ8048 vector. (B) Calcium influx time course measurements performed in Fura-2-loaded DML1 cells expressing 10His-Strep-TEV-^Δ23GDT1 or transformed with the empty vector pNZ8048 (C). After 3 h of induction, the cells were washed and resuspended in Ca²⁺-free assay medium pH 7.4. The fluorescence ratio (340/380) was recorded every 10 sec and converted into the [Ca²⁺]_cyt using the equation derived by Liao et al.⁴¹. The arrow indicates addition of 0.5 mM CaCl₂. (C) Effect of the external Ca²⁺ concentration (right axis; mM) on Ca²⁺ accumulation in DML1 L. lactis cells expressing 10His-Strep-TEV-^Δ23GDT1 at an extracellular pH of 7.4. (D) Effect of the external pH on Ca²⁺ accumulation in DML1 cells expressing 10His-Strep-TEV-^Δ23GDT1 or transformed with the empty vector pNZ8048 (C) after addition of 0.5 mM CaCl₂.