Figure 3.

Ca²⁺ and Mn²⁺ transport mediated by truncated TMEM165 expressed in L. lactis. A, production of the truncated TMEM165 in L. lactis. The cells were grown to an A₆₀₀ of 0.5 and incubated in the presence (+nis) or not (−nis) of nisin (2.5 μg/l) for 2 h to induce the expression of ^Δ55TMEM165 or ^Δ78TMEM165. The tagged proteins (N terminus) were detected in membrane protein extracts by Western blotting with anti-HA antibodies. ∅, cells with the empty pNZ8048 vector. Coomassie Blue–stained polyvinylidene fluoride membrane was used as loading control. B, time-course measurement of the fluorescence emitted by Fura-2 in L. lactis DML1 expressing the truncated versions of TMEM165 or containing the empty pNZ8048 vector (∅) in the presence of 75 μm CaCl₂ (left panel) or 75 μm MnCl₂ (right panel). The cells were grown to an A₆₀₀ of 0.5, at which the truncated TMEM165 expression was initiated by addition of 2.5 μg/liter nisin. After a 2-h postinduction time, the cells were washed and incubated for 2 h in the presence of Fura-2/AM. Left panel, time-course measurements of the ratio of the fluorescence emitted at 510 nm after excitations at 340 and 380 nm (340/380). Right panel, time-course measurements of the quenching by Mn²⁺ of the fluorescence emitted at 510 nm after excitation at 360 nm normalized to the initial fluorescence. CaCl₂ or MnCl₂ were added after 120 s of measurement (arrow), and the curves represent the means (n = 3 ± S.D.).