The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast

Abstract

Nonessential metal ions such as cadmium are most likely transported across plant membranes via transporters for essential cations. To identify possible pathways for Cd2+ transport we tested putative plant cation transporters for Cd2+ uptake activity by expressing cDNAs in Saccharomyces cerevisiae and found that expression of one clone, LCT1, renders the growth of yeast more sensitive to cadmium. Ion flux assays showed that Cd2+ sensitivity is correlated with an increase in Cd2+ uptake. LCT1-dependent Cd2+ uptake is saturable, lies in the high-affinity range (apparent KM for Cd2+ = 33 microM) and is sensitive to block by La3+ and Ca2+. Growth assays demonstrated a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations. LCT1-dependent increase in Ca2+ uptake correlated with the observed phenotype. Furthermore, LCT1 complements a yeast disruption mutant in the MID1 gene, a non-LCT1-homologous yeast gene encoding a membrane Ca2+ influx system required for recovery from the mating response. We conclude that LCT1 mediates the uptake of Ca2+ and Cd2+ in yeast and may therefore represent a first plant cDNA encoding a plant Ca2+ uptake or an organellar Ca2+ transport pathway in plants and may contribute to transport of the toxic metal Cd2+ across plant membranes.

Figure 1

Effect of LCT1 expression on cadmium sensitivity of yeast. INVSc1 cells transformed with either the empty pYES2 plasmid (Control) or LCT1 in pYES2 were grown on arginine-phosphate medium without Cd²⁺ (Left) or with 50 μM Cd²⁺ (Right).

Figure 2

Effect of LCT1 expression on cadmium uptake in yeast. INVSc1 cells, transformed with LCT1 in pYES2 (■, •) and with the empty control pYES2 plasmid (□, ○) were grown in arginine-phosphate medium to an OD of ≈0.2 and assayed for cadmium uptake with ¹⁰⁹Cd. Cells were incubated in uptake solution, containing 30 μM Cd²⁺, at 30°C (•, ○) or 0°C (■, □). Aliquots were taken at different time points and radioactivity was measured. Bars = SE; n = 8 for each data point.

Figure 3

Concentration-dependence of LCT1-dependent uptake. Cd²⁺ uptake rates of INVSc1 control cells (carrying the empty pYES2 plasmid) were subtracted from Cd²⁺ uptake rates of LCT1-expressing cells to determine the LCT1-dependent Cd²⁺ uptake rate at different Cd²⁺ concentrations. Bars = SE; n = 3. The inset shows a Lineweaver–Burke plot of the uptake data. K_M = 32.9 μM.

Figure 4

Competition of Ca²⁺ and Mg²⁺ with LCT1-dependent Cd²⁺ uptake. Cd²⁺ uptake rates were determined for INVSc1 controls (○) and LCT1-expressing cells (•) in the presence of different Mg²⁺ (A) and Ca²⁺ (B) concentrations. LCT1-dependent Cd²⁺ uptake (■) was determined by subtracting the rates of control cells from the rates of LCT1-expressing cells. Bars = SE; n = 4.

Figure 5

Ca²⁺ sensitivity of LCT1-expressing cells. LCT1-expressing cells (■, •) and INVSc1 control cells (□, ○) were grown in arginine-phosphate medium containing either 10 μM Ca²⁺ (•, ○) or 3 mM Ca²⁺ (■, □).

Figure 6

Effect of LCT1 expression on calcium uptake in yeast. INVSc1 cells transformed with LCT1 in pYES2 (■, •) and with the empty pYES2 control plasmid (□, ○) were grown in arginine-phosphate medium to an OD of ≈0.2 and assayed for calcium uptake with ⁴⁵Ca²⁺. Cells were incubated in uptake solution, containing 100 μM Ca²⁺, at 30°C (•, ○) or 0°C (■, □). Aliquots were taken at different time points and radioactivity was measured. Bars = SE; n = 7 for each data point.

Figure 7

Effect of different divalent cations on LCT1-dependent Ca²⁺ uptake. Ca²⁺ uptake of LCT1-expressing cells (■) and INVSc1 control cells (□) was measured in the presence of other cations. The external Ca²⁺ concentration was 100 μM, the concentration of Pb²⁺ was 10 μM, the concentration of the other cations was 100 μM. The uptake rates are shown as % of controls that had no competing metal added to the uptake assay. Native background Ca²⁺ uptake (□) is compared with LCT1-dependent Ca²⁺ uptake (■). LCT1-dependent Ca²⁺ uptake was determined by subtracting the rate of control cells from the rate determined for LCT1-expressing cells. The average Ca²⁺ uptake rate in these experiments was 213.0 (±20.1) pmol⋅min⁻¹⋅mg⁻¹ d.w. for control cells and 444.0 (±26.4) pmol⋅min⁻¹⋅mg⁻¹ dry weight for LCT1-expressing cells. Error bars represent SE, n = 3 for each condition.

Figure 8

Expression of LCT1 in the mid1 mutant prevented cell death in response to mating pheromone compared with non-LCT1-expressing mid1 cells. Yeast viability was determined by using the methylene blue liquid method. Cells were incubated for 3 hr in the presence of 3 μM α-factor. Four hundred yeast cells were scored for uptake of methylene blue by microscopic observation for each strain-plasmid combination [□, C699–5 (pYES2); ▧, C699–5 (LCT1); ░⃞, C699–5Δmid1 (pYES2); ■, C699–5Δmid1 (LCT1)] in this experiment and two independent transformants were tested for each with the same result. Bars = SD.