DIVERSITY OF CALCIUM-EFFLUX TRANSPORTERS IN WHEAT ALEURONE CELLS

The ability of plant cells to regulate cellular calcium is dependent upon the action of calcium-transport proteins. Although significant progress has been made in identifying calcium-transporter activities (CaTs) in a number of plant tissues, very little is known about the diversity of CaTs in any single differentiated cell type. We have used isolated membrane vesicles from wheat (Triticum aestivum L.) aleurone cells to identify and characterize the principal CaTs that are responsible for efflux of Ca2+ from the cytosol of this highly differentiated cell. Based on the sensitivity of transport to protonophores and on their buoyant density in isopycnic sucrose density gradients, our data show that at least three prominent CaTs can be distinguished in membrane vesicles from aleurone cells. Two of these CaTs, which we have named Type I and Type III, were insensitive to protonophores and were inhibited by vanadate or erythrosin B (EB). Type I was associated with a membrane fraction enriched in endoplasmic reticulum whereas Type III was associated with the plasma membrane. The third prominent CaT, Type II, was inhibited by protonophores and by nitrate and was associated with a membrane fraction enriched in tonoplast. The three CaTs differed significantly in their intrinsic properties. Type I had the highest affinity for Ca2+ (K-m, 0.15 mu M), was stimulated by oxalate, inhibited by vanadate and erythrosin B, and was unaffected by exogenous calmodulin. Type II exhibited complex kinetics with regard to Ca2+ and was best described as a combination of a low-affinity (K-m, 21 mu M) and a high-affinity component (K-m, 0.2 mu M). Type II CaT was stimulated by calmodulin (1 mu M) and oxalate, and was inhibited by dicyclohexylcarbodiimide (500 mu M). Finally, Type III had an intermediate affinity for Ca2+ (K-m, 2.0 mu M), was not stimulated by calmodulin or by oxalate, and was inhibited by low concentrations of erythrosin B (0.1 mu M). These data provide one of the first comparative investigations of the Ca2+-transport activities in a single highly differentiated cell type. They indicate that at least three CaTs function to regulate cellular Ca2+ and we speculate that cytosolic Ca2+ may be buffered primarily by Type II and Type III CaTs which are associated with an acidic intracellular compartment, probably the vacuole, and with the plasma membrane.