DIVERSITY OF CALCIUM-EFFLUX TRANSPORTERS IN WHEAT ALEURONE CELLS

The ability of plant cells to regulate cellular calcium is dependent u pon the action of calcium-transport proteins. Although significant pro gress has been made in identifying calcium-transporter activities (CaT s) in a number of plant tissues, very little is known about the divers ity of CaTs in any single differentiated cell type. We have used isola ted 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 ve sicles from aleurone cells. Two of these CaTs, which we have named Typ e I and Type III, were insensitive to protonophores and were inhibited by vanadate or erythrosin B (EB). Type I was associated with a membra ne fraction enriched in endoplasmic reticulum whereas Type III was ass ociated with the plasma membrane. The third prominent CaT, Type II, wa s inhibited by protonophores and by nitrate and was associated with a membrane fraction enriched in tonoplast. The three CaTs differed signi ficantly in their intrinsic properties. Type I had the highest affinit y for Ca2+ (K-m, 0.15 mu M), was stimulated by oxalate, inhibited by v anadate and erythrosin B, and was unaffected by exogenous calmodulin. Type II exhibited complex kinetics with regard to Ca2+ and was best de scribed as a combination of a low-affinity (K-m, 21 mu M) and a high-a ffinity component (K-m, 0.2 mu M). Type II CaT was stimulated by calmo dulin (1 mu M) and oxalate, and was inhibited by dicyclohexylcarbodiim ide (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 Ca2and we speculate that cytosolic Ca2+ may be buffered primarily by Type II and Type III CaTs which are associated with an acidic intracellula r compartment, probably the vacuole, and with the plasma membrane.