Tonoplast localization of a calmodulin-stimulated Ca 2+-pump from maize roots

Abstract

The subcellular localization of a calmodulin-stimulated calcium (Ca 2+)-ATPase activity from maize roots ( Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca 2+ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure 45Ca 2+ transport in sealed vesicles. Most of the ATP-dependent Ca 2+ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H +/Ca 2+ antiport) and the plasma membrane. When the Ca 2+ transport was measured in the presence of exogenous calmodulin (1 μM), a 3–5-fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, rulling out a direct effect of calmodulin on the H +/Ca 2+ antiport. In conclusion, four different Ca 2+ transporters are present in young maize root cells. A Ca 2+/H + antiport system is present on the tonoplast, whereas, the plasma membrane and the ER posses each a calmodulin-insensitive Ca 2+-ATPase. Finally, a calmodulin-stimulated Ca 2+-ATPase is associated with the tonoplast.