A Thapsigargin-Sensitive Ca2+ Pump Is Present in the Pea Golgi Apparatus Membrane

The Golgi apparatus behaves as a bona fide Ca 2+ store in animal cells and yeast ( Saccharomyces cerevisiae ); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca 2+ accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca 2+ uptake in subcellular fractions isolated from the elongating zone of etiolated pea ( Pisum sativum ) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca 2+ uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca 2+ uptake of 2.5 nmol mg min −1 and an apparent K m for Ca 2+ of 209 nm. The ATP-dependent Ca 2+ uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I 50 ] = 126 μm) and cyclopiazonic acid (I 50 = 0.36 nmol mg protein −1 ) and was not stimulated by calmodulin (1 μm). Addition of Cd 2+ and Cu 2+ at nanomolar concentration inhibited the Ca 2+ uptake, whereas Mn 2+ , Fe 2+ , and Co 2+ had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I 50 = 88 nm), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca 2+ ATPase from mammalian cells. A thapsigargin-sensitive Ca 2+ uptake activity was also detected in a cauliflower ( Brassica oleracea ) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca 2+ pumps. To our knowledge, this is the first report of a plant Ca 2+ pump activity that shows sensitivity to low concentrations of thapsigargin.