A thapsigargin-sensitive Ca²⁺ pump is present in the pea Golgi apparatus membrane

The Golgi apparatus behaves as a bona fide Ca²⁺ 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 Ca²⁺ accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca²⁺ 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 Ca²⁺ uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca²⁺ uptake of 2.5 nmol mg min^-1 and an apparent K_m for Ca²⁺ of 209 nM. The ATP-dependent Ca²⁺ uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I₅₀] = 126 μM) and cyclopiazonic acid (I₅₀ = 0.36 nmol mg protein^-1) and was not stimulated by calmodulin (1 μM). Addition of Cd²⁺ and Cu²⁺ at nanomolar concentration inhibited the Ca²⁺ uptake, whereas Mn²⁺, Fe²⁺, and Co²⁺ had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I₅₀ = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca²⁺ ATPase from mammalian cells. A thapsigargin-sensitive Ca²⁺ uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca²⁺ pumps. To our knowledge, this is the first report of a plant Ca²⁺ pump activity that shows sensitivity to low concentrations of thapsigargin.