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

Abstract

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 Km 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.