The Arabidopsis thaliana AtHMA1 protein is a member of the P IB -ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplas- Plant Ca 2ϩ and heavy metal-ATPases belong to the superfamily of P-ATPases (1, 2). Their common characteristic is the presence of a phosphorylated intermediary in the catalytic cycle. In Arabidopsis, the heavy metal-ATPases belong to the P IB subfamily and normally have eight predicted transmembrane domains, whereas the Ca 2ϩ -ATPases are part of the P IIA and P IIB subfamilies and have ten predicted transmembrane domains (2). A common feature among the P IB -ATPases is the presence of a CPX motif, which is though to play a role in metal translocation, as well as putative metal-binding domains located at the amino or carboxyl terminus (3). On the other hand, P IIB ATPases have a calmodulin-binding domain that regulate their activity; however, the P IIA do not have this domain (4). The most important difference between the calcium and heavy metal-ATPases is their substrate specificity (1). However, in vitro metal transport studies performed with membrane fractions isolated from seedlings suggest that one or more members of the superfamily of P-ATPases are capable of transporting calcium and heavy metals. The studies showed competitive inhibition between active transport of heavy metals (such as copper and cadmium) and calcium.mic2 Interestingly, both transport activities were inhibited by the sesquiterpene lactone thapsigargin, a potent and specific inhibitor of SERCA 3 -type pumps (5-7). To date no one has described plant ion pumps that transport calcium and heavy metals in biochemical terms, nor have scientists described genes encoding for ion pumps inhibited by thapsigargin or plant mutants with thapsigargin-sensitive/tolerant phenotypes. Based on the results of biochemical assays suggesting the existence of thapsigargin-sensitive Ca 2ϩ /heavy metal-ATPase, we searched for potential candidate proteins in the Arabidopsis genome. This was made possible by the highly conserved "stalk segment" or S3 sequence adjacent to the third transmembrane segment of the SERCA pumps (8, 9) composed of amino acids DEF-GEQLSK (5-7). This sequence was almost complete and was annotated as a stalk segment (using the topology prediction software ARAMEMNON) in the Arabidopsis heavy metal pump AtHMA1 (At4g37270). This pump belongs to the subclass of zinc/cobalt/cadmium/lead-ATPases and is the most divergent metal pump of the Arabidopsis P IB -ATPases (1, 2, 10, 11) (see Fig. 1). It lacks an amino-terminal heavy metal-binding domain, such as GMXCXXC or GICC(T/S)SE, which is often found in other members of the group. It has an intramembranous SPC instead of the CP(C/H/S) motif located at the putative metal transporting site of P IB -ATPases (11, 12). The pump possesses other structural characteristics related to heavy metal binding and transport, such as a poly-H motif commonly found in zinc-binding pro...

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.

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