AtHMA1 Is a Thapsigargin-sensitive Ca2+/Heavy Metal Pump

Abstract

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...

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