A High-Affinity Ca(2+) Pump, ECA1, from the Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but Not by Thapsigargin

To identify and characterize individual Ca(2+) pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca(2+)-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca(2+) pump and grows very poorly on Ca(2+)-depleted medium. Membranes isolated from the mutant showed high H(+)/Ca(2+)-antiport but no Ca(2+)-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca(2+)-depleted medium. (45)Ca(2+) pumping into vesicles from ECA1 transformants was detected after the H(+)/Ca(2+)-antiport activity was eliminated with bafilomycin A(1) and gramicidin D. The pump had a high affinity for Ca(2+) (K(m) = 30 nm) and displayed two affinities for ATP (K(m) of 20 and 235 μm). Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, inhibited Ca(2+) transport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin (3 μm) did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca(2+)-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca(2+) and replenishing Ca(2+) in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca(2+) pumps from eukaryotes.