A HIGH-AFFINITY CA2-RETICULUM IS INHIBITED BY CYCLOPIAZONIC ACID BUT NOT BY THAPSIGARGIN( PUMP, ECA1, FROM THE ENDOPLASMIC)

To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-A TPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on Ca2+-depleted medium. Membranes isolated fro m the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity. E xpression of ECA1 in endomembranes increased mutant growth by 10- to 2 0-fold in Ca2+-depleted medium. Ca-45(2+) pumping into vesicles from E CA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A(1) and gramicidin D. The pump had a hig h affinity for Ca2+ (K-m = 30 nM) and displayed two affinities for ATP (K-m of 20 and 235 mu M). Cyclopiazonic acid, a specific blocker of a nimal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited Ca2+ t ransport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin ( 3 mu M) did not. Transport was insensitive to calmodulin. These result s suggest that this endoplasmic reticulum-type Ca2+-ATPase could suppo rt cell growth in plants as in yeast by maintaining submicromolar leve ls of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartment s. This study demonstrates that the yeast K616 mutant provides a power ful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes.