ECA1 complements yeast mutants defective in Ca(2+) pumps and encodes an endoplasmic reticulum-type Ca(2+)-ATPase in Arabidopsis thaliana

To understand the structure, role, and regulation of individual Ca(2+) pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca(2+) pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca(2+) pumps. Yeast mutants defective in a Golgi Ca(2+) pump (pmr1) or both Golgi and vacuolar Ca(2+) pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn(2+). Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca(2+)-pumping ATPases. In the presence of [γ-(32)P]ATP, ECA1p formed a Ca(2+)-dependent [(32)P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca(2+) pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca(2+) pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn(2+), and the formation of a Mn(2+)-dependent phosphoprotein suggested that ECA1p may also regulate Mn(2+) homeostasis by pumping Mn(2+) into endomembrane compartments of plants.