RT Journal Article SR Electronic T1 A High-Affinity Ca2+ Pump, ECA1, from the Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but Not by Thapsigargin JF Plant Physiology JO Plant Physiol. FD American Society of Plant Biologists SP 817 OP 825 DO 10.1104/pp.118.3.817 VO 118 IS 3 A1 Liang, Feng A1 Sze, Heven YR 1998 UL http://www.plantphysiol.org/content/118/3/817.abstract AB To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding anendoplasmic reticulum-typeCa2+-ATPase 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 from the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca2+-depleted medium. 45Ca2+ pumping into vesicles fromECA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A1 and gramicidin D. The pump had a high affinity for Ca2+ (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 Ca2+-ATPase, inhibited Ca2+ 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 Ca2+-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes. BTP1,3-bis[tris(hydroxymethyl)methylamino]propanePMplasma membraneSC-URAsynthetic complete medium minus uracilSERsarcoplasmic/ERSERCASER Ca2+ ATPase