Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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A High-Affinity Ca2+ Pump, ECA1, from the Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but Not by Thapsigargin1

Feng Liang2 and Heven Sze*

Department of Cell Biology and Molecular Genetics, H.J. Patterson Hall, University of Maryland, College Park, Maryland 20742

To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-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 from ECA1 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+ (Km = 30 nM) and displayed two affinities for ATP (Km 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.

1   This work was supported in part by Department of Energy grant no. DE-95ER20200 and by Maryland Agricultural Experiment Station grant no. MD-J-151 (to H.S.).
2   Present address: The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850.
*   Corresponding author; e-mail hs29{at}umail.umd.edu; fax 1-301-314-9082.

Plant Physiol. (1998) 118: 817-825
Copyright Clearance Center:   0032-0889/98/118//09
© 1998 American Society of Plant Physiologists




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