A high-affinity Ca2+ pump, ECA1, from the endoplasmic reticulum is inhibited by cyclopiazonic acid but not by thapsigargin.
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 microM). 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 microM) 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.
Liang
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F
Department of Cell Biology and Molecular Genetics, H.J. Patterson Hall, University of Maryland, College Park, Maryland 20742, USA.
Sze
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H
D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000494:pharmacology; D002384:Catalysis; D003470:Culture Media; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D012441:Saccharomyces cerevisiae / Q000235:genetics; D019284:Thapsigargin / Q000494:pharmacology*
D002147:Calmodulin; D003470:Culture Media; D007211:Indoles; D011994:Recombinant Proteins; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid