A high-affinity Ca{sup 2+} pump, ECA1, from the endoplasmic reticulum is inhibited by cyclopiazonic acid but not by thapsigargin
- Univ. of Maryland, College Park, MD (United States). Dept. of Cell Biology and Molecular Genetics
To identify and characterize individual Ca{sup 2+} pumps, the authors have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca{sup 2+}-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca{sup 2+} pump and grows very poorly on Ca{sup 2+}-depleted medium. Membranes isolated from the mutant showed high H{sup +}/Ca{sup 2+}-antiport but no Ca{sup 2+}-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca{sup 2+}-depleted medium. {sup 45}Ca{sup 2+} pumping into vesicles from ECA1 transformants was detected after the H{sup +}/Ca{sup 2+}-antiport activity was eliminated with bafilomycin A{sub 1} and gramicidin D. The pump had a high affinity for Ca{sup 2+} (K{sub m} = 30 nM) and displayed two affinities for ATP. Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca{sup 2+}-ATPase, inhibited Ca{sup 2+} transport but thapsigargin did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca{sup 2+}-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca{sup 2+} and replenishing Ca{sup 2+} in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca{sup 2+} pumps from eukaryotes.
- Sponsoring Organization:
- USDOE, Washington, DC (United States)
- OSTI ID:
- 305469
- Journal Information:
- Plant Physiology (Bethesda), Journal Name: Plant Physiology (Bethesda) Journal Issue: 3 Vol. 118; ISSN 0032-0889; ISSN PLPHAY
- Country of Publication:
- United States
- Language:
- English
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