Liang F, Sze H
Department of Cell Biology and Molecular Genetics, H.J. Patterson Hall, University of Maryland, College Park, Maryland 20742, USA.
Plant Physiol. 1998 Nov;118(3):817-25. doi: 10.1104/pp.118.3.817.
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.
为了鉴定和表征单个钙离子泵，我们在酵母（酿酒酵母）突变体K616中表达了拟南芥ECA1基因，该基因编码一种内质网型钙离子ATP酶同源物。该突变体（pmc1pmr1cnb1）缺乏高尔基体和液泡膜钙离子泵，在缺钙培养基上生长非常差。从该突变体分离的膜显示出高的H⁺/Ca²⁺反向转运活性，但没有钙离子泵活性。在内膜中表达ECA1可使突变体在缺钙培养基中的生长增加10至20倍。在用巴弗洛霉素A1和短杆菌肽D消除H⁺/Ca²⁺反向转运活性后，检测到来自ECA1转化体的囊泡中有⁴⁵Ca²⁺泵入。该泵对Ca²⁺具有高亲和力（Km = 30 nM），并对ATP表现出两种亲和力（Km分别为20和235 μM）。环匹阿尼酸是动物肌浆网/内质网钙离子ATP酶的特异性阻滞剂，可抑制Ca²⁺转运（50%抑制剂量 = 3 nmol/mg蛋白质），但毒胡萝卜素（3 μM）则无此作用。转运对钙调蛋白不敏感。这些结果表明，这种内质网型钙离子ATP酶可以像在酵母中一样，通过维持亚微摩尔水平的胞质Ca²⁺并在内膜区室中补充Ca²⁺来支持植物细胞生长。这项研究表明，酵母K616突变体提供了一个强大的表达系统，用于研究真核生物钙离子泵的结构/功能关系。
