Liang F, Cunningham K W, Harper J F, Sze H
Department of Plant Biology, University of Maryland, College Park, MD 20742, USA.
Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8579-84. doi: 10.1073/pnas.94.16.8579.
To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [gamma-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.
为了解植物中单个钙离子泵的结构、作用和调控机制，我们利用酵母作为异源表达系统来测试拟南芥（ECA1）一个基因的功能。ECA1编码一个116 kDa的多肽，该多肽具有P型钙离子泵（EC 3.6.1.38）共有的所有保守结构域。与肌浆网/内质网钙离子泵（53%）相比，该氨基酸序列与质膜钙离子泵（32%）的同源性更高。在高尔基体钙离子泵（pmr1）或高尔基体和液泡钙离子泵（pmr1 pmc1 cnb1）均有缺陷的酵母突变体，在含有10 mM EGTA或3 mM Mn2+的培养基上生长敏感。ECA1的表达恢复了任一突变体在EGTA上的生长。从用ECA1转化的pmr1 pmc1 cnb1突变体中分离出膜，以确定ECA1多肽（ECA1p）是否可作为钙离子泵ATP酶反应循环的中间体被磷酸化。在存在[γ-32P]ATP的情况下，ECA1p形成了一种106 kDa的钙离子依赖性[32P]磷蛋白，该磷蛋白对羟胺敏感。动物肌浆网/内质网钙离子泵的阻滞剂环匹阿尼酸抑制了磷蛋白的形成，而毒胡萝卜素则没有。用针对羧基末端的抗体进行免疫印迹表明，ECA1p主要与从拟南芥植物中分离出的内质网膜相关。这些结果支持了ECA1在拟南芥中编码内质网型钙离子泵的模型。ECA1p恢复突变体pmr1在含有Mn2+的培养基上生长的能力，以及形成Mn2+依赖性磷蛋白，表明ECA1p也可能通过将Mn2+泵入植物内膜区室来调节Mn2+的稳态。
