Biochemical Characterization of Arabidopsis Calcium Antiporter, CAX1p

9604246 Rea在刺激偶联的胞质游离Ca2+升高后重新建立静息状态需要从植物细胞的胞质中快速去除Ca2+。研究者与博士合作。怀特黑德生物医学研究所的Hirschi和Fink最近从拟南芥中分离出了两个cdna， CAXl和CAX2。这些cDNA抑制了酿酒酵母液泡Ca2+积累缺陷菌株的突变（Hirschi et al., 1996）。对酵母中表达CAXl或CAX2的空泡膜富集囊泡的实验表明，这些基因分别编码高效率和低效率的H+/Ca2+交换体。CAXl基因产物的特性表明，它是一种高容量转运体，通过催化pH梯度激活的液泡Ca2+积累，在植物细胞中维持低胞质游离Ca2+浓度。在克隆了CAXl和CAX2并广泛定义了它们在酵母中的翻译产物的功能特征后，研究者现在的目标是扩展他对其中一个的研究，CAXlp，假定的液泡H+/Ca2+反转运蛋白，以更好地定义其在完整植物中的运输能力和膜定位。研究计划的主要目标有四个方面：(1)测定与CAXlp相关的植物膜组分。研究者的数据强烈表明，CAXlp对应于液泡H+/Ca2+反转运蛋白，但这仍有待直接测试。(2)建立了CAXlp的纯化和重组方法。这对于研究CAXlp对H+/Ca2+反转运的充分性以及其他反转运功能的充分性是必要的，并为未来研究转运化学计量学和结构-功能关系提供了材料。(3)阐明了caxlp介导的H+/Ca2+交换的电原性和转运化学计量学。知道每Ca2+交换的H+离子的数量是至关重要的，因为这决定了在体内普遍条件下CAXlp可以实现的最大积累比。反过来，这将为CAXlp的生理功能提供指示。(4)检测CAXlp运输Na+、Ca2+或Na+以及H+的能力。研究者已经表明，CAXlp催化Ca2+转运优先于其他二价阳离子，但不知道Na+是否可以替代H+作为驱动离子，或者CAXlp是否胜任Na+/H+反转运以及H+/Ca2+反转运，就像一些非植物H+/Ca2+和Na+/Ca2+反转运一样。了解其膜定位、充足性、可能的生理平衡和离子选择性，将为研究者和其他人未来研究CAXlp对植物一般离子稳态和Ca2+依赖性刺激-反应偶联的调节、结构-功能特征和生理影响提供坚实的基础。植物细胞对光、重力、触觉和寒冷等刺激的反应是通过胞质钙离子（Ca2+）浓度的变化来调节的。在这些过程中，一些Ca2+的主要细胞内储存库是液泡。由于液泡Ca2+浓度通常比细胞质高10,000倍，并且维持低细胞质浓度对正常细胞功能至关重要，因此需要在液泡中浓缩Ca2+的有效系统。其中之一是转运蛋白，一种钙质子反转运蛋白，位于液泡周围的膜上。这种膜蛋白利用膜上氢离子（H+）浓度梯度所含的电化学能量，通过将液泡中的H+与细胞质中的Ca2+交换，驱动Ca2+从细胞质转运到液泡中。本研究旨在了解这种转运体的分子机制。

9604246 Rea Re-establishment of the resting state after stimulus-coupled elevations of cytosolic free Ca2+ requires the rapid removal of Ca2+ from the cytosol of plant cells. The investigator in collaboration with Drs. Hirschi and Fink, Whitehead Institute for Biomedical Research, has recently isolated two cDNAs, CAXl and CAX2, from Arabidopsis thaliana. These cDNA's suppress a mutation in a Saccharomyces cerevisiae strain defective in vacuolar Ca2+ accumulation (Hirschi et al., 1996). Experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing CAXl or CAX2 demonstrate that these genes encode high efficiency and low efficiency H+/Ca2+ exchangers, respectively. The properties of the CAXl gene product indicate that it is the high capacity transporter responsible for contributing to the maintenance of low cytosolic free Ca2+ concentrations in plant cells by catalyzing pH gradient-energized vacuolar Ca2+ accumulation. Having cloned CAXl and CAX2 and broadly defined the functional characteristics of their translation products in yeast, the investigator aims now to extend his studies of one of these, CAXlp, the putative vacuolar H+/Ca2+ antiporter, to better defining its transport capabilities and membrane localization in the intact plant. The main objectives of the research program are be four-fold: (1) Determination of the plant membrane fraction(s) with which CAXlp is associated. The investigator's data strongly indicate that CAXlp corresponds to the vacuolar H+/Ca2+ antiporter but this remains to be tested directly. (2) The development of procedures for the purification and reconstitution of CAXlp. This is necessary for studies of the sufficiency of CAXlp for H+/Ca2+ antiport, and possibly other antiport functions, and the provision of material for future investigations of transport stoichiometry and structure-function relations. (3) Elucidation of the electrogenicity, and thence transport stoichiometry, of CAXlp-mediated H+/Ca2+ exchange. It is critical to know the numbe r of H+ ions exchanged per Ca2+ since this determines the maximum accumulation ratio that can be achieved by CAXlp under the conditions prevailing in vivo. This, in turn, will provide indications of the physiological function of CAXlp. (4) Examination of the capacity of CAXlp for the transport of Na+ as well as Ca2+ or Na+ as well as H+. The investigator has shown that CAXlp catalyzes Ca2+ transport in preference to other divalent cations but it is not known if Na+ can substitute for H+ as driver ion or whether CAXlp is competent in Na+/H+ antiport as well as H+/Ca2+ antiport, as is the case for some non-plant H+/Ca2+ and Na+/Ca2+ antiporters. Knowledge of its membrane localization, sufficiency, likely physiological poise and ion-selectivity will provide a firm foundation for future investigations, by both the investigator and others, of the regulation, structure-function characteristics and physiological impact of CAXlp on general ion homeostasis and Ca2+-dependent stimulus-response coupling in plants. The coordination of many plant cell responses to stimuli, such as light, gravity, touch and cold, is mediated by changes in cytoplasmic calcium ion ( Ca2+) concentration. A major intracellular reservoir for some of the Ca2+ involved in these processes is the vacuole. Since the vacuolar concentration of Ca2+ is often 10,000 times higher than that of the cytoplasm, and the maintenance of low cytoplasmic concentrations is critical for normal cell function, efficient systems for concentrating Ca2+ in the vacuole are required. One of these is a transporter, a calcium-proton antiporter, located in the membrane surrounding the vacuole. This membrane protein harnesses the electrochemical energy contained in the hydrogen ion (H+) concentration gradient across this membrane to drive the transport of Ca2+ from the cytoplasm into the vacuole by exchanging vacuolar H+ for cytoplasmic Ca2+. This research is concerned with understanding the molecular mechanism of this transporter.