Biochemical Characterization of Arabidopsis Calcium Antiporter, CAX1p

9604246 Rea细胞质游离Ca 2+的刺激偶联升高后静息状态的重建需要从植物细胞的细胞质中快速除去Ca 2+。研究人员与Whitehead生物医学研究所的Hirschi和Fink博士合作，最近从拟南芥中分离出两种cDNA，CAX1和CAX2。 这些cDNA抑制酿酒酵母菌株中液泡Ca2+积累缺陷的突变（Hirschi等人，1996年）。对从表达CAX1或CAX2的酵母分离的液泡膜富集囊泡的实验证明，这些基因分别编码高效和低效H +/Ca2+交换剂。CAX1基因产物的性质表明，它是负责通过催化pH梯度激发的液泡Ca 2+积累来维持植物细胞中低胞质游离Ca 2+浓度的高容量转运蛋白。在克隆了CAX1和CAX2并广泛定义了它们在酵母中的翻译产物的功能特征之后，研究者现在的目标是扩展他对其中之一CAXlp（推定的液泡H +/Ca 2+反向转运蛋白）的研究，以更好地定义其在完整植物中的运输能力和膜定位。该研究计划的主要目标有四个：（1）确定与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+反向转运蛋白，但这仍有待直接测试。(2)CAXlp的纯化和重建程序的开发。这对于研究CAXlp对H +/Ca 2+反向转运的充分性以及可能的其他反向转运功能以及为未来运输化学计量和结构-功能关系的研究提供材料是必要的。(3)阐明CAXlp介导的H +/Ca2+交换的产电性，以及由此的转运化学计量。知道每个Ca2+交换的H+离子的数目是至关重要的，因为这决定了在体内主要条件下通过CAXlp可以实现的最大积累率。这反过来将提供CAXlp的生理功能的指示。(4)检查CAXlp转运Na+以及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，但尚不清楚Na+是否可以取代H+作为驱动离子，或者CAXlp是否能够在Na +/H+反向转运以及H +/Ca2+反向转运中胜任，就像一些非植物H +/Ca2+和Na +/Ca2+反向转运体的情况一样。其膜定位，充分性，可能的生理平衡和离子选择性的知识将提供一个坚实的基础，为未来的调查，由研究者和其他人，的监管，结构-功能特性和生理影响的CAXlp一般离子稳态和Ca2+依赖的刺激-响应耦合在植物中。 许多植物细胞对刺激（如光、重力、触摸和冷）的响应的协调是由细胞质钙离子（Ca 2+）浓度的变化介导的。一个主要的细胞内水库的一些Ca2+参与这些过程是液泡。由于液泡中的Ca 2+浓度通常比细胞质高10，000倍，并且维持低细胞质浓度对于正常细胞功能至关重要，因此需要有效的系统来浓缩液泡中的Ca 2+。其中之一是转运蛋白，一种钙质子反向转运蛋白，位于液泡周围的膜中。这种膜蛋白利用跨膜的氢离子（H+）浓度梯度中所含的电化学能，通过将液泡H+交换为细胞质Ca 2+来驱动Ca 2+从细胞质转运到液泡中。本研究旨在了解这种转运蛋白的分子机制。

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.