Geisler M, Frangne N, Gomès E, Martinoia E, Palmgren M G
Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Plant Physiol. 2000 Dec;124(4):1814-27. doi: 10.1104/pp.124.4.1814.
Several lines of evidence suggest that regulation of intracellular Ca(2+) levels is crucial for adaptation of plants to environmental stress. We have cloned and characterized Arabidopsis auto-inhibited Ca(2+)-ATPase, isoform 4 (ACA4), a calmodulin-regulated Ca(2+)-ATPase. Confocal laser scanning data of a green fluorescent protein-tagged version of ACA4 as well as western-blot analysis of microsomal fractions obtained from two-phase partitioning and Suc density gradient centrifugation suggest that ACA4 is localized to small vacuoles. The N terminus of ACA4 contains an auto-inhibitory domain with a binding site for calmodulin as demonstrated through calmodulin-binding studies and complementation experiments using the calcium transport yeast mutant K616. ACA4 and PMC1, the yeast vacuolar Ca(2+)-ATPase, conferred protection against osmotic stress such as high NaCl, KCl, and mannitol when expressed in the K616 strain. An N-terminally modified form of ACA4 specifically conferred increased NaCl tolerance, whereas full-length ATPase had less effect.
多条证据表明，细胞内Ca(2+)水平的调节对于植物适应环境胁迫至关重要。我们已经克隆并鉴定了拟南芥自抑制Ca(2+)-ATPase亚型4（ACA4），一种钙调蛋白调节的Ca(2+)-ATPase。对绿色荧光蛋白标记的ACA4进行共聚焦激光扫描数据以及对通过两相分配和蔗糖密度梯度离心获得的微粒体部分进行蛋白质印迹分析表明，ACA4定位于小液泡。通过钙调蛋白结合研究和使用钙转运酵母突变体K616的互补实验证明，ACA4的N末端包含一个带有钙调蛋白结合位点的自抑制结构域。当在K616菌株中表达时，ACA4和酵母液泡Ca(2+)-ATPase PMC1赋予了对高NaCl、KCl和甘露醇等渗透胁迫的抗性。ACA4的N末端修饰形式特异性地赋予了更高的NaCl耐受性，而全长ATPase的作用较小。
