n nnnTo examine the role of the tonoplast in plant salt toleranceand identify proteins involved in the regulation of transportersfor vacuolar Na+ sequestration, we exploited a targeted quantitativeproteomics approach. Two-dimensional differential in-gel electrophoresisanalysis of free flow zonal electrophoresis separated tonoplastfractions from control, and salt-treated Mesembryanthemum crystallinumplants revealed the membrane association of glycolytic enzymesaldolase and enolase, along with subunits of the vacuolar H+-ATPaseV-ATPase. Protein blot analysis confirmed coordinated salt regulationof these proteins, and chaotrope treatment indicated a strongtonoplast association. Reciprocal coimmunoprecipitation studiesrevealed that the glycolytic enzymes interacted with the V-ATPasesubunit B VHA-B, and aldolase was shown to stimulate V-ATPaseactivity in vitro by increasing the affinity for ATP. To investigatea physiological role for this association, the Arabidopsis thalianacytoplasmic enolase mutant, los2, was characterized. These plantswere salt sensitive, and there was a specific reduction in enolaseabundance in the tonoplast from salt-treated plants. Moreover,tonoplast isolated from mutant plants showed an impaired abilityfor aldolase stimulation of V-ATPase hydrolytic activity. Theassociation of glycolytic proteins with the tonoplast may notonly channel ATP to the V-ATPase, but also directly upregulateH+-pump activity.展开▼
机译:ABSTRACTn FONT> TH> TR> TABLE> n
n TOP n <字体颜色= 464c53>抽象 FONT> n 介绍 n 结果 n 讨论 n 方法 n 参考文献 n FONT> TH> TR> TABLE> n nnn研究液泡膜在植物耐盐性中的作用 SUP>和ide nt化蛋白参与液泡状Na + SUP>螯合的转运蛋白 SUP>的蛋白质,我们开发了一种靶向定量 SUP>蛋白质组学方法。二维微分凝胶电泳 SUP>分析自由流区带电泳分离的液泡膜 SUP>级分和盐处理过的菊花膜 I> SUP>植物揭示了糖酵解酶 SUP>醛缩酶和烯醇酶的膜结合,以及液泡H + SUP> -ATPase SUP> V-ATPase的亚基。蛋白质印迹分析证实了这些蛋白质的协调盐调节 SUP>,而离液剂处理表明强烈的 SUP>液泡质缔合。相互免疫沉淀研究 SUP>显示糖酵解酶与V-ATPase SUP> B亚基VHA-B相互作用,醛缩酶被证明可刺激V-ATPase SUP>通过增加对ATP的亲和力来提高活性为了研究 SUP>与此关联的生理作用,表征了拟南芥 I> SUP>胞质烯醇化酶突变体 los2 I>。这些植物 SUP>对盐敏感,并且盐处理过的植物的液泡中烯醇酶 SUP>的丰度有所降低。此外,从突变植物中分离出的 SUP>质膜对醛缩酶刺激V-ATPase水解活性的能力 SUP>受损。糖酵解蛋白与液泡膜的 SUP>关联可能不仅 SUP>仅将ATP引导至V-ATPase,而且还直接上调 SUP> H + < / SUP>-泵活动。 SUP>
展开▼