Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance
Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance
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DOI：
10.1105/tpc.109.069211
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发表时间：
2009-12-01
期刊：
影响因子：
11.6
通讯作者：
Pantoja, Omar
中科院分区：
文献类型：
--
作者：
Barkla, Bronwyn J.;Vera-Estrella, Rosario;Pantoja, Omar
为了研究液泡膜在植物耐盐性中的作用，并确定参与调节液泡Na+螯合转运蛋白的蛋白质，我们利用了一种有针对性的定量蛋白质组学方法。自由流动区带电泳的二维差异凝胶电泳分析分离出的液泡膜组分从控制，盐处理的Mesembryocarcinum stack um植物揭示了糖酵解酶醛缩酶和烯醇化酶，沿着与液泡H+-ATP酶V-ATP酶的亚基的膜协会。蛋白质印迹分析证实了这些蛋白质的协调盐调节，离液剂处理表明一个强大的液泡膜协会。相互免疫共沉淀研究表明，糖酵解酶与V-ATP酶亚基B VHA-B相互作用，并且醛缩酶显示通过增加对ATP的亲和力来刺激V-ATP酶活性。为了研究这种关联的生理作用，拟南芥胞质烯醇化酶突变体，LOS 2，其特征在于。这些植物是盐敏感的，并有一个特定的减少烯醇化酶丰度的液泡膜从盐处理的植物。此外，从突变体植物中分离的液泡膜表现出对V-ATPase水解活性的醛缩酶刺激能力受损。糖酵解蛋白与液泡膜的结合不仅可以将ATP传递给V-ATPase，而且可以直接上调H+泵的活性。
To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity.