Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana
Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana
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DOI：
10.1093/pcp/pch099
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发表时间：
2004-06-01
影响因子：
4.9
通讯作者：
Mimura, T
中科院分区：
文献类型：
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作者：
Shimaoka, T;Ohnishi, M;Mimura, T
液泡膜中的大量蛋白质，包括泵、载体、离子通道和受体，支持植物液泡的各种功能。迄今为止，很少有蛋白质参与这些活动已被确定在分子水平上。在这项研究中，蛋白质组学分析被用来确定新的液泡膜蛋白。通过蛋白质组学进行任何细胞器分析的主要要求是分离的细胞器的纯度需要高。利用悬浮培养的拟南芥细胞（拟南芥Co 1 -0细胞悬浮液），开发了一种用于分离完整的高度纯化的液泡的方法。在分离的空泡组分的Western印迹中没有检测到质膜蛋白，只有少数来自高尔基体和内质网的蛋白。纯化的液泡膜的蛋白质组学分析涉及通过SDS-PAGE和LC-MS/MS分析的蛋白质的分级分离。使用这种方法，有可能确定163个蛋白质。这些包括特征良好的液泡膜蛋白，如V型H+-ATP酶和V型H+-PP酶，以及其他功能合理预期与液泡膜相关的蛋白。还有许多蛋白质的功能尚未推导出来。
A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Co1-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+-ATPases and V-type H+-PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.