A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture
A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture
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DOI：
10.1074/mcp.m600250-mcp200
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发表时间：
2007-03-01
影响因子：
7
通讯作者：
Bourguignon, Jacques
中科院分区：
文献类型：
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作者：
Jaquinod, Michel;Villiers, Florent;Bourguignon, Jacques
为了更好地理解细胞运输、各种代谢物的储存及其最终降解的机制，建立了拟南芥液泡蛋白质组。为了这个目的，开发了一种程序来制备高度纯化的液泡从原生质体分离拟南芥细胞培养物使用Ficoll密度梯度。基于空泡标记物α-甘露糖苷酶的比活性，估计空泡的富集因子接近42倍，平均产率为2.1%。使用针对叶绿体、线粒体、质膜和内质网的特异性标记物的抗体，通过Western印迹法验证了其他细胞隔室的显著污染的缺失。基于这些结果，液泡制剂显示了蛋白质组学研究所需的纯度。因此，蛋白质组学的方法来确定蛋白质组分存在于膜和可溶性组分的拟南芥细胞液泡。该方法包括以下步骤：（i）温和的氧化步骤，导致半胱氨酸残基转化为磺基丙氨酸，蛋氨酸转化为蛋氨酸亚砜，（ii）非常疏水的蛋白质的溶液中蛋白水解消化，以及（iii）通过SDS-PAGE进行短暂迁移的蛋白质预分级分离，然后通过液相色谱-串联质谱法进行分析。该程序允许鉴定超过650种蛋白质，其中三分之二与膜疏水部分共纯化，三分之一与可溶性部分共纯化。在从膜组分鉴定的416种蛋白质中，基于一个或多个预测的跨膜结构域的存在，195种被认为是完整的膜蛋白，并且鉴定了110种转运蛋白和相关蛋白质（91种推定的转运蛋白和19种与V-ATP酶泵相关的蛋白质）。关于功能，约20%的蛋白质鉴定以前已知与液泡活动。所鉴定的蛋白质参与离子和代谢物运输（26%）、应激反应（9%）、信号转导（7%）和代谢（6%），或者已被描述为参与典型的液泡活动，如蛋白质和糖水解。几个假定的液泡蛋白的亚细胞定位证实了瞬时表达的绿色荧光蛋白融合结构。
To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker a-mannosidase, the enrichment factor of the vacuoles was estimated at similar to 42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs.