Assaying protein palmitoylation in plants.
Assaying protein palmitoylation in plants.
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DOI：
10.1186/1746-4811-4-2
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发表时间：
2008-01-11
期刊：
影响因子：
5.1
通讯作者：
Grierson, Claire S
中科院分区：
文献类型：
--
作者：
Hemsley, Piers A;Taylor, Laura;Grierson, Claire S
蛋白质S酰化(又称棕榈酰化)是蛋白质中的半胱氨酸残基通过硫代酯键发生的可逆的翻译后加成反应。它允许与膜有很强的结合。虽然已有S酰化反应的预测方法，但预测并不完善。现有的演示植物蛋白S酰化反应的方法要么费时费力，要么昂贵。我们描述了一种测定植物蛋白S酰化反应的生物素开关方法。我们演示了这项技术，证明了异三聚体G蛋白亚基AGG2是S酰化的，正如诱变实验所预测的那样。我们还表明，拟南芥α-微管蛋白亚单位池的一部分在PlantA中是S酰化的。这可能解释了观察到的植物微管的膜结合。由于α-微管蛋白普遍表达，无论研究中的细胞类型如何，它们都有可能被用作S酰化试验的阳性对照。我们提供强大的生物素切换方案，允许使用标准实验室技术快速分析植物中的蛋白质S酰化状态，而不需要昂贵或专业的设备。我们建议将α-微管蛋白作为该方案的有效阳性对照。
Protein S-acylation (also known as palmitoylation) is the reversible post-translational addition of acyl lipids to cysteine residues in proteins through a thioester bond. It allows strong association with membranes. Whilst prediction methods for S-acylation exist, prediction is imperfect. Existing protocols for demonstrating the S-acylation of plant proteins are either laborious and time consuming or expensive. We describe a biotin switch method for assaying the S-acylation of plant proteins. We demonstrate the technique by showing that the heterotrimeric G protein subunit AGG2 is S-acylated as predicted by mutagenesis experiments. We also show that a proportion of the Arabidopsis alpha-tubulin subunit pool is S-acylated in planta. This may account for the observed membrane association of plant microtubules. As alpha-tubulins are ubiquitously expressed they can potentially be used as a positive control for the S-acylation assay regardless of the cell type under study. We provide a robust biotin switch protocol that allows the rapid assay of protein S-acylation state in plants, using standard laboratory techniques and without the need for expensive or specialised equipment. We propose alpha-tubulin as a useful positive control for the protocol.