Phosphoproteome Exploration Reveals a Reformatting of Cellular Processes in Response to Low Sterol Biosynthetic Capacity in Arabidopsis
Phosphoproteome Exploration Reveals a Reformatting of Cellular Processes in Response to Low Sterol Biosynthetic Capacity in Arabidopsis
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DOI：
10.1021/pr201127u
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发表时间：
2012-02-01
影响因子：
4.4
通讯作者：
Schaller, Hubert
中科院分区：
文献类型：
--
作者：
Heintz, Dimitri;Gallien, Sebastien;Schaller, Hubert
甾醇是细胞活力和生长所需的膜结合类异戊二烯脂质。在植物中，通常认为3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）是其生物合成的关键元件，但该途径的分子调控在很大程度上是未知的。在试图确定监管机构的生物合成流量从酰基辅酶A对植物甾醇，我们比较了野生型拟南芥和突变体的膜磷酸化蛋白质组缺乏HMGR 1。我们进行了一个N-末端标记的微粒体肽与三甲氧基苯基膦（TMPP）衍生物，然后用光谱计数法的磷酸肽的定量评估。肽的TMPP衍生化导致改进的LC-MS/MS检测，这是由于色谱中的疏水性和电喷雾中的电离效率增加。磷酸化蛋白质组覆盖率高出40%。我们进一步发现，与野生型相比，hmgr 1 -1突变体中有31个蛋白质处于不同的磷酸化状态。这些蛋白质中有三分之一是基于新型磷酸肽鉴定的。这种方法揭示了拟南芥膜蛋白质组中的磷酸化变化靶向主要的细胞过程，如运输，钙稳态，光形态建成和碳水化合物合成。这些过程的重新格式化似乎是遗传减少的甾醇生物合成的反应。
Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type Arabidopsis thaliana and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC-MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the hmgr1-1 mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the Arabidopsis membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis.