REGULATION OF PHOSPHOINOSITIDE METABOLISM & EARLY RESPONSE TO OSMOTIC STRESS

我们项目的一个目标是了解 延伸因子1A(EEF-1A)调节磷脂酰肌醇4-激酶 活动。EEF-1A是一种多功能蛋白质。已知的有三个 EEF-1A的翻译后修饰可能潜在地 影响其功能。除了磷酸化，其他的 翻译后修饰尚未在植物中得到证实。 我们用MALDI-MS证明胡萝卜EEF-1A含有一个 磷酸甘油乙醇胺(PGE)的翻译后修饰和 鉴定被修饰的多肽片段。虽然我们可以 体内标记研究表明，EEF-1A已被 [14C]乙醇胺只有通过使用质谱学才能 完全是翻译后修饰的特征。我们的数据 表明单独的PGE翻译后修饰不能 影响EEF-1A的功能；然而，与其他 翻译后修饰，前列腺素E脂修饰可以 影响EEF-1a的分布从而影响其功能 在细胞内。我们正在继续我们的研究，使用现场定向 突变以敲除翻译后修饰的氨基酸。 质谱学数据对这项工作至关重要。

One goal of our project is to understand the mechanism by which elongation factor 1A (eEF-1A) regulates phosphatidylinositol 4-kinase activity. eEF-1A is a multifunctional protein. There are three known posttranslational modifications of eEF-1A which could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. We used MALDI-MS to show that carrot eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification and to identify the peptide fragment which is modified. While we could demonstrate by in vivo labeling studies that eEF-1A incorporated [14C]ethanolamine it was only by using mass spectrometry that we could definitely characterize the postranslational modification. Our data indicate that the PGE posttranslational modification alone does not affect the function of eEF-1A; however, in combination with other posttranslational modifications, the PGE lipid modification could affect the distribution and thereby affect the function of eEF-1A within the cell. We are continuing our studies using site-directed mutagenesis to knock out the posttranslationally modified amino acid. The mass spectra data were essential for this work.