Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex
Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex
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DOI：
10.1002/jcb.20658
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发表时间：
2006-02-15
影响因子：
4
通讯作者：
Chen, KY
中科院分区：
文献类型：
--
作者：
Jao, DLE;Chen, KY
真核起始因子 5A (eIF5A) 是自然界中唯一含有马尿苷的蛋白质，马尿苷是一种不寻常的氨基酸，由脱氧马尿苷合酶和脱氧马尿苷羟化酶在翻译后分两步形成。编码 eIF5A 奥尔脱氧马尿碱合酶的基因对于细胞存活和增殖至关重要。为了确定 eIF5A 的生理功能，我们采用串联亲和纯化 (TAP) 方法和质谱法来寻找和鉴定潜在的 eIF5A 相互作用蛋白。 TAP 标签与染色体 TIF51A 基因框内融合，并以其天然水平表达的 eIF5A-TAP 融合蛋白用作诱饵，以钓出其相互作用的伙伴。在 150 mM 的盐浓度下，脱氧马尿苷合酶是唯一与 eIF5A 结合的蛋白质。当盐浓度降低至 125 mM 或更低时，eIF5A 与一组蛋白质相互作用，这些蛋白质被鉴定为 80S 核糖体复合物的组成部分。 eIF5A-核糖体相互作用对 RNase 和 EDTA 处理敏感，表明相互作用需要 RNA 以及 40S 和 60S 核糖体亚基的连接。重要的是，hypusine 到精氨酸的单一突变完全消除了 eIF5A-核糖体相互作用。对数细胞与稳定期细胞和 eIF3 突变株的蔗糖梯度沉降分析表明，内源性 eIF5A 与主动翻译的 80S 核糖体和多聚核糖体以 RNase 和 EDTA 敏感的方式共沉降。我们的研究首次证明 eIF5A 以 hypusine 依赖性方式与分子复合物而不是单个蛋白质相互作用，这表明 eIF5A 的基本功能很可能是通过其与主动翻译核糖体的相互作用介导的。
Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally in two steps by deoxyhypusine synthase and deoxyhyphusine hydroxylase. Genes encoding eIF5A ordeoxyhypusine synthase are essential for cell Survival and proliferation. To determine the physiological function of eIF5A, we have employed the tandem affinity purification (TAP) method and mass spectrometry to search for and identify the potential eIF5A-interacting proteins. The TAP-tag was fused in-frame to chromosomal TIF51A gene and eIF5A-TAP fusion protein expressed at its natural level was used as the bait to fish Out its interacting partners. At salt concentrations of 150 mM, deoxyhypusine synthase was the only protein bound to eIF5A. As salt concentrations were lowered to 125 mM or less, eIF5A interacted with a set of proteins, which were identified as the components of the 80S ribosome complex. The eIF5A-ribosome interaction was sensitive to RNase and EDTA treatments, indicating the requirement of RNA and the joining of 40S and 60S ribosomal subunits for the interaction. Importantly, a single mutation of hypusine to arginine completely abolished the eIF5A-ribosome interaction. Sucrose gradient sedimentation analysis of log versus stationary phase cells and eIF3 mutant strain showed that the endogenous eIF5A co-sedimented with the actively translating 80S ribosomes and polyribosomes in an RNase- and EDTA-sensitive manner. Our Study demonstrates for the first time that eIF5A interacts in a hypusine-dependent manner with a molecular complex rather than a single protein, suggesting that the essential function of eIF5A is mostly likely mediated through its interaction with the actively translating ribosomes.