Institute of Natural Sciences, Massey University, Auckland, New Zealand.
FEBS J. 2012 May;279(10):1761-76. doi: 10.1111/j.1742-4658.2012.08553.x. Epub 2012 Apr 10.
Adjusting protein synthesis by phosphorylating eukaryotic translation initiation factor 2 (eIF2α) is a major mechanism by which eukaryotes adapt to and overcome stress. The eIF2α kinase Gcn2 is essential for overcoming amino acid starvation in all eukaryotes. We have shown that to sense starvation, the Gcn2 RWD domain must directly contact its effector protein, Gcn1, and both must bind to the ribosome, suggesting that starvation is sensed within a Gcn1-Gcn2-ribosome complex. The mammalian protein IMPACT, highly expressed in neurons, and its yeast orthologue yeast IMPACT homologue (Yih1) harbour an RWD domain with Gcn1-binding activity. We have shown that Yih1 downregulates Gcn2 by competing with Gcn2 for Gcn1-binding. Here, we provide evidence that Yih1 forms a complex with ribosomes. In velocity sedimentation assays, overexpressed glutathione S-transferase (GST)-tagged Yih1 cosedimented with polyribosomes independently of Gcn1. Reduction of polyribosomes to monosomes concomitantly decreased GST-Yih1 sedimentation in the heavy fractions where polyribosomes are normally found. Furthermore, GST-Yih1 coprecipitated large ribosomal protein Rpl39 independently of Gcn1. GST-Yih1 overexpression did not significantly affect Gcn1-ribosome or Gcn2-ribosome cosedimentation. myc-tagged Yih1 expressed from its own promoter cosedimented with polyribosomes independently of Gcn1, indicating that Yih1-ribosome interaction occurs under physiological conditions. GST-IMPACT cosedimented with yeast ribosomes and coprecipitated Rpl39 in a Gcn1-independent fashion, suggesting that Yih1/IMPACT-ribosome association is evolutionarily conserved. Moreover, GST-IMPACT coprecipitated actin as found for GST-Yih1. Taken together, our findings strongly suggest that IMPACT/Yih1 associates with ribosomes and that these ribosomes may simultaneously carry Gcn1 and Gcn2. Close physical proximity of Yih1 to the Gcn1-Gcn2-ribosome complex would allow cells to quickly inhibit Gcn2 whenever or wherever necessary.
通过磷酸化真核翻译起始因子 2（eIF2α）来调节蛋白质合成是真核生物适应和克服应激的主要机制。在所有真核生物中，eIF2α 激酶 Gcn2 对于克服氨基酸饥饿是必不可少的。我们已经表明，为了感知饥饿，Gcn2 的 RWD 结构域必须直接与效应蛋白 Gcn1 接触，并且两者都必须与核糖体结合，这表明饥饿是在 Gcn1-Gcn2-核糖体复合物中被感知的。哺乳动物蛋白 IMPACT，在神经元中高度表达，其酵母直系同源物（酵母 IMPACT 同源物（Yih1））具有与 Gcn1 结合活性的 RWD 结构域。我们已经表明，Yih1 通过与 Gcn2 竞争 Gcn1 结合来下调 Gcn2。在这里，我们提供了证据表明 Yih1 与核糖体形成复合物。在速度沉降测定中，过表达的谷胱甘肽 S-转移酶（GST）标记的 Yih1 与多核糖体一起共沉降，与 Gcn1 无关。多核糖体还原为单核糖体，同时 GST-Yih1 在重级分中的沉降减少，多核糖体通常在重级分中。此外，GST-Yih1 与核糖体大亚基蛋白 Rpl39 共沉淀，与 Gcn1 无关。GST-Yih1 的过表达并没有显著影响 Gcn1-核糖体或 Gcn2-核糖体共沉降。来自其自身启动子的 myc 标记的 Yih1 与多核糖体一起共沉降，与 Gcn1 无关，表明 Yih1-核糖体相互作用发生在生理条件下。GST-IMPACT 与酵母核糖体共沉降，并以 Gcn1 非依赖性方式共沉淀 Rpl39，表明 Yih1/IMPACT-核糖体的结合在进化上是保守的。此外，GST-IMPACT 共沉淀肌动蛋白，如 GST-Yih1 一样。综上所述，我们的研究结果强烈表明，IMPACT/Yih1 与核糖体结合，并且这些核糖体可能同时携带 Gcn1 和 Gcn2。Yih1 与 Gcn1-Gcn2-核糖体复合物的紧密物理接近将使细胞能够在任何时候和任何地方快速抑制 Gcn2，只要有必要。
