Polyribosome binding by GCN1 is required for full activation of eukaryotic translation initiation factor 2α kinase GCN2 during amino acid starvation
Polyribosome binding by GCN1 is required for full activation of eukaryotic translation initiation factor 2α kinase GCN2 during amino acid starvation
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DOI：
10.1074/jbc.m414566200
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发表时间：
2005-04-22
影响因子：
4.8
通讯作者：
Hinnebusch, AG
中科院分区：
文献类型：
--
作者：
Sattlegger, E;Hinnebusch, AG
蛋白激酶GCN 2通过磷酸化真核翻译起始因子2 α介导氨基酸饥饿细胞中基因表达的翻译控制。在酿酒酵母中，饥饿细胞中不带电荷的tRNA激活GCN 2需要它与GCN 1中心点GCN 20调节复合物和核糖体直接相互作用。GCN 1也与细胞提取物中的核糖体相互作用，但尚不清楚这种活性是否对其在饥饿细胞中刺激GCN 2功能的能力至关重要。我们描述了GCN 1的两个保守的，不连续的片段中的点突变，其导致活细胞中GCN 1中心点GCN 20的多聚核糖体缔合减少，而不减少GCN 1表达或其与GCN 20的相互作用。与单独突变一个片段相比，同时突变两个片段可使GCN 1中心点GCN 20与多核糖体结合的降低程度更大，并使真核生物翻译起始因子2 α磷酸化的降低程度更强。这些发现提供了强有力的证据，证明GCN 1的核糖体结合是其作为GCN 2的正调节剂所必需的。GCN 1结构域中的一个特定突变，在序列上与翻译延伸因子3（eEF 3）相关，降低GCN 2激活的程度远远超过它降低GCN 1与核糖体结合的程度。因此，eEF 3样结构域似乎在GCN 2活化中具有效应子功能。这一结论支持了GCN 1的eEF 3相关活性影响饥饿细胞中不带电荷的tRNA对核糖体解码位点的占据的模型。
The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2 alpha. In Saccharomyces cerevisiae, activation of GCN2 by uncharged tRNAs in starved cells requires its direct interaction with both the GCN1 center dot GCN20 regulatory complex and ribosomes. GCN1 also interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1 center dot GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1 center dot GCN20 and a stronger decrease in eukaryotic translation initiation factor 2 alpha phosphorylation than did mutating in one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells.