Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of polysome enrichment in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or ‘moonlighting’ function that modulates the ISR independent of its aspartate aminotransferase activity.

中文翻译：

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细胞溶质天冬氨酸氨基转移酶在氧化应激期间作为 Gcn2 活性的核糖体结合调节剂

翻译调节是细胞对快速变化的外部条件做出反应的一个基本方面。特定的 RNA 结合蛋白 (RBP) 在压力期间协调不同 mRNA 队列的翻译调节。为了鉴定在翻译控制中具有先前未被重视的作用的 RBP，我们使用多核糖体分析和质谱来鉴定和量化与无应激酵母细胞中以及氧化应激和氨基酸饥饿期间翻译核糖体相关的蛋白质，这两者都会诱导综合应激反应。 ISR）。超过 800 种蛋白质在 polysome 梯度级分中被鉴定出来，包括核糖体蛋白质、翻译因子和许多其他没有先前描述的翻译相关作用的蛋白质，包括许多代谢酶。我们确定了无应激和应激细胞中多核糖体富集模式的变化，并确定了应激期间重多核糖体富集的蛋白质。对富含多核糖体的 RBP 进行遗传筛选，发现细胞溶质天冬氨酸转氨酶 Aat2 是一种核糖体相关蛋白，其缺失赋予生长对氧化应激的敏感性。Aat2 的缺失通过增强的 eIF2α 磷酸化和GCN4激活。重要的是，非催化AAT2突变体保留了多核糖体关联并且没有表现出增强的应激敏感性。因此，Aat2 具有单独的与核糖体相关的翻译调节或“moonlighting”功能，可独立于其天冬氨酸氨基转移酶活性调节 ISR。