Braun Katherine A, Vaga Stefania, Dombek Kenneth M, Fang Fang, Palmisano Salvator, Aebersold Ruedi, Young Elton T
Department of Biochemistry, University of Washington, 1705 Northeast Pacific Street, Seattle, WA 98195-7350, USA.
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, CH-8057 Zurich, Switzerland.
Sci Signal. 2014 Jul 8;7(333):ra64. doi: 10.1126/scisignal.2005000.
Stresses, such as glucose depletion, activate Snf1, the Saccharomyces cerevisiae ortholog of adenosine monophosphate-activated protein kinase (AMPK), enabling adaptive cellular responses. In addition to affecting transcription, Snf1 may also promote mRNA stability in a gene-specific manner. To understand Snf1-mediated signaling, we used quantitative mass spectrometry to identify proteins that were phosphorylated in a Snf1-dependent manner. We identified 210 Snf1-dependent phosphopeptides in 145 proteins. Thirteen of these proteins are involved in mRNA metabolism. Of these, we found that Ccr4 (the major cytoplasmic deadenylase), Dhh1 (an RNA helicase), and Xrn1 (an exoribonuclease) were required for the glucose-induced decay of Snf1-dependent mRNAs that were activated by glucose depletion. Unexpectedly, deletion of XRN1 reduced the accumulation of Snf1-dependent transcripts that were synthesized during glucose depletion. Deletion of SNF1 rescued the synthetic lethality of simultaneous deletion of XRN1 and REG1, which encodes a regulatory subunit of a phosphatase that inhibits Snf1. Mutation of three Snf1-dependent phosphorylation sites in Xrn1 reduced glucose-induced mRNA decay. Thus, Xrn1 is required for Snf1-dependent mRNA homeostasis in response to nutrient availability.
诸如葡萄糖耗竭等应激会激活Snf1，它是酿酒酵母中与单磷酸腺苷激活的蛋白激酶（AMPK）同源的蛋白，可实现适应性细胞反应。除了影响转录外，Snf1还可能以基因特异性方式促进mRNA稳定性。为了解Snf1介导的信号传导，我们使用定量质谱法鉴定以Snf1依赖性方式磷酸化的蛋白质。我们在145种蛋白质中鉴定出210个Snf1依赖性磷酸肽。其中13种蛋白质参与mRNA代谢。在这些蛋白质中，我们发现Ccr4（主要的细胞质去腺苷酸化酶）、Dhh1（一种RNA解旋酶）和Xrn1（一种外切核糖核酸酶）是葡萄糖诱导的由葡萄糖耗竭激活的Snf1依赖性mRNA降解所必需的。出乎意料的是，XRN1的缺失减少了在葡萄糖耗竭期间合成的Snf1依赖性转录本的积累。SNF1的缺失挽救了同时缺失XRN1和REG1（编码一种抑制Snf1的磷酸酶的调节亚基）的合成致死性。Xrn1中三个Snf1依赖性磷酸化位点的突变减少了葡萄糖诱导的mRNA降解。因此，Xrn1是响应营养可用性的Snf1依赖性mRNA稳态所必需的。
