A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae.
A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae.
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DOI：
10.1080/21541248.2022.2141019
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发表时间：
2022-01
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雷帕霉素（mTOR）复合物的机制靶点负责协调真核细胞生长的营养有效性。氨基酸信号通过Rag/Gtr异源二聚体传递到mTOR。由于Rag/Gtr GTPases的强制性异二聚体结构，研究它们的生化特性一直具有挑战性。在这里，我们描述了一种更新的检测方法，使我们能够探测酿酒酵母Gtr异源二聚体的鸟嘌呤核苷酸结合亲和力和动力学。我们首先确定了Gtr2p缺乏使交联的结构元素。通过使用基于序列保守的突变，我们恢复了Gtr2p与结合核苷酸之间的交联。使用这种结构，我们确定了Gtr异源二聚体的核苷酸结合亲和力，并发现它在亚基间通信的不同形式下运作，而不是人类Rag GTPases。我们的研究定义了营养感知的Gtr/Rag-mTOR轴的进化分化。
The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.