一种新的研究酿酒葡萄球菌Gtr异源二聚体鸟嘌呤核苷酸结合的交联试验。

Q2 Biochemistry, Genetics and Molecular Biology
Dylan D Doxsey, Kristen Veinotte, Kuang Shen
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引用次数: 1

摘要

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。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of <i>S. cerevisiae</i>.

A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of <i>S. cerevisiae</i>.

A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of <i>S. cerevisiae</i>.

A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae.

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.

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来源期刊
Small GTPases
Small GTPases Biochemistry, Genetics and Molecular Biology-Biochemistry
CiteScore
6.10
自引率
0.00%
发文量
6
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