Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, United States.
Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, United States.
Methods Enzymol. 2022;675:131-158. doi: 10.1016/bs.mie.2022.07.007. Epub 2022 Aug 19.
The mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient levels in the cell and based on the availability, regulates cellular growth and proliferation. Its activity is tightly modulated by two GTPase units, the Rag GTPases and the Rheb GTPase. The Rag GTPases are the central hub of amino acid sensing as they summarize the amino acid signals from upstream regulators and control the subcellular localization of mTORC1. Unique from canonical signaling GTPases, the Rag GTPases are obligatory heterodimers, and the two subunits coordinate their nucleotide loading states to regulate their functional states. Robust biochemical analysis is indispensable to understanding the molecular mechanism governing the GTPase cycle. This chapter discusses protocols for purifying and biochemically characterizing the Rag GTPase heterodimer. We described two purification protocols to recombinantly produce the Rag GTPase heterodimer in large quantities. We then described assays to quantitatively measure the nucleotide binding and hydrolysis by the Rag GTPases. These assays allow for a thorough investigation of this unique heterodimeric GTPase, and they could be applicable to investigations of other noncanonical GTPases.
雷帕霉素复合物 1 （mTORC1）的机械靶点感知细胞内的营养水平，并根据可用性调节细胞生长和增殖。其活性受到两种 GTPase 单元，即 Rag GTPases 和 Rheb GTPase 的严格调节。Rag GTPases 是氨基酸感应的中心枢纽，因为它们总结了来自上游调节剂的氨基酸信号，并控制 mTORC1 的亚细胞定位。与典型的信号转导 GTPase 不同，Rag GTPases 是必需的异源二聚体，两个亚基协调它们的核苷酸加载状态以调节它们的功能状态。强大的生化分析对于理解调节 GTPase 循环的分子机制是必不可少的。本章讨论了纯化和生化表征 Rag GTPase 异源二聚体的方案。我们描述了两种纯化方案，以大量重组产生 Rag GTPase 异源二聚体。然后，我们描述了定量测量 Rag GTPases 核苷酸结合和水解的测定法。这些测定法允许对这种独特的异源二聚体 GTPase 进行彻底研究，并且它们可能适用于其他非典型 GTPase 的研究。
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