Purification and biochemical characterization of the Rag GTPase heterodimer

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.