Figure 1.

Schematic diagram of method to isolate endogenous mTORC1 using recombinant Rag GTPase proteins.A, GST-tagged RagA and RagC are expressed and purified from E. coli. B, GST-Rag GTPases are individually set into the high-mTORC1-affinity states: GST-RagA-GTPγS and GST-RagC-GDP. C, GST-RagA-GTPγS and GST-RagC-GDP are combined at a 1:1 ratio to form a high-mTORC1-affinity dimer. D, GST-Rag GTPase dimers are incubated with cell lysate containing mTORC1. E, GST-Rag GTPases are isolated using glutathione beads, which bind the GST tag. In this way, mTORC1 is captured on the beads and separated from other lysate proteins. F, mTORC1 is eluted by switching the nucleotide state of the GST-Rag GTPases to a low-mTORC1-affinity state. GTPγS, GTPgammaS; mTORC1, mechanistic target of rapamycin complex 1.