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. Author manuscript; available in PMC: 2015 Jan 15.
Published in final edited form as: Science. 2015 Jan 7;347(6218):188–194. doi: 10.1126/science.1257132

Figure 3.

Figure 3

Stable overexpression of full-length SLC38A9.1 or its N-terminal Ragulator-binding domain makes the mTORC1 pathway insensitive to amino acid deprivation. (A) Stable overexpression of FLAG-SLC38A9.1 largely restores mTORC1 signaling during total amino acid starvation and completely restores it upon deprivation of leucine or arginine. HEK-293T cells transduced with lentiviruses encoding the specified proteins were deprived for 50 min of all amino acids, leucine, or arginine and, where indicated, re-stimulated for 10 min with the missing amino acid(s). Cell lysates were analyzed for the levels of the specified proteins and the phosphorylation states of S6K1, ULK1, and 4E-BP1. (B and C) Overexpression of neither SLC38A9.2 nor a point mutant of SLC38A9.1 that fails to bind Ragulator rescues mTORC1 signaling during amino acid starvation. Experiment was performed as in (A) except that cells were stably expressing SLC38A9.2 (B) or SLC38A9.1 I68A (C). (D) Stable overexpression of the Ragulator-binding domain of SLC38A9.1 largely restores mTORC1 signaling during total amino acid starvation and completely rescues it upon deprivation of leucine or arginine. Experiment was performed as in (A) except cells were stably expressing FLAG-SLC38A9.1 1-119. (E) The ability of SLC38A9.1 overexpression to rescue mTORC1 signaling during amino acid starvation is eliminated by co-expression of RagBT54N-RagCQ120L, a Rag heterodimer locked in the nucleotide configuration associated with amino acid deprivation. Effects of expressing the indicated proteins on mTORC1 signaling were monitored by the phosphorylation state of co-expressed FLAG-S6K1. (F) Effects of concanamycin A and Torin1 on mTORC1 signaling in cells stably expressing SLC38A9.1. HEK-293T cells stably expressing the indicated FLAG-tagged proteins were treated with the DMSO vehicle or the specified small molecule inhibitor during the 50 min starvation for and, where indicated, the 10 min stimulation with amino acids.