The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.

Broad Institute of Harvard and the Massachusetts Institute of Technology Cambridge MA 02142

Brown Center on the Biology of Aging Brown University Providence RI 02903

Center for Genomic Medicine Massachusetts General Hospital Boston MA 02114

Department of Biology Massachusetts Institute of Technology Cambridge MA 02139

Department of Medicine Massachusetts General Hospital Boston MA 02114

Department of Pathology and Laboratory Medicine Brown University Providence RI 02903

Department of Pathology Massachusetts General Hospital Boston MA 02114

Department of Surgery Massachusetts General Hospital Boston MA 02114

Harvard Medical School Boston MA 02115

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague 166 10 Czech Republic

Legorreta Cancer Center Brown University Providence RI 02903

Whitehead Institute for Biomedical Research Cambridge MA 02142

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Structural basis for the dynamic regulation of mTORC1 by amino acids