Abstract
Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to Ras(Ser-17) abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from (32)P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive [(32)P]GTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.
Highlights
Signaling by mTOR complex 1 requires its amino acid-stimulated binding to Rag GTPase heterodimers
We examine whether the ability of amino acids to regulate mammalian target of rapamycin complex 1 (mTORC1) binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging
The finding that RagC/D is preferentially occupied by [32P]GDP indicates that, in contrast to the lack of detectable GTPase activity in short-term incubations in vitro, RagC/D in cells is capable of GTP hydrolysis, whether intrinsic or GAP-mediated. This result should be compared with the finding of Jeong et al [25], who observed that in the x-ray crystal structure of a recombinant wild-type Gtr1-Gtr2 complex isolated from Escherichia coli, Gtr1 contained only GTP, whereas Gtr2 contained only GDP. These studies provide two unexpected findings that will require revision of hypotheses concerning the mechanisms by which amino acids regulate Rag function and the manner in which Rags contribute to mTORC1 activation
Summary
Signaling by mTOR complex 1 requires its amino acid-stimulated binding to Rag GTPase heterodimers. Rags reside in this late endosomal/lysosomal compartment through a noncovalent association with the Ragulator complex, an assembly of five polypeptides anchored to the membrane by the myristoylated/palmitoylated protein p18/LAMTOR1 [17, 18] These results support the conclusion that amino acids, presumably by their ability to promote the binding of raptor to the Rag heterodimer, translocate mTORC1 into the membrane compartment containing Rheb, the proximate activator of mTORC1. We examined the ability of wild-type and mutant Rag heterodimers to bind and hydrolyze guanyl nucleotides in vitro and the extent of Rag GTP charging in amino acid-replete and amino acid-deprived cells. Given the constitutively high RagA/B GTP charging, amino acid sufficiency must, as with raptor binding, promote these other RagA/B interactions independently of altered Rag GTP charging
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