Crystal structure of the Gtr1p-Gtr2p complex reveals new insights into the amino acid-induced TORC1 activation - PubMed (original) (raw)

Crystal structure of the Gtr1p-Gtr2p complex reveals new insights into the amino acid-induced TORC1 activation

Rui Gong et al. Genes Dev. 2011.

Abstract

The target of rapamycin (TOR) complex 1 (TORC1) is a central cell growth regulator in response to a wide array of signals. The Rag GTPases play an essential role in relaying amino acid signals to TORC1 activation through direct interaction with raptor and recruitment of the TORC1 complex to lysosomes. Here we present the crystal structure of the Gtr1p-Gtr2p complex, the Rag homologs from Saccharomyces cerevisiae, at 2.8 Å resolution. The heterodimeric GTPases reveal a pseudo-twofold symmetric organization. Structure-guided functional analyses of RagA-RagC, the human homologs of Gtr1p-Gtr2p, show that both G domains (N-terminal GTPase domains) and dimerization are important for raptor binding. In particular, the switch regions of the G domain in RagA are indispensible for interaction with raptor, and hence TORC1 activation. The dimerized C-terminal domains of RagA-RagC display a remarkable structural similarity to MP1/p14, which is in a complex with lysosome membrane protein p18, and directly interact with p18, therefore recruiting mTORC1 to the lysosome for activation by Rheb. Our results reveal a structural model for the mechanism of the Rag GTPases in TORC1 activation and amino acid signaling.

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Figures

Figure 1.

Overall structure of the Gtr1p–Gtr2p. (A) Overall structure of the Gtr1p–Gtr2p complex as a ribbon representation in two different views. G domains of Gtr1p and Gtr2p bound to GMPPNP are colored in blue and red, respectively, and CTDs are colored in green and orange, respectively. GMPPNP is shown as a ball-and-stick representation, and magnesium atoms are shown as black balls. (B,C) Dimerization is required for RagA–RagC to interact with raptor. Different RagA and RagC constructs were cotransfected with raptor into HEK293 cells. Protein interaction was determined by coimmunoprecipitation. RagAQL and RagCSN are mutants restricted to RagAGTP (Q66L) and RagCGDP(S75N), respectively. (A-N) G domain of RagA; (C-N) G domain of RagC; (IP) immunoprecipitation; (WB) Western blot. (D) The G domain of RagC is required to promote the interaction between RagA and raptor. (CSN) RagCSN (RagCGDP); (C-C) CTD of RagC. (E) The CTD of RagA is required for both basal and RagC-enhanced activity to stimulate TORC1. TORC1 activity was indirectly measured by the level of S6K phosphorylation. RagA and RagC constructs were cotransfected with HA-S6K into HEK293 cells. For amino acid starvation, cells were starved for amino acids for 1 h before harvesting. Amino acid starvation is denoted as AA−. Sample cultured in complete medium is denoted as AA+. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies.

Figure 2.

Mapping the raptor-interacting surface on RagA. (A) RagA plays a major role in raptor binding. The interaction between raptor and cotransfected RagA or RagC was examined by coimmunoprecipitation. 3A is the T90A/L93A/T96A mutation of RagC, in which mutations of the corresponding residues in Ras have been shown to abolish the effector binding. (B) G domain of Gtr1p is shown in a ribbon representation (left panel) and a surface representation (right panel). Corresponding residues involved in composite RagA mutations (M1–M4) are indicated with a stick representation and are colored in green, and residues mutated in M5–M13 are colored in cyan on the surface, as shown in Supplemental Figure S1 and Supplemental Table S2. All mutants were generated based on RagAQL(RagAGTP). (C) The regions close to switch I and II in the RagA G domain are important for raptor interaction. Interaction between raptor and cotransfected RagA mutants was examined by coimmunoprecipitation. (D) The raptor interaction-defective RagA mutants cannot activate TORC1. RagA mutants were cotransfected with HA-S6K into HEK293 cells, and phosphorylation of HA-S6K in the absence of amino acids (indicating the activity of RagA) was determined.

Figure 3.

Mapping the dimerization critical residues. (A) Detailed interaction of Gtr1p–Gtr2p CTDs. Residues involved in hydrogen bond formation are connected with a dashed line directly, and hydrophobic interactions are linked by the central boxes with the detailed interactions shown. Critical residues for dimer formation are indicated as black stars. (B) Sequence alignment of critical regions for dimer formation in the CTDs of Rag GTPases. Composite RagA and RagC mutations are indicated above the sequence and are summarized in Supplemental Table S3. (C) The α8 and β9 of RagC are critical for dimer formation with RagA. Different RagA or RagC mutants were cotransfected as indicated. The interaction was determined by coimmunoprecipitation and Western blot. (SE) Short exposure; (LE) long exposure; (AWT) wild-type RagA; (CWT) wild-type RagC; [AWT(low)] transfection of 100 ng of DNA; [AWT(high)] transfection of 200 ng of DNA.

Figure 4.

The CTDs of Rag GTPases share similar structures with p14/MP1 and are responsible for p18 interaction and lysosomal localization. (A) Structure comparison of Gtr1p–Gtr2p CTDs and the p14/MP1 complex. The structures are shown in a ribbon representation, and Gtr1p and Gtr2p CTDs are colored green and orange, respectively, while p14 and MP1 are colored pink and light blue, respectively. (B) The CTD dimer of RagA–RagC interacts with p18. RagA and RagC constructs were cotransfected with the p18 construct and the protein interaction was determined by coimmunoprecipitation. (C) The CTDs of RagA and RagC are necessary and sufficient for lysosomal localization. Different deletion mutants were transfected in 293 cells. The transfected Flag-RagA or HA-RagC was stained (red) along with DNA (blue) and lysosomal marker LAMP2 (green). (D) Working model of Rag GTPases in TORC1 activation. In the cartoon, the Rag CTDs interact with p18 and p14/MP1 to target the GTPases to lysosomes. The G domains of Rag associate with raptor, thus recruiting TORC1 to lysosomes for activation.

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Crystal structure of the Gtr1p-Gtr2p complex reveals new insights into the amino acid-induced TORC1 activation - PubMed (original) (raw)