GATOR2 regulates the nutrient-dependent recruitment of GATOR1 to lysosomes

Technical Summary: GATOR2 Regulates the Nutrient-Dependent Recruitment of GATOR1 to Lysosomes

Problem Statement
The Target of Rapamycin Complex 1 (TORC1) is a central regulator of cellular metabolism, integrating signals from nutrients, growth factors, and energy status. Its activation is contingent upon recruitment to the lysosomal surface by the Rag GTPases. This recruitment is governed by the GATOR complex, which comprises two subcomplexes: GATOR1, a GTPase-activating protein (GAP) that inhibits TORC1 by converting RagA to its inactive GDP-bound state during starvation, and GATOR2, which antagonizes GATOR1 to promote TORC1 activation under nutrient-replete conditions. While the structural components of these complexes are known, the precise mechanism by which GATOR2 inhibits GATOR1's GAP activity and how nutrient status dynamically regulates the assembly and recruitment of these complexes to the lysosome in a multicellular organism remains undefined. Specifically, it is unclear whether the regulatory hierarchies observed in mammalian tissue culture cells (such as the requirement for the KICSTOR complex) are conserved in Drosophila, and how the GATOR1-GATOR2 supercomplex behaves dynamically in response to nutrient stress in vivo.

Methodology
To address these questions, the authors developed a comprehensive genetic and imaging toolkit in Drosophila melanogaster.

1. Genomic Tagging: Using CRISPR/Cas9, the authors generated homozygous viable fly lines with endogenous tags (e.g., V5, FLAG, T7, GFP11) on key pathway components, including GATOR1 subunits (iml1, nprl2, nprl3), GATOR2 subunits (mio, wdr24, wdr59), and Rag GTPases (ragA, ragC). This ensured native expression levels and avoided artifacts associated with overexpression.
2. Split-GFP System: To visualize endogenous proteins in specific cell types without background from surrounding tissues, the authors employed a split-GFP strategy. Genomically tagged proteins were fused to GFP11, while GFP1-10 was expressed under tissue-specific drivers (e.g., nos-Gal4 for the germline, slbo-Gal4 for border cells). Reconstitution of the GFP signal allowed for high-resolution subcellular localization.
3. Loss-of-Function Genetics: The authors generated homozygous null mutant clones in the germline for various pathway components to assess the dependency of lysosomal recruitment among GATOR1, GATOR2, and the Rag GTPases.
4. Biochemical Assays: Immunoprecipitation (IP) followed by mass spectrometry and Western blotting was used to analyze protein-protein interactions and complex stability in wild-type versus wdr24 mutant ovaries under fed and starved conditions.
5. Fluorescence Recovery After Photobleaching (FRAP): The authors performed lysosomal FRAP on whole cultured ovaries to measure the dynamic exchange rates (mobile fractions) of tagged GATOR1, GATOR2, and Rag GTPase components between the lysosome and the cytoplasm under fed and starved conditions.
6. Cross-Species Validation: Parallel FRAP experiments were conducted in transiently transfected HeLa cells to test the conservation of observed dynamics in mammalian systems.

Key Contributions and Results

• Conservation of Regulatory Hierarchies: The study confirms that in Drosophila, the Rag GTPase is required for the lysosomal recruitment of both GATOR1 and GATOR2. Conversely, GATOR1 and GATOR2 are mutually dependent for their lysosomal localization. Notably, the authors found that Drosophila lacks homologs of the KICSTOR complex (required for GATOR1 recruitment in mammals), yet the core recruitment logic remains conserved.
• GATOR2 Maintains GATOR1 in an Inactive State: Biochemical analysis revealed that in the absence of GATOR2 (specifically in wdr24 mutants), the association between the catalytic subunit of GATOR1 (Nprl2) and the inhibitory subunit (Iml1/DEPDC5) is significantly reduced. In wild-type conditions, GATOR2 promotes a high-affinity association between Iml1 and the Nprl2/Nprl3 heterodimer. This suggests GATOR2 stabilizes GATOR1 in a GAP-inactive conformation where Iml1 binds RagA, preventing GTP hydrolysis.
• Nutrient-Dependent Decoupling of the Supercomplex: FRAP analysis provided dynamic evidence that GATOR1 and GATOR2 function as a supercomplex in nutrient-replete conditions, exhibiting similar lysosomal exchange rates. However, upon starvation, their behaviors diverge:
  • GATOR1: The mobile fraction of GATOR1 components (Nprl2, Iml1) decreases significantly, indicating they become more stably associated with the lysosome (or less exchangeable), consistent with the transition to a GAP-active state that engages RagA-GTP.
  • GATOR2: In contrast, the mobile fraction of core GATOR2 components (Mio, Wdr24) increases upon starvation, suggesting a dissociation from the lysosomal supercomplex.
• The Unique Role of Wdr59: The study identifies Wdr59 as a critical regulator that does not behave like a core GATOR2 component. In fed conditions, Wdr59 dynamics mirror the GATOR1-GATOR2 supercomplex. However, upon starvation, Wdr59 dynamics shift to resemble GATOR1 (reduced mobility) rather than GATOR2. Furthermore, in wdr59 mutants, the starvation-induced decoupling of GATOR1 and GATOR2 is abolished; GATOR1 remains in a state where its lysosomal recruitment does not change with nutrient status. This suggests Wdr59 is required to relieve GATOR2-mediated inhibition and facilitate the transition of GATOR1 to its active state.
• Conservation in Mammals: FRAP experiments in HeLa cells recapitulated the Drosophila findings: starvation decreased the lysosomal recruitment of the GATOR1 component NPRL2 while increasing the recruitment of the GATOR2 component WDR24, and WDR59 behaved similarly to GATOR1.

Significance
The paper establishes a versatile in vivo toolkit for dissecting TORC1 regulation at the single-cell and subcellular levels in Drosophila. The authors propose a refined model for GATOR regulation: under nutrient-rich conditions, GATOR2 stabilizes GATOR1 in a GAP-inactive supercomplex that transiently cycles on and off the lysosome. Upon nutrient starvation, this supercomplex dissociates. This dissociation is facilitated by Wdr59 and allows GATOR1 to adopt a GAP-active conformation, where the catalytic subunit Nprl2 engages RagA to inhibit TORC1. The study highlights that while the core regulatory logic is conserved between mammals and Drosophila, specific components like Wdr59 play nuanced, context-dependent roles in remodeling the GATOR complex to respond to nutrient stress.