# SEA and GATOR 10 Years Later
## 5 Function of the SEA and GATOR in Nutrient Sensing and Responding
### SEACIT/GATOR1 as GAP for EGO/RAG  
Two papers published simultaneously in 2013 reported the results that have dramatically increased the significance of the SEA/GATOR complex in the regulation of mTORC1 pathway. The laboratory of [PERSON] found that in _S. cerevisiae_, the SEA subcomplex, which was subsequently named SEACIT (SEAC subcomplex inhibiting TORC1 signaling) [14], acts as a GAP for Gtr1 and, thus, inhibits TORC1 [11]. In a parallel study, [PERSON]'s laboratory characterized for the first time the human homologue of the SEA complex, and also found the GAP activity of the SEACIT analogue, which received the GATOR1 name (GTPase activating protein activity towards RAGA) [12]. In both yeast and humans, SEACAT/GATOR2 acts upstream of SEACIT/GATOR1, suppressing its GAP activity, thus being \"an inhibitor of an inhibitor\", although how exactly this suppression is achieved is completely unknown. A molecular mechanism of how SEACIT/GATOR1 acts as a GAP has been addressed in several functional and structural studies, but a complete consensus of how exactly the GAP function is exerted has not yet been achieved. Indeed, in an initial study by the [PERSON] group, it was demonstrated that in _S. cerevisiae_, Iml1/Seal can co-precipitate with Gtr1 in the presence but substantially less in the absence of other SEACIT subunits. Yet, in the in vitro binding and GAP essays, Iml1/Seal could directly bind to Gtr1 and promote GTP hydrolysis in the absence of Npr2 and Npr3. GAPs often supply a catalytic amino acid residue (Arg, Asp or Gln) in their active sites, thus forming an \"arginine finger\" or \"Gln/Asn thumb\" that can be inserted into nucleotide-binding pocket of a GTPase [109]. In the highly conserved Iml1/Seal domain, essential for its GAP activity (aa 929-952), a conserved Arg\({}^{943}\) was critical for GAP activity both in vitro and in yeast cells. Human DEPDC5 could partially complement TORC1 inhibition defect in _iml1_A cells, suggesting a conserved role of Iml1/Seal and DEPDC5 across the species. Therefore, when the cryo-EM structure of GATOR1 (Figure 2A) and GATOR1 in the complex with RAG GTPases was solved, it came as a surprise because it revealed a very unexpected mode of interaction between GTPases and GAPs [29]. For the structural studies, GATOR1 was copurified with RAG GTPase heterodimer, containing wild type RAGA and mutant RAGC, which can bind GTP, but not GDP. In addition, this heterodimer was loaded with GDP and non-hydrolysable GTP analogue (GppNHp) to create the most favorable nucleotide-binding configuration for interaction with GATOR1. The structure demonstrated that the overall conformation of the GATOR1 in a complex with RAG GTPases is similar to a free GATOR1 (see above). The SHEN domain of DEPDC5 can contact directly with a site proximal to nucleotide binding pocket of GTP analogue-bound RAGA. However, quite surprisingly, this interaction did not appear to be responsible for the stimulation of GTP hydrolysis. The kinetic analysis of GTP hydrolysis of DEPDC5 alone with RAGA or NPRL2/NPRL3 dimer with RAGA revealed that it is rather NPRL2/NPRL3, which has GAP activity. Moreover, a conserved Arg\({}^{78}\) localized on the loop of NPRL2 longin domain is the \"arginine finger\", responsible for GAP activity [110]. However, this Arg\({}^{78}\) is located far away and is opposite to the RAGs binding interface of DEPDC5. Moreover, an earlier study from [PERSON] laboratory showed that amino acid stimulation enhances the interaction of RAGA with both endogenous DEPDC5 and NPRL3 [111]. To explain these rather contradictory observations, a two-state model of GATOR1 interaction with RAG GTPases was proposed: in the inhibitory mode, DEPDC5 SHEN domain interacts strongly with RAGs and GAP activity of GATOR1 is weak; alternatively, a low affinity interaction dependent on NPRL2/NPRL3 stimulates GAP activity. Such bi-modal activity has not been previously observed between a GAP and a GTPase.