## 5 A New Level of Regulation: Role of the GAT Domain in Forming Filamentous Structures  
In 2010, CTPS was found to form filamentous structures termed cytoophidia (meaning \"cellular snakes\" in Greek) in _Drosophila melanogaster_ using laser-scanning confocal microscopy [113]. Cytoophidia are widely distributed in cells from many tissues including ovaries, testes, larval brains, guts, trachea, lymph glands, and accessory glands [113; 114]. Subsequent studies have shown that CTPS forms cytoophidia in bacteria [55], budding yeast [115], fission yeast [116], plants [91], archaea [117], zebrafish [118], mice [54], andhumans [119], indicating that filament formation is an ancient and evolutionarily-conserved property of CTPS (Figure 4). In order to explore the molecular biological basis of cytophidia, [PERSON] and co-workers purified _Dm_CTPS and found that the protein can form two types of filamentous structures in vitro with different conformations in the presence of substrates ATP, UTP, or product CTP (Figure 5A,B) [66]. In these two types of filaments, _Dm_CTPS is present in tetrameric units,  
Figure 4: CTPS forms cytophidia in various organisms. Intracellular filamentous structures of CTPS known as cytophidia (green) are formed in a variety or organisms, including the _Drosophila melanogaster_ egg chamber (**A**) [113], _Hidarcula bispanica_ (**B**), conventional confocal image; (**C**), STED image) [117], _S Schizosaccharomyces pombe_ (**D**) [116], and mouse thymus (**E**) [120]. (**A**) _Drosophila_ egg chamber (CTPS, green; DNA, magenta); (**B**) CTPS, green; (**C**) CTPS, red; (**D**) CTPS, green; DNA, magenta; (**E**) CTPS, green; DNA, blue; 5-ethynyl-2\({}^{\prime}\)-deoxyuridine (EdU, which labels proliferating cells), red. Reprinted with permission from Refs. [113], [117], and [120]. Copyright 2010, 2020, and 2021 Elsevier. which interact with each other through an interface in the GAT domain to form the stable filamentous structures. By comparing amino acid sequences of CTPSs, [PERSON] and colleagues discovered that these structures are conserved among multiple species including human, fly, and yeast. At the same time, cryo-EM structures of the two CTPS isoforms in human, hCTPS1 and hCTPS2 [56; 61], and the two CTPS isoforms in budding yeast, URA7 and URA8 [121], revealed that these CTPSs are assembled into filamentous structures through the same region (Figure 5C). Interestingly, there is an insertion of about 15 amino acids between \(\beta\) sheets 2 and 3 of the GAT domains from _Drosophila_ and human CTPSs compared with the GAT domain from other class I Gln-dependent amidotransferases, which makes _Drosophila_ and human GAT domains form an additional short helix and helix2 extensions. In _Dm_CTPS, the adjacent GAT domains are used to form filamentous structures through the interaction of helix2 and surrounding amino acids. Indeed, a single point mutation yielding the H355A _Dm_CTPS variant is sufficient to disrupt this interaction and affect the formation of filamentous structure (Figure 5D) [66]. This role of the GAT domain in filament forming ability obtained through amino acid insertion undoubtedly provides a new level for the regulation of the enzyme's catalytic activity [56; 61; 66; 121]; however, the role of filament formation on the GAT domain itself  
Figure 5: Structural features of _Dm_CTPS filaments. (**A**) Atom model display for CTPS filament assembly of tetramers for product-bound _Dm_CTPS. Protomers of a tetramer are colored differently and CTP is shaded pink. (**B**) Atom model display for CTPS filament assembly of tetramers for substrate-bound _Dm_CTPS. Protomers of a tetramer are colored differently and GTP is shaded cyan. (**C**) Comparison of the filament interaction region between \(\beta\) sheets 2 and 3 among different species as shown in the color key. Models are aligned by the GAT domain. \(\beta\)-Sheets 2 and 3 are colored in light cyan. (**D**) _Dm_CTPS filament interface. Residues at the interface are displayed and labeled. Sulfur atoms are shown as yellow spheres.