Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

Abstract

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

Structural basis for the helical filament formation of Escherichia coli glutamine synthetase

“…Many metabolic regulatory mechanisms are known to be controlled by enzyme filamentation, 27–29 such as those mediated by yeast glutamate dehydrogenase, 27 glutamine synthetase, 16 asparagine synthetase, 30 and CTP synthase 31 . Here, we have demonstrated how metal ions can drive polymerization of EcGS.…”

“…Intracellular yeast GS filamentation is known to silence glutamine synthesis and nitrogen assimilation, 16 and filamentous yeast CTP synthase displays reduced catalytic activity. 31 Thus, GS filamentation may be a universal mechanism to prevent continuous glutamine production in vivo, which would otherwise consume energy excessively and promote cellular proliferation. 32 Alternatively, F I G U R E 7 Evolutionary analysis of GS indicates a specific H5/H13 subgroup.…”

“…Nevertheless, Ni 2+ ‐induced EcGS filaments cannot convert glutamate to glutamine, as the reagents glutamate and ATP immediately dissociate polymeric EcGS, even though the catalytic funnel for loading reaction substrate glutamate and cofactor ATP remains open. Intracellular yeast GS filamentation is known to silence glutamine synthesis and nitrogen assimilation, 16 and filamentous yeast CTP synthase displays reduced catalytic activity 31 . Thus, GS filamentation may be a universal mechanism to prevent continuous glutamine production in vivo, which would otherwise consume energy excessively and promote cellular proliferation 32 .…”

Molecular Crowding Facilitates Bundling of IMPDH Polymers and Cytoophidium Formation

“…Electrostatic interactions often require high protein concentrations and changes in salt or pH. The formation of multiple filamentous macrostructures in budding yeast have been shown to be induced by the change of pH, implying that the cytoophidium is assembled through the self-association of filamentous polymers (Hansen et al, 2021; Petrovska et al, 2014). Our findings support this notion as the presence of molecular crowders is sufficient to trigger purified human IMPDH2 proteins to reconstitute cytoophidium-like macrostructures (Figure 1).…”

Structural basis of dynamic P5CS filaments

“…This filamentous structure is membraneless and termed the cytoophidium for its appearance (Liu, 2010;Liu, 2016). The cytoophidium has emerged as a mechanism for the regulation of metabolic enzymes (Hansen et al, 2021;Liu, 2016;Zhou et al, 2021). Recently, we have shown that Drosophila P5CS forms cytoophidia in vivo and forms individual filaments in vitro (Zhang et al, 2020).…”