Filamentation of asparagine synthetase in Saccharomyces cerevisiae.

Asparagine synthetase (ASNS) and CTP synthase (CTPS) are two metabolic enzymes crucial for glutamine homeostasis. A genome-wide screening in Saccharomyces cerevisiae reveal that both ASNS and CTPS form filamentous structures termed cytoophidia. Although CTPS cytoophidia were well documented in recent years, the filamentation of ASNS is less studied. Using the budding yeast as a model system, here we confirm that two ASNS proteins, Asn1 and Asn2, are capable of forming cytoophidia in diauxic and stationary phases. We find that glucose deprivation induces ASNS filament formation. Although ASNS and CTPS form distinct cytoophidia with different lengths, both structures locate adjacently to each other in most cells. Moreover, we demonstrate that the Asn1 cytoophidia colocalize with the Asn2 cytoophidia, while Asn2 filament assembly is largely dependent on Asn1. In addition, we are able to alter Asn1 filamentation by mutagenizing key sites on the dimer interface. Finally, we show that ASN1D330V promotes filamentation. The ASN1D330V mutation impedes cell growth in an ASN2 knockout background, while growing normally in an ASN2 wild-type background. Together, this study reveals a connection between ASNS and CTPS cytoophidia and the differential filament-forming capability between two ASNS paralogs.

Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.

Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filament-forming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frame-GFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases (Ura7p and Ura8p) and two asparagine synthetases (Asn1p and Asn2p) form filaments both in the cytoplasm and in the nucleus. Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated.

Nucleotide synthesis is regulated by cytoophidium formation during neurodevelopment and adaptive metabolism.

The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. Recently, it has been demonstrated that the enzymatic activity of CTPsyn is attenuated by incorporation into cytoophidia in bacteria and yeast cells. Here we demonstrate that CTPsyn is regulated in a similar manner in Drosophila tissues in vivo. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.

CTP synthase forms cytoophidia in the cytoplasm and nucleus.

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase.