Original researchThe proline synthesis enzyme P5CS forms cytoophidia in Drosophila
Introduction
Cell metabolism manages energy mobilization and utilization in each cell, by coordinating hundreds of thousands of metabolic reactions occur simultaneously at any time. Spatial and temporal segregation of different reactions is critical for keeping the cell functionally normal. In eukaryotes, many membrane-bound organelles provide such a way to compartmentalize metabolic pathways. For example, mitochondrion is the place for oxidative reaction. Lysosomes house numeral digestive enzymes for the degradation of organelles and other molecules. Various post-translational protein modifications take place in the Golgi apparatus, while endoplasmic reticulum lies in the crossroad of protein synthesis.
However, the membrane-bound organelles do not account for the segregation of all metabolic reactions in the cell. The traditional view of cytosol as a homogeneous soup has recently been challenged. Many membraneless structures, such as P bodies, purinosomes, and U bodies have been identified in the cytoplasm (Sheth and Parker, 2006; Liu and Gall, 2007; An et al., 2008). In 2010, three studies independently reported that the metabolic enzyme CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium (which translates as cellular snake in Greek) in bacteria, yeast, and fruit flies (Ingerson-Mahar et al., 2010; Liu, 2010; Noree et al., 2010). Subsequently, CTPS-containing cytoophidia have been found in humans (Carcamo et al., 2011; Chen et al., 2011; Gou et al., 2014; Huang et al., 2017; Sun and Liu, 2019, Sun and Liu, 2019), fission yeast (Zhang et al., 2014; Li et al., 2018; Andreadis et al., 2019; Zhang and Liu, 2019), plants (Daumann et al., 2018) and archaea (Zhou et al., 2020), suggesting filament-forming is an evolutionarily conserved property of CTPS (Liu, 2011; Aughey and Liu, 2015, Liu, 2016). In addition, genome-wide analysis and functional studies have shown that many more metabolic enzymes can form filamentous or dot structures in response to specific developmental stages or environmental stimuli (Noree et al., 2010; Aughey et al., 2014; Barry et al., 2014; Strochlic et al., 2014; Wang et al., 2015; Shen et al., 2016; Zhang et al., 2018; Wu and Liu, 2019).
CTPS catalyzes the rate-limiting step of the de novo biosynthesis of CTP, an essential nucleotide for the synthesis of RNA, DNA, and sialoglycoproteins (Higgins et al., 2007). It also plays an important role in the synthesis of membrane phospholipids (McDonough et al., 1995; Hatch and McClarty, 1996; Ostrander et al., 1998). CTPS catalyzes the ATP-dependent phosphorylation of UTP, followed by a glutaminase reaction that transfers the amide nitrogen to the C4 position of UTP to generate CTP (Lieberman, 1956; Long CW, 1967). CTPS activity is important to cell proliferation and has been demonstrated to be upregulated in cancers (Williams et al., 1978; Van Den Berg et al., 1993, 1995; Verschuur et al., 1998; Martin et al., 2014). In Drosophila, CTPS functionally links to the oncogene Myc (Aughey et al., 2016). Cytoophidia have been found to be enriched in various human cancers (Chang et al., 2017). Other studies have implicated abnormal CTPS activity with a number of human cancers, as well as with viral infection and parasitic diseases (Kizaki et al., 1980; Weber et al., 1980; Gharehbaghi et al., 2000; Verschuur et al., 2000, 2001; De Clercq, 2001).
Aiming to identify proteins interacting with CTPS, we carry out a genome-wide yeast two-hybrid screen and identify a putative CTPS-interacting protein, △1-pyrroline-5-carboxylate synthase (P5CS). P5CS, a bifunctional enzyme that encompasses simultaneously glutamate kinase and γ-glutamyl phosphate reductase activities, catalyzes the reduction of glutamate to △1-pyrroline-5-carboxylate, which is subsequently converted to proline by P5C reductase (P5CR) (Vogel and Davis, 1952; Smith et al., 1980; Merrill et al., 1989; Hu et al., 1992, 1999). P5CS controls the rate-limiting step in proline synthesis and is negatively regulated by proline (Hu et al., 1992; Zhang et al., 1995; Hong et al., 2000). Defects in P5CS cause a connective tissue disorder characterized by lax skin and joint dislocations (Baumgartner et al., 2000; Baumgartner et al., 2005; Bicknell et al., 2008; Hu et al., 2008). In plants, P5CS is a stress-inducible gene and involved in salt and drought tolerance (Rai and Penna, 2013).
Here we identify P5CS as a novel filament-forming protein in Drosophila and reveal coordinated filamentation between P5CS and CTPS. Although these two enzymes have not been connected in previous biochemical studies, this study provides evidence supporting that P5CS and CTPS are coordinated spatially.
Section snippets
P5CS forms cytoophidia in Drosophila cells
Using the full-length Drosophila CTPS as bait, we carried out a yeast two-hybrid analysis by screening a genome-wide library of Drosophila peptides for potential interacting partners for CTPS. To this end, we retrieved a pool of 19 genes (Table S1), which encode putative CTPS-interacting proteins. Interestingly, most of these proteins are metabolic enzymes.
Then, we focused on one of the genes in our list, CG7470, which codes for a 776-aa protein. Bioinformatics analysis revealed that CG7470 is
Discussion
Using the Drosophila follicle cell as a model system, we demonstrate that CTPS and P5CS form cytoophidia interdependently. Both CTPS and P5CS cytoophidia show very similar patterns. However, we provide evidence that they are not the same structure. First, we observe that P5CS filaments entangle with CTPS filaments. Image analysis shows that these strings are very close but not identical. Second, the ends of P5CS filaments frequently associate with the cell cortex, whereas CTPS does not show
Yeast two-hybrid screen
A yeast two-hybrid screen was carried out by Hybrigenics Services (Cambridge, MA, USA). The full-length Drosophila melanogaster CG6854 protein was used as bait. The screen was performed on Drosophila whole-embryo cDNA library using two different fusions of N-LexA-CG6854-C and N-Gal4-CG6854-C. The screen identified P5CS as an interacting protein with CTPS.
Drosophila melanogaster stocks and genetics
All stocks were raised at 21°C on standard cornmeal. The stocks used were as follows: hsFLP;UAS-GFPnls;UAS-dcr2; tub<Gal80>Gal4/SM5, Cy-TM6, Tb
Acknowledgments
This work was supported by ShanghaiTech University, the UK Medical Research Council (Grant No. MC_UU_12021/3 and MC_U137788471), and National Natural Science Foundation of China (Grant No. 31771490). We are grateful to Andrew Bassett and Mayte Siswick for technical support and to Christos Andreadis and Chia Chun Chang for reading the manuscript. We thank Ying Han for assistance with mass spectrometry equipment, Xiaoming Li for training on the use of the confocal microscope and software
References (69)
- et al.
The TOR pathway modulates cytoophidium formation in Schizosaccharomyces pombe
J. Biol. Chem.
(2019) - et al.
CTP synthase forms the cytoophidium in human hepatocellular carcinoma
Exp. Cell Res.
(2017) - et al.
Glutamine analogs promote cytoophidium assembly in human and Drosophila cells
J. Genet. Genomics
(2011) - et al.
Sensitizing human colon carcinoma HT-29 cells to cisplatin by cyclopentenylcytosine, in vitro and in vivo
Life Sci.
(2000) - et al.
CTP synthase forms cytoophidia in the cytoplasm and nucleus
Exp. Cell Res.
(2014) - et al.
Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblasts by cytidine 5′-triphosphate
J. Bio. Chem.
(1996) - et al.
Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3
J. Biol. Chem.
(2007) - et al.
Molecular enzymology of mammalian Δ1-pyrroline-5-carboxylate synthase alternative splice donor utilization generates isoforms with different sensitivity to ornithine inhibition
J. Biol. Chem.
(1999) - et al.
Critical roles of CTP synthase N-terminal in cytoophidium assembly
Exp. Cell Res.
(2017) Enzymatic amination of uridine triphosphate to cytidine triphosphate
J. Biol. Chem.
(1956)
Intracellular compartmentation of CTP synthase in Drosophila
J. Genet. Genomics
Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by CTP
J. Biol. Chem.
Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH
J. Biol. Chem.
Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae
J. Biol. Chem.
Filamentation of metabolic enzymes in Saccharomyces cerevisiae
J. Genet. Genomics
Targeting of aberrant mRNAs to cytoplasmic processing bodies
Cell
mTOR-S6K1 pathway mediates cytoophidium assembly
J. Genet. Genomics
CTP synthase is required for optic lobe homeostasis in Drosophila
J. Genet. Genomics
Cytidine triphosphate (CTP) synthetase activity during cell cycle progression in normal and malignant T-lymphocytic cells
Eur. J. Canc.
Cyclopentenyl cytosine induces apoptosis and increases cytarabine-induced apoptosis in a T-lymphoblastic leukemic cell-line
Leuk. Res.
Cyclopentenyl cytosine inhibits cytidine triphosphate synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy
Eur. J. Cancer
Enxymology of human colon tumors
Life Sci.
Cytoophidia respond to nutrient stress in Drosophila
Exp. Cell Res.
Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants
J. Biol. Chem.
Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe
J. Genet. Genomics
Drosophila CTP synthase can form distinct substrate- and product-bound filaments
J. Genet. Genomics
Reversible compartmentalization of de novo purine biosynthetic complexes in living cells
Science
The interplay between Myc and CTP synthase in Drosophila
PLoS Genet.
Nucleotide synthesis is regulated by cytoophidium formation during neurodevelopment and adaptive metabolism
Biol. Open
Metabolic regulation via enzyme filamentation
Crit. Rev. Biochem. Mol. Biol.
Only one isoform of Drosophila melanogaster CTP synthase forms the cytoophidium
PLoS Genet.
Large-scale filament formation inhibits the activity of CTP synthetase
eLife
Hyperammonemia with reduced ornithine, citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding Δ1-pyrroline-5-carboxylate synthase
Hum. Mol. Genet.
Δ 1-pyrroline-5-carboxylate synthase deficiency: neurodegeneration, cataracts and connective tissue manifestations combined with hyperammonaemia and reduced ornithine, citrulline, arginine and proline
Eur. J. Pediatr.
Cited by (20)
CTP synthase does not form cytoophidia in Drosophila interfollicular stalks
2022, Experimental Cell ResearchDrosophila intestinal homeostasis requires CTP synthase
2021, Experimental Cell ResearchCitation Excerpt :The product CTP serves as a building block for RNA and DNA, and functions in making phospholipids and sialoglycoproteins [1,2]. Since 2010, CTPS has been found to form filamentous subcellular structures termed cytoophidia in many organisms, such as archaea [3], bacteria [4], yeast [5–13], fruit fly [5,14–25], zebrafish [26, 27], plant [28] and mammalian cells [16,29–37]. Research has shown that cytoophidia are abundant in mouse embryonic stem cells.
CTPS forms the cytoophidium in zebrafish
2021, Experimental Cell ResearchCitation Excerpt :Cytidine triphosphate synthase (CTPS) and inosine 5′-monophosphate dehydrogenase (IMPDH) are two of the best known cytoophidium-forming proteins. More recently, a number of metabolic enzymes have joined the list with detailed characterization, such as phosphoribosyl pyrophosphate synthetase 1 (PRPS1), asparagine synthetase (ASNS) and delta-1-pyrroline-5-carboxylate synthase (P5CS), among others [3–5]. With an accumulation of evidence, polymerization and forming larger aggregates are believed to represent a common mechanism for many enzymes.
Histone transcription regulator Slm9 is required for cytoophidium biogenesis
2021, Experimental Cell ResearchCitation Excerpt :It was first discovered in fruit flies [1], bacteria [2], and yeast [3] by three groups independently in 2010. Previous studies have shown that cytoophidia exist in archaea [4], bacteria [2], budding yeast [3,5–10], fission yeast [11–14], plants [15], fruit flies [1,16–29] and human cell lines [16,18,30–33]. Multiple factors are involved in the formation and degradation of cytoophidia, including temperature, nutritional stress, cell division, and gene expression [5,9,14,18,21,24,31,34–42].
Polarised maintenance of cytoophidia in Drosophila follicle epithelia
2021, Experimental Cell ResearchThe atlas of cytoophidia in Drosophila larvae
2020, Journal of Genetics and GenomicsCitation Excerpt :A genome-wide screening of Saccharomyces cerevisiae revealed that 23 proteins, mostly metabolic enzymes, can form filamentous structures (Shen et al., 2016). The proline synthesis enzyme P5CS can form cytoophidia in Drosophila (Zhang et al., 2020) and asparagine synthetase forms cytoophidia in Saccharomyces cerevisiae (Zhang et al., 2018). These results suggest that formation of cytoophidia represents a general strategy for cellular regulation and metabolic control.
- 1
These authors contributed equally to this work.
- 2
Current address: The Francis Crick Institute, Midland Road, London NW1 1AT, United Kingdom.