The proline synthesis enzyme P5CS forms cytoophidia in Drosophila

Bo Zhang^{a, b, c, 1},
Ömür Y. Tastan^{d, 1},
Xian Zhou^{a, 1},
Chen-Jun Guo^a,
Xuyang Liu^{a, e},
Aaron Thind^d,
Huan-Huan Hu^a,
Suwen Zhao^{a, e},
Ji-Long Liu^{a, d
, ,}

a.
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
b.
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
c.
University of Chinese Academy of Sciences, Beijing, 100049, China
d.
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom
e.
iHuman Institute, ShanghaiTech University, Shanghai, 201210, China

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Corresponding author: E-mail address: jilong.liu@dpag.ox.ac.uk (Ji-Long Liu)

These authors contributed equally to this work.

摘要

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Abstract: Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism. In 2010, three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium. In searching for CTPS-interacting proteins, here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein, △¹-pyrroline-5-carboxylate synthase (P5CS). Using the Drosophila follicle cell as the in vivo model, we confirm that P5CS forms cytoophidia, which are associated with CTPS cytoophidia. Overexpression of P5CS increases the length of CTPS cytoophidia. Conversely, filamentation of CTPS affects the morphology of P5CS cytoophidia. Finally, in vitro analyses confirm the filament-forming property of P5CS. Our work links CTPS with P5CS, two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis, respectively.
- CTPS /
- Cytoophidium /
- Glutamate /
- P5CS /
- Proline
These authors contributed equally to this work.
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Fig. 1. Venus-P5CS forms filamentous structures in Drosophila follicle cells. Transgenic fly was generated with Venus-P5CS. A: Confocal images of Venus-P5CS filaments (green) in follicle cells of a stage 9 egg chamber. DNA was labeled with Hoechst 33342 (blue). Cell membrane was labeled with Hu-li tai shao (red). Scale bar, 20 μm. B: Representative linear or curly P5CS filaments and ring-shaped structures at one end of linear filaments are shown. P5CS, △¹-pyrroline-5-carboxylate synthase.

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Fig. 2. P5CS forms cytoophidia in multiple tissues. Tissues were derived from offspring of UAS-Venus-P5CS crossed with da-GAL4 lines. DNA was stained with Hoechst 33342 (magenta). A: Wing disc. B: Larval ventral nerve cord. C: Larval midgut. D: Larval salivary gland. E: Adult trachea. F: Adult hindgut. G: Adult accessory gland. H: Adult ejaculatory duct. Scale bars, 10 μm.

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Fig. 3. Association of P5CS and CTPS cytoophidia in Drosophila follicle cells. A: Immunostaining results of P5CS and CTPS and the merged picture. The yellow color indicates the overlap bulk of CTPS and P5CS filaments. Scale bar, 10 μm. B: Zoom-in views of some parts of CTPS, P5CS filaments and merged results from A. The green ring-shaped structure suggests that it contains only P5CS filament. Scale bar, 5 μm. C: Fluorescence intensity of the overlapping part of two types of filaments and the ring-shaped structure of P5CS filament was measured. Green, P5CS signal; red, CTPS signal.

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Fig. 4. P5CS cytoophidia are anchored on cell cortex. A: Confocal images of P5CS, CTPS filaments and merged results (cell membrane, white). B: The relationship between P5CS, CTPS filaments, and cell membrane. C: Zoom-in view of A. Scale bar, 10 μm. CTPS, CTP synthase; P5CS, △¹-pyrroline-5-carboxylate synthase.

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Fig. 5. CTPS filamentation affects the morphology of P5CS cytoophidia. A, C and E: Conformational results of CTPS and P5CS filaments and merged images under CTPS cytoophidia wild-type (WT), disruption (SD), and overexpression (OE) levels in follicle cells, respectively. Scale bar, 10 μm. B, D and F: Fluorescence intensity of P5CS and CTPS filaments was measured under different CTPS levels. Green, P5CS signal; red, CTPS signal. G and H: Quantification analysis of length and curvature of P5CS filaments under different CTPS levels. ∗, P < 0.05, ∗∗∗,P < 0.001, Error bars show the SEM. SEM, standard error of mean; CTPS, CTP synthase; P5CS, △¹-pyrroline-5-carboxylate synthase.

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Fig. 6. Overexpressing P5CS increases the length of CTPS cytoophidia. A, B, D and E: Representative images of CTPS filaments in mCherry control and P5CS overexpression lines. Both UAS-mCherry control and UAS-Venus-P5CS flies were crossed with Actin5c-GAL4 driver. C and F: Zoom-in views of B and E, respectively. Scale bars, 10 μm. G: CTPS cytoophidium areas of mCherry control and P5CS overexpression line were quantified. ∗∗∗∗, P < 0.0001. Error bars show SEM. SEM, standard error of mean; CTPS, CTP synthase; P5CS, △¹-pyrroline-5-carboxylate synthase.

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Fig. 7. P5CS dimerization interface mutants disrupt filament formation. A: Homology model of P5CS dimer with human P5CS structure as the template and P5CS dimerization interface. Key residues in dimerization interface are labeled, and mutation sites that may have a strong effect on dimerization of P5CS are highlighted in blue (N708 and F713). B: Venus-P5CS, Venus-P5CS^N708A, and Venus-P5CS^F713A were cloned into pAc 5.1 vector and transfected into S2 cells. Representative confocal images of cells are presented. P5CS signal is shown in green. DNA was labeled with Hoechst 33342 (blue). Scale bar, 10 μm. P5CS, △¹-pyrroline-5-carboxylate synthase.

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Fig. 8. P5CS forms filamentous structures in vitro. A: Negative stain electron microscopy images of P5CS conformation in apo and substrate-bound states (glutamate, ATP, and NADPH). A magnified filament image is shown in lower panel. B: Quantification analysis of filaments length under different conditions by removing glutamate, ATP, and NADPH successively from the complete reaction. ∗∗, P < 0.01; ns, no significant. Error bars show the SEM.C–F: Two-dimensional classification of two kinds of filaments. G and H: Quantification and comparison between the two types of filaments on their periodicity and diameter. Error bars show the SEM. I: A representative image of filaments under the condition with substrates of CTPS catalytic reaction (glutamine, ATP, UTP, and GTP) and substrates of P5CS catalytic reaction (glutamate, ATP, and NADPH). Two kinds of filaments are indicated by red and black arrows, respectively. SEM, standard error of mean; CTPS, CTP synthase; P5CS, △¹-pyrroline-5-carboxylate synthase.

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