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Hong J, Li J, Zhang Y, Wang J, Li C, Liu JL, Liu J. Integrative role of CTPS cytoophidia in polyploid tissue growth and nutrient adaptation. INSECT SCIENCE 2025. [PMID: 40287929 DOI: 10.1111/1744-7917.70060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/16/2025] [Revised: 03/19/2025] [Accepted: 03/24/2025] [Indexed: 04/29/2025]
Abstract
Tissue growth and development are fundamental to organismal survival, requiring precise coordination of metabolic processes, nutrient availability, and signaling pathways. Cytidine triphosphate synthase (CTPS) is a rate-limiting enzyme in nucleotide biosynthesis and assembles filamentous cytoophidia, conserved across species. Despite increasing interest in cytoophidia, how CTPS filaments integrate metabolic and signaling cues to drive cell size and tissue growth remains incompletely understood. Using RNA interference and clustered regularly interspaces short palindromic repeats (CRISPR) / CRISPR-associate nuclease 9 gene editing, we generated CTPS-knockdown and point-mutated mutants to investigate the role of cytoophidia in cell growth. Specifically, we introduced the H355A mutation, which disrupts CTPS filament formation without affecting its enzymatic activity. Our findings revealed that CTPS depletion or filament disruption significantly impairs growth in polyploid organs, such as the fat body and salivary glands, underscoring the pivotal role of CTPS cytoophidia in cell growth regulation. Mutants lacking cytoophidia exhibited reduced DNA replication activity and smaller cell sizes compared to wild-type controls. Mechanistically, we found that nutrient-sensing pathways, particularly insulin-PI3K-Akt signaling pathway, regulate CTPS expression and cytoophidia formation in response to nutrient availability. Activation of the sterol regulatory element-binding protein partially rescued the growth defects caused by CTPS depletion. These findings provide new insights into the molecular mechanisms of the regulation of CTPS filaments, highlighting their role as critical mediators of tissue growth by integrating environmental demands, metabolism, and signaling pathways to regulate cell size and nutrient adaptation.
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Affiliation(s)
- Jiayi Hong
- College of Life Sciences, Shanghai Normal University, Shanghai, China
| | - Jiamin Li
- College of Life Sciences, Shanghai Normal University, Shanghai, China
| | - Yuanbing Zhang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Jing Wang
- College of Life Sciences, Shanghai Normal University, Shanghai, China
| | - Chengui Li
- College of Life Sciences, Shanghai Normal University, Shanghai, China
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Jingnan Liu
- College of Life Sciences, Shanghai Normal University, Shanghai, China
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2
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Liu L, Chen W, Luo H, Zhang W, Zhang Z, Huang X, Fu X. HSPD1-facilitated formation of CTPS cytoophidia promotes proliferation in C2C12 cells. Exp Cell Res 2025; 446:114462. [PMID: 39971178 DOI: 10.1016/j.yexcr.2025.114462] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2024] [Revised: 01/16/2025] [Accepted: 02/16/2025] [Indexed: 02/21/2025]
Abstract
CTP synthase (CTPS) is a rate-limiting enzyme that controls CTP synthesis and can polymerize to form a filamentous structure called cytoophidia. The presence of cytoophidia affects the enzymatic activity of CTPS. However, whether CTPS can form cytoophidia in C2C12 cells and whether it affects the proliferation of skeletal muscle satellite cells needs to be further studied. In this study, we found that CTPS could form cytoophidia during C2C12 cell proliferation, and that overexpression of CTPS significantly promoted the formation of CTPS cytoophidia and increased the viability and proliferation rate of C2C12 cells. However, the CTPS H355A mutation hindered the formation of CTPS cytoophidia and inhibited the viability and proliferation of C2C12 cells. In addition, we found that the HSPD1 protein could interact with the CTPS protein and interference with Hspd1 gene expression inhibited the formation of CTPS cytoophidia, even with the overexpression of the CTPS gene. Subsequently, it inhibited C2C12 cells proliferation. Thus, these findings reveal the role of CTPS cytoophidia formation in C2C12 cells proliferation.
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Affiliation(s)
- Lili Liu
- Department of Life Science and Agroforestry, Qiqihar University, No. 42 Wenhua Street, JianHua District, Qiqihar, Heilongjiang Province, China
| | - Wen Chen
- Department of Life Science and Agroforestry, Qiqihar University, No. 42 Wenhua Street, JianHua District, Qiqihar, Heilongjiang Province, China
| | - Haijing Luo
- Department of Life Science and Agroforestry, Qiqihar University, No. 42 Wenhua Street, JianHua District, Qiqihar, Heilongjiang Province, China
| | - Weiwei Zhang
- Department of Life Science and Agroforestry, Qiqihar University, No. 42 Wenhua Street, JianHua District, Qiqihar, Heilongjiang Province, China.
| | - Zhenzhu Zhang
- Department of Life Science and Agroforestry, Qiqihar University, No. 42 Wenhua Street, JianHua District, Qiqihar, Heilongjiang Province, China; Heilongjiang Agricultural Engineering Vocational College, Haerbing, No.2, Qunying Street, Limin Avenue, Harbin City, China
| | - Xin Huang
- Department of Life Science and Agroforestry, Qiqihar University, No. 42 Wenhua Street, JianHua District, Qiqihar, Heilongjiang Province, China
| | - Xuepeng Fu
- Department of Life Science and Agroforestry, Qiqihar University, No. 42 Wenhua Street, JianHua District, Qiqihar, Heilongjiang Province, China
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3
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Hugener J, Xu J, Wettstein R, Ioannidi L, Velikov D, Wollweber F, Henggeler A, Matos J, Pilhofer M. FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis. Cell 2024; 187:3303-3318.e18. [PMID: 38906101 DOI: 10.1016/j.cell.2024.04.026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2023] [Revised: 02/06/2024] [Accepted: 04/19/2024] [Indexed: 06/23/2024]
Abstract
Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.
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Affiliation(s)
- Jannik Hugener
- Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland; Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland; Max Perutz Labs, University of Vienna, 1030 Vienna, Austria
| | - Jingwei Xu
- Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland
| | - Rahel Wettstein
- Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland; Max Perutz Labs, University of Vienna, 1030 Vienna, Austria
| | - Lydia Ioannidi
- Max Perutz Labs, University of Vienna, 1030 Vienna, Austria
| | - Daniel Velikov
- Max Perutz Labs, University of Vienna, 1030 Vienna, Austria; Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, 1030 Vienna, Austria
| | - Florian Wollweber
- Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland
| | - Adrian Henggeler
- Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland; Max Perutz Labs, University of Vienna, 1030 Vienna, Austria
| | - Joao Matos
- Institute of Biochemistry, ETH Zürich, 8093 Zürich, Switzerland; Max Perutz Labs, University of Vienna, 1030 Vienna, Austria.
| | - Martin Pilhofer
- Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland.
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Yin Y, Yu H, Wang X, Hu Q, Liu Z, Luo D, Yang X. Cytoophidia: a conserved yet promising mode of enzyme regulation in nucleotide metabolism. Mol Biol Rep 2024; 51:245. [PMID: 38300325 DOI: 10.1007/s11033-024-09208-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2023] [Accepted: 01/02/2024] [Indexed: 02/02/2024]
Abstract
Nucleotide biosynthesis encompasses both de novo and salvage synthesis pathways, each characterized by significant material and procedural distinctions. Despite these differences, cells with elevated nucleotide demands exhibit a preference for the more intricate de novo synthesis pathway, intricately linked to modes of enzyme regulation. In this study, we primarily scrutinize the biological importance of a conserved yet promising mode of enzyme regulation in nucleotide metabolism-cytoophidia. Cytoophidia, comprising cytidine triphosphate synthase or inosine monophosphate dehydrogenase, is explored across diverse biological models, including yeasts, Drosophila, mice, and human cancer cell lines. Additionally, we delineate potential biomedical applications of cytoophidia. As our understanding of cytoophidia deepens, the roles of enzyme compartmentalization and polymerization in various biochemical processes will unveil, promising profound impacts on both research and the treatment of metabolism-related diseases.
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Affiliation(s)
- Yue Yin
- School of Queen Mary, Jiangxi Medical College, Nanchang University, Jiangxi, China
| | - Huanhuan Yu
- First School of Clinical Medicine, Jiangxi Medical College, Nanchang University, Jiangxi, China
| | - Xinyi Wang
- Thyroid Surgery Center, West China Hospital of Sichuan University, Chengdu, China
| | - Qiaohao Hu
- The 1st Affiliated Hospital, Jiangxi Medical College, Nanchang University, Jiangxi, China
| | - Zhuoqi Liu
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Jiangxi Medical College, Nanchang University, Jiangxi, China
| | - Daya Luo
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Jiangxi Medical College, Nanchang University, Jiangxi, China.
| | - Xiaohong Yang
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Jiangxi Medical College, Nanchang University, Jiangxi, China.
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Weng RY, Zhang L, Liu JL. Connecting Hippo Pathway and Cytoophidia in Drosophila Posterior Follicle Cells. Int J Mol Sci 2024; 25:1453. [PMID: 38338731 PMCID: PMC10855297 DOI: 10.3390/ijms25031453] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 01/18/2024] [Accepted: 01/19/2024] [Indexed: 02/12/2024] Open
Abstract
CTP synthase (CTPS), the rate-limiting enzyme in the de novo synthesis of CTP, assembles into a filamentous structure termed the cytoophidium. The Hippo pathway regulates cell proliferation and apoptosis. The relationship of the nucleotide metabolism with the Hippo pathway is little known. Here, we study the impact of the Hippo pathway on the cytoophidium in Drosophila melanogaster posterior follicle cells (PFCs). We find that the inactivation of the Hippo pathway correlates with reduced cytoophidium length and number within PFCs. During the overexpression of CTPS, the presence of Hippo mutations also reduces the length of cytoophidia in PFCs. In addition, we observe that knocking down CTPS mitigates hpo (Hippo)-associated over-proliferation. In summary, our results suggest that there is a connection between the Hippo pathway and the nucleotide biosynthesis enzyme CTPS in PFCs.
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Affiliation(s)
- Rui-Yu Weng
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; (R.-Y.W.)
- State Key Laboratory of Cell Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Lei Zhang
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; (R.-Y.W.)
- State Key Laboratory of Cell Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
- Sheng Yushou Center of Cell Biology and Immunology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; (R.-Y.W.)
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Yoon J, Min CW, Kim J, Baek G, Kim D, Jang JW, Gupta R, Kim ST, Cho LH. Quantitative Proteomic Analysis Deciphers the Molecular Mechanism for Endosperm Nuclear Division in Early Rice Seed Development. PLANTS (BASEL, SWITZERLAND) 2023; 12:3715. [PMID: 37960070 PMCID: PMC10650807 DOI: 10.3390/plants12213715] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/11/2023] [Revised: 10/26/2023] [Accepted: 10/27/2023] [Indexed: 11/15/2023]
Abstract
Understanding the molecular mechanisms underlying early seed development is important in improving the grain yield and quality of crop plants. We performed a comparative label-free quantitative proteomic analysis of developing rice seeds for the WT and osctps1-2 mutant, encoding a cytidine triphosphate synthase previously reported as the endospermless 2 (enl2) mutant in rice, harvested at 0 and 1 d after pollination (DAP) to understand the molecular mechanism of early seed development. In total, 5231 proteins were identified, of which 902 changed in abundance between 0 and 1 DAP seeds. Proteins that preferentially accumulated at 1 DAP were involved in DNA replication and pyrimidine biosynthetic pathways. Notably, an increased abundance of OsCTPS1 was observed at 1 DAP; however, no such changes were observed at the transcriptional level. We further observed that the inhibition of phosphorylation increased the stability of this protein. Furthermore, in osctps1-2, minichromosome maintenance (MCM) proteins were significantly reduced compared with those in the WT at 1 DAP, and mutations in OsMCM5 caused defects in seed development. These results highlight the molecular mechanisms underlying early seed development in rice at the post-transcriptional level.
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Affiliation(s)
- Jinmi Yoon
- Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea;
- Department of Biological Sciences and Bioengineering, Industry-Academia Interactive R&E Center for Bioprocess Innovation, Inha University, Incheon 22212, Republic of Korea
| | - Cheol Woo Min
- Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Republic of Korea;
| | - Jiyoung Kim
- Department of Plant Bioscience, College of Natural Resources and Life Science, Pusan National University, Miryang 50463, Republic of Korea; (J.K.); (G.B.); (D.K.); (J.W.J.)
| | - Gibeom Baek
- Department of Plant Bioscience, College of Natural Resources and Life Science, Pusan National University, Miryang 50463, Republic of Korea; (J.K.); (G.B.); (D.K.); (J.W.J.)
| | - Dohyeon Kim
- Department of Plant Bioscience, College of Natural Resources and Life Science, Pusan National University, Miryang 50463, Republic of Korea; (J.K.); (G.B.); (D.K.); (J.W.J.)
| | - Jeong Woo Jang
- Department of Plant Bioscience, College of Natural Resources and Life Science, Pusan National University, Miryang 50463, Republic of Korea; (J.K.); (G.B.); (D.K.); (J.W.J.)
| | - Ravi Gupta
- College of General Education, Kookmin University, Seoul 02707, Republic of Korea;
| | - Sun Tae Kim
- Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Republic of Korea;
- Department of Plant Bioscience, College of Natural Resources and Life Science, Pusan National University, Miryang 50463, Republic of Korea; (J.K.); (G.B.); (D.K.); (J.W.J.)
| | - Lae-Hyeon Cho
- Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Republic of Korea;
- Department of Plant Bioscience, College of Natural Resources and Life Science, Pusan National University, Miryang 50463, Republic of Korea; (J.K.); (G.B.); (D.K.); (J.W.J.)
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7
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Nasalingkhan C, Sirinonthanawech N, Noree C. Robust assembly of the aldehyde dehydrogenase Ald4p in Saccharomyces cerevisiae. Biol Open 2023; 12:bio060070. [PMID: 37767855 PMCID: PMC10602002 DOI: 10.1242/bio.060070] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Accepted: 09/26/2023] [Indexed: 09/29/2023] Open
Abstract
As part of our studies of yeast aldehyde dehydrogenase (Ald4p) assembly, we identified a population of transformants (SWORD strain) that show more robust filament formation of GFP-tagged Ald4p (Ald4p-GFP) than that of a wild type ALD4::GFP strain. Sequencing of the ALD4 gene in the SWORD strain showed that the increased assembly was not due to changes to the ALD4 coding sequence, suggesting that a second mutation site was altering Ald4p assembly. Using short-read whole-genome sequencing, we identified spontaneous mutations in FLO9. Introduction of the SWORD allele of FLO9 into a wild-type ALD4::GFP yeast strain revealed that the changes to FLO9 were a contributor to the increased length of Ald4p-GFP filaments we observe in the SWORD strain and that this effect was not due to an increase in Ald4p protein levels. However, the expression of the FLO9 (SWORD) allele in wild-type yeast did not fully recapitulate the length control defect we observed in SWORD strains, arguing that there are additional genes contributing to the filament length phenotype. For our future work, this FLO9 from SWORD will be tested whether it could show global effect, promoting the assembly of some other filament-forming enzymes.
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Affiliation(s)
- Channarong Nasalingkhan
- Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom 73170Thailand
| | - Naraporn Sirinonthanawech
- Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom 73170Thailand
| | - Chalongrat Noree
- Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom 73170Thailand
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Zhong J, Cui J, Liu J, Zhong C, Hu F, Dong J, Cheng J, Hu K. Fine-mapping and candidate gene analysis of the Mcgy1 locus responsible for gynoecy in bitter gourd (Momordica spp.). TAG. THEORETICAL AND APPLIED GENETICS. THEORETISCHE UND ANGEWANDTE GENETIK 2023; 136:81. [PMID: 36952023 DOI: 10.1007/s00122-023-04314-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Accepted: 01/28/2023] [Indexed: 06/18/2023]
Abstract
The Mcgy1 locus responsible for gynoecy was fine-mapped into a 296.94-kb region, in which four single-nucleotide variations and six genes adjacent to them might be associate with sex differentiation in bitter gourd. Gynoecy plays an important role in high-efficiency hybrid seed production, and gynoecious plants are excellent materials for dissecting sex differentiation in Cucurbitaceae crop species, including bitter gourd. However, the gene responsible for gynoecy in bitter gourd is unknown. Here, we first identified a gynoecy locus designated Mcgy1 using the F2 population (n = 291) crossed from the gynoecious line S156G and the monoecious line K8-201 via bulked segregant analysis with whole-genome resequencing (BSA-seq) and molecular marker linkage analysis. Then, a large S156G × K8-201 F2 population (n = 5,656) was used for fine-mapping to delimit the Mcgy1 locus into a 296.94-kb physical region on pseudochromosome MC01, where included 33 annotated genes different from any homologous gynoecy genes previously reported in Cucurbitaceae species. Within this region, four underlying single-nucleotide variations (SNVs) that might cause gynoecy were identified by multiple genomic sequence variation analysis, and their six neighbouring genes were considered as potential candidate genes for Mcgy1. Of these, only MC01g1681 showed a significant differential expression at two-leaf developmental stage between S156G and its monoecious near-isogenic line S156 based on RNA sequencing (RNA-seq) and qRT-PCR analyses. In addition, transcriptome analysis revealed 21 key differentially expressed genes (DEGs) and possible regulatory pathways of the formation of gynoecy in bitter gourd. Our findings provide a new clue for researching on gynoecious plants in Cucurbitaceae species and a theoretical basis for breeding gynoecious bitter gourd lines by the use of molecular markers-assisted selection.
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Affiliation(s)
- Jian Zhong
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs/Guangdong Vegetables Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, 510642, China
| | - Junjie Cui
- Department of Horticulture, Foshan University, Foshan, 528225, China
| | - Jia Liu
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs/Guangdong Vegetables Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, 510642, China
| | - Chunfeng Zhong
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs/Guangdong Vegetables Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, 510642, China
| | - Fang Hu
- Henry Fok School of Biology and Agricultural, Shaoguan University, Shaoguan, 512023, China
| | - Jichi Dong
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs/Guangdong Vegetables Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, 510642, China
| | - Jiaowen Cheng
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs/Guangdong Vegetables Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, 510642, China.
| | - Kailin Hu
- Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs/Guangdong Vegetables Engineering Research Center, College of Horticulture, South China Agricultural University, Guangzhou, 510642, China.
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Andreadis C, Li T, Liu JL. Ubiquitination regulates cytoophidium assembly in Schizosaccharomyces pombe. Exp Cell Res 2022; 420:113337. [PMID: 36087798 DOI: 10.1016/j.yexcr.2022.113337] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2022] [Revised: 08/26/2022] [Accepted: 08/27/2022] [Indexed: 12/30/2022]
Abstract
CTP synthase (CTPS), a metabolic enzyme responsible for the de novo synthesis of CTP, can form filamentous structures termed cytoophidia, which are evolutionarily conserved from bacteria to humans. Here we used Schizosaccharomyces pombe to study the cytoophidium assembly regulation by ubiquitination. We tested the CTP synthase's capacity to be post-translationally modified by ubiquitin or be affected by the ubiquitination state of the cell and showed that ubiquitination is important for the maintenance of the CTPS filamentous structure in fission yeast. We have identified proteins which are in complex with CTPS, including specific ubiquitination regulators which significantly affect CTPS filamentation, and mapped probable ubiquitination targets on CTPS. Furthermore, we discovered that a cohort of deubiquitinating enzymes is important for the regulation of cytoophidium's filamentous morphology. Our study provides a framework for the analysis of the effects that ubiquitination and deubiquitination have on the formation of cytoophidia.
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Affiliation(s)
- Christos Andreadis
- School of Life Science and Technology, ShanghaiTech University, 201210, Shanghai, China
| | - Tianhao Li
- School of Life Science and Technology, ShanghaiTech University, 201210, Shanghai, China
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, 201210, Shanghai, China; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom.
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GTP-Dependent Regulation of CTP Synthase: Evolving Insights into Allosteric Activation and NH3 Translocation. Biomolecules 2022; 12:biom12050647. [PMID: 35625575 PMCID: PMC9138612 DOI: 10.3390/biom12050647] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2022] [Revised: 04/21/2022] [Accepted: 04/21/2022] [Indexed: 12/24/2022] Open
Abstract
Cytidine-5′-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible “wing” region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.
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11
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Connecting Ras and CTP synthase in Drosophila. Exp Cell Res 2022; 416:113155. [DOI: 10.1016/j.yexcr.2022.113155] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 04/03/2022] [Accepted: 04/08/2022] [Indexed: 11/04/2022]
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Krämer M, Dörfer E, Hickl D, Bellin L, Scherer V, Möhlmann T. Cytidine Triphosphate Synthase Four From Arabidopsis thaliana Attenuates Drought Stress Effects. FRONTIERS IN PLANT SCIENCE 2022; 13:842156. [PMID: 35360303 PMCID: PMC8960734 DOI: 10.3389/fpls.2022.842156] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/23/2021] [Accepted: 02/08/2022] [Indexed: 06/14/2023]
Abstract
Cytidine triphosphate synthase (CTPS) catalyzes the final step in pyrimidine de novo synthesis. In Arabidopsis, this protein family consists of five members (CTPS1-5), and all of them localize to the cytosol. Specifically, CTPS4 showed a massive upregulation of transcript levels during abiotic stress, in line with increased staining of CTPS4 promoter:GUS lines in hypocotyl, root and to lesser extend leaf tissues. In a setup to study progressive drought stress, CTPS4 knockout mutants accumulated less fresh and dry weight at days 5-7 and showed impaired ability to recover from this stress after 3 days of rewatering. Surprisingly, a thorough physiological characterization of corresponding plants only revealed alterations in assimilation and accumulation of soluble sugars including those related to drought stress in the mutant. Bimolecular fluorescence complementation (BiFC) studies indicated the interaction of CTPS4 with other isoforms, possibly affecting cytoophidia (filaments formed by CTPS formation. Although the function of these structures has not been thoroughly investigated in plants, altered enzyme activity and effects on cell structure are reported in other organisms. CTPS activity is required for cell cycle progression and growth. Furthermore, drought can lead to the accumulation of reactive oxygen species (ROS) and by this, to DNA damage. We hypothesize that effects on the cell cycle or DNA repair might be relevant for the observed impaired reduced drought stress tolerance of CTPS4 mutants.
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13
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Miura N. Condensate Formation by Metabolic Enzymes in Saccharomyces cerevisiae. Microorganisms 2022; 10:232. [PMID: 35208686 PMCID: PMC8876316 DOI: 10.3390/microorganisms10020232] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Revised: 01/17/2022] [Accepted: 01/18/2022] [Indexed: 12/31/2022] Open
Abstract
Condensate formation by a group of metabolic enzymes in the cell is an efficient way of regulating cell metabolism through the formation of "membrane-less organelles." Because of the use of green fluorescent protein (GFP) for investigating protein localization, various enzymes were found to form condensates or filaments in living Saccharomyces cerevisiae, mammalian cells, and in other organisms, thereby regulating cell metabolism in the certain status of the cells. Among different environmental stresses, hypoxia triggers the spatial reorganization of many proteins, including more than 20 metabolic enzymes, to form numerous condensates, including "Glycolytic body (G-body)" and "Purinosome." These individual condensates are collectively named "Metabolic Enzymes Transiently Assembling (META) body". This review overviews condensate or filament formation by metabolic enzymes in S. cerevisiae, focusing on the META body, and recent reports in elucidating regulatory machinery of META body formation.
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Affiliation(s)
- Natsuko Miura
- Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599-8531, Japan
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14
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Liu M, Chen X, Xia J. Multienzyme Catalysis in Phase-Separated Protein Condensates. Methods Mol Biol 2022; 2487:345-354. [PMID: 35687245 DOI: 10.1007/978-1-0716-2269-8_20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, and understanding how phase separation regulates multienzyme catalysis may need the help of in vitro investigations. Recently we have constructed synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. Here, we describe the methods for checking the enzyme assembly using fluorescent microscopy and centrifugation assay. We further provide steps for analysis of the cascade enzyme catalytic efficiencies inside the condensates, using enzymes from terpene biosynthesis pathway.
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Affiliation(s)
- Miao Liu
- Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, SAR, China
| | - Xi Chen
- Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, SAR, China
| | - Jiang Xia
- Department of Chemistry, The Chinese University of Hong Kong, Hong Kong, SAR, China.
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15
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Bellin L, Scherer V, Dörfer E, Lau A, Vicente AM, Meurer J, Hickl D, Möhlmann T. Cytosolic CTP Production Limits the Establishment of Photosynthesis in Arabidopsis. FRONTIERS IN PLANT SCIENCE 2021; 12:789189. [PMID: 34917117 PMCID: PMC8669480 DOI: 10.3389/fpls.2021.789189] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Accepted: 11/02/2021] [Indexed: 06/14/2023]
Abstract
CTP synthases (CTPS) comprise a protein family of the five members CTPS1-CTPS5 in Arabidopsis, all located in the cytosol. Specifically, downregulation of CTPS2 by amiRNA technology results in plants with defects in chlorophyll accumulation and photosynthetic performance early in development. CTP and its deoxy form dCTP are present at low levels in developing seedlings. Thus, under conditions of fast proliferation, the synthesis of CTP (dCTP) can become a limiting factor for RNA and DNA synthesis. The higher sensitivity of ami-CTPS2 lines toward the DNA-Gyrase inhibitor ciprofloxacin, together with reduced plastid DNA copy number and 16S and 23S chloroplast ribosomal RNA support this view. High expression and proposed beneficial biochemical features render CTPS2 the most important isoform for early seedling development. In addition, CTPS2 was identified as an essential enzyme in embryo development before, as knock-out mutants were embryo lethal. In line with this, ami-CTPS2 lines also exhibited reduced seed numbers per plant.
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Affiliation(s)
- Leo Bellin
- Plant Physiology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany
| | - Vanessa Scherer
- Plant Physiology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany
| | - Eva Dörfer
- Plant Physiology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany
| | - Anne Lau
- Plant Physiology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany
| | - Alexandre Magno Vicente
- Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany
| | - Jörg Meurer
- Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany
| | - Daniel Hickl
- Plant Physiology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany
| | - Torsten Möhlmann
- Plant Physiology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany
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16
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Yoon J, Cho L, Kim S, Tun W, Peng X, Pasriga R, Moon S, Hong W, Ji H, Jung K, Jeon J, An G. CTP synthase is essential for early endosperm development by regulating nuclei spacing. PLANT BIOTECHNOLOGY JOURNAL 2021; 19:2177-2191. [PMID: 34058048 PMCID: PMC8541778 DOI: 10.1111/pbi.13644] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/03/2021] [Revised: 05/04/2021] [Accepted: 05/22/2021] [Indexed: 06/12/2023]
Abstract
Cereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild-type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10-16 clumped nuclei while wild-type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild-type nuclei were evenly distributed by microtubules while the enl2-2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.
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Affiliation(s)
- Jinmi Yoon
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
- Department of Plant BioscienceCollege of Natural Resources and Life SciencePusan National UniversityMiryangRepublic of Korea
| | - Lae‐Hyeon Cho
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
- Department of Plant BioscienceCollege of Natural Resources and Life SciencePusan National UniversityMiryangRepublic of Korea
| | - Sung‐Ryul Kim
- Gene Identification and Validation GroupGenetic Design and Validation UnitInternational Rice Research Institute (IRRI)Metro ManilaPhilippines
| | - Win Tun
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
| | - Xin Peng
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
- Institution of Genomics and BioinformaticsSouth China Agricultural UniversityGuangzhouChina
| | - Richa Pasriga
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
| | - Sunok Moon
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
| | - Woo‐Jong Hong
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
| | - Hyeonso Ji
- National Institute of Agricultural Sciences, Rural Development AdministrationJeonjuRepublic of Korea
| | - Ki‐Hong Jung
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
| | - Jong‐Seong Jeon
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
| | - Gynheung An
- Crop Biotech Institute and Graduate School of BiotechnologyKyung Hee UniversityYonginRepublic of Korea
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17
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Chang CC, Keppeke GD, Antos CL, Peng M, Andrade LEC, Sung LY, Liu JL. CTPS forms the cytoophidium in zebrafish. Exp Cell Res 2021; 405:112684. [PMID: 34129847 DOI: 10.1016/j.yexcr.2021.112684] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2021] [Revised: 05/30/2021] [Accepted: 06/01/2021] [Indexed: 12/26/2022]
Abstract
Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.
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Affiliation(s)
- Chia-Chun Chang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Gerson Dierley Keppeke
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, SP 04023-062, Brazil
| | - Christopher L Antos
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Min Peng
- Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan
| | - Luis Eduardo Coelho Andrade
- Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, SP 04023-062, Brazil
| | - Li-Ying Sung
- Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom.
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18
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Simonet JC, Burrell AL, Kollman JM, Peterson JR. Freedom of assembly: metabolic enzymes come together. Mol Biol Cell 2021; 31:1201-1205. [PMID: 32463766 PMCID: PMC7353150 DOI: 10.1091/mbc.e18-10-0675] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Many different enzymes in intermediate metabolism dynamically assemble filamentous polymers in cells, often in response to changes in physiological conditions. Most of the enzyme filaments known to date have only been observed in cells, but in a handful of cases structural and biochemical studies have revealed the mechanisms and consequences of assembly. In general, enzyme polymerization functions as a mechanism to allosterically tune enzyme kinetics, and it may play a physiological role in integrating metabolic signaling. Here, we highlight some principles of metabolic filaments by focusing on two well-studied examples in nucleotide biosynthesis pathways—inosine-5’-monophosphate (IMP) dehydrogenase and cytosine triphosphate (CTP) synthase.
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Affiliation(s)
| | - Anika L Burrell
- Department of Biochemistry, University of Washington, Seattle, Washington 98195
| | - Justin M Kollman
- Department of Biochemistry, University of Washington, Seattle, Washington 98195
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19
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Hickl D, Scheuring D, Möhlmann T. CTP Synthase 2 From Arabidopsis thaliana Is Required for Complete Embryo Development. FRONTIERS IN PLANT SCIENCE 2021; 12:652434. [PMID: 33936137 PMCID: PMC8082242 DOI: 10.3389/fpls.2021.652434] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/12/2021] [Accepted: 03/08/2021] [Indexed: 06/12/2023]
Abstract
Pyrimidine de novo synthesis is an essential pathway in all organisms. The final and rate-limiting step in the synthesis of the nucleotide cytidine triphosphate (CTP) is catalyzed by CTP synthase (CTPS), and Arabidopsis harbors five isoforms. Single mutant lines defective in each one of the four isoforms do not show apparent phenotypical alterations in comparison to wild-type plants. However, Arabidopsis lines that contain T-DNA insertions in the CTPS2 gene were unable to produce homozygous offspring. Here, we show that CTPS2 exhibits a distinct expression pattern throughout embryo development, and loss-of-function mutants are embryo lethal, as siliques from +/ctps2 plants contained nearly 25% aborted seeds. This phenotype was rescued by complementation with CTPS2 under control of its endogenous promoter. CTPS2::GFP lines revealed expression only in the tip of columella cells in embryo root tips of the heart and later stages. Furthermore, CTPS2 expression in mature roots, most pronounced in the columella cells, shoots, and vasculature tissue of young seedlings, was observed. Filial generations of +/ctps2 plants did not germinate properly, even under external cytidine supply. During embryo development, the CTPS2 expression pattern resembled the established auxin reporter DR5::GFP. Indeed, the cloned promoter region we used in this study possesses a repeat of an auxin response element, and auxin supply increased CTPS2 expression in a cell-type-specific manner. Thus, we conclude that CTPS2 is essential for CTP supply in developing embryos, and loss-of-function mutants in CTPS2 are embryo lethal.
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Affiliation(s)
- Daniel Hickl
- Plant Physiology, University of Kaiserslautern, Kaiserslautern, Germany
| | - David Scheuring
- Plant Pathology, University of Kaiserslautern, Kaiserslautern, Germany
| | - Torsten Möhlmann
- Plant Physiology, University of Kaiserslautern, Kaiserslautern, Germany
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20
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Jang S, Xuan Z, Lagoy RC, Jawerth LM, Gonzalez IJ, Singh M, Prashad S, Kim HS, Patel A, Albrecht DR, Hyman AA, Colón-Ramos DA. Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo. Biophys J 2021; 120:1170-1186. [PMID: 32853565 PMCID: PMC8059094 DOI: 10.1016/j.bpj.2020.08.002] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Revised: 07/31/2020] [Accepted: 08/05/2020] [Indexed: 02/08/2023] Open
Abstract
Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.
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Affiliation(s)
- SoRi Jang
- Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut
| | - Zhao Xuan
- Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut
| | - Ross C Lagoy
- Department of Biomedical Engineering and Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts
| | - Louise M Jawerth
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Ian J Gonzalez
- Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut
| | - Milind Singh
- Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut
| | - Shavanie Prashad
- Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut
| | - Hee Soo Kim
- Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut
| | - Avinash Patel
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Dirk R Albrecht
- Department of Biomedical Engineering and Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts
| | - Anthony A Hyman
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
| | - Daniel A Colón-Ramos
- Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut; Instituto de Neurobiología, Universidad de Puerto Rico, San Juan, Puerto Rico.
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21
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Surasiang T, Noree C. Effects of A6E Mutation on Protein Expression and Supramolecular Assembly of Yeast Asparagine Synthetase. BIOLOGY 2021; 10:biology10040294. [PMID: 33916846 PMCID: PMC8065433 DOI: 10.3390/biology10040294] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/01/2021] [Revised: 03/25/2021] [Accepted: 03/31/2021] [Indexed: 11/16/2022]
Abstract
Simple Summary Certain mutations causing extremely low abundance of asparagine synthetase (the enzyme responsible for producing asparagine, one of the amino acids required for normal growth and development) have been identified in humans with neurological problems and small head and brain size. Currently, yeast is becoming more popular in modeling many human diseases. In this study, we incorporate a mutation, associated with human asparagine synthetase deficiency, into the yeast asparagine synthetase gene to demonstrate that this mutation can also show similar effects as those observed in humans, leading to very low abundance of yeast asparagine synthetase and slower yeast growth rate. This suggests that our yeast system can be alternatively used to initially screen for any drugs that can help rescue the protein levels of asparagine synthetase before applying them to further studies in mammals and humans. Furthermore, this mutation might specifically be introduced into the asparagine synthetase gene of the target cancer cells in order to suppress the overproduction of asparagine synthetase within these abnormal cells, therefore inhibiting the growth of cancer, which might be helpful for patients with blood cancer to prevent them developing any resistance to the conventional asparaginase treatment. Abstract Asparagine synthetase deficiency (ASD) has been found to be caused by certain mutations in the gene encoding human asparagine synthetase (ASNS). Among reported mutations, A6E mutation showed the greatest reduction in ASNS abundance. However, the effect of A6E mutation has not yet been tested with yeast asparagine synthetase (Asn1/2p). Here, we constructed a yeast strain by deleting ASN2 from its genome, introducing the A6E mutation codon to ASN1, along with GFP downstream of ASN1. Our mutant yeast construct showed a noticeable decrease of Asn1p(A6E)-GFP levels as compared to the control yeast expressing Asn1p(WT)-GFP. At the stationary phase, the A6E mutation also markedly lowered the assembly frequency of the enzyme. In contrast to Asn1p(WT)-GFP, Asn1p(A6E)-GFP was insensitive to changes in the intracellular energy levels upon treatment with sodium azide during the log phase or fresh glucose at the stationary phase. Our study has confirmed that the effect of A6E mutation on protein expression levels of asparagine synthetase is common in both unicellular and multicellular eukaryotes, suggesting that yeast could be a model of ASD. Furthermore, A6E mutation could be introduced to the ASNS gene of acute lymphoblastic leukemia patients to inhibit the upregulation of ASNS by cancer cells, reducing the risk of developing resistance to the asparaginase treatment.
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22
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Abstract
Live-cell imaging is widely used by researchers to study cellular dynamics and obtain a deep understanding of cell biological processes. Keeping cells in the proper growing environment and immobilizing the cells are essential for the imaging of live yeast cells. Here we describe a protocol for monitoring cytoophidia in Saccharomyces cerevisiae and Schizosaccharomyces pombe using inverted confocal fluorescence microscopy. This protocol includes yeast culture, sample preparation, fluorescence imaging, and data analysis.
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Affiliation(s)
- Shanshan Zhang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China
| | - Hui Li
- School of Systems Science, Beijing Normal University, Beijing, China
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
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23
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Noree C, Sirinonthanawech N. Nuclear targeted Saccharomyces cerevisiae asparagine synthetases associate with the mitotic spindle regardless of their enzymatic activity. PLoS One 2020; 15:e0243742. [PMID: 33347445 PMCID: PMC7751962 DOI: 10.1371/journal.pone.0243742] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Accepted: 11/28/2020] [Indexed: 12/14/2022] Open
Abstract
Recently, human asparagine synthetase has been found to be associated with the mitotic spindle. However, this event cannot be seen in yeast because yeast takes a different cell division process via closed mitosis (there is no nuclear envelope breakdown to allow the association between any cytosolic enzyme and mitotic spindle). To find out if yeast asparagine synthetase can also (but hiddenly) have this feature, the coding sequences of green fluorescent protein (GFP) and nuclear localization signal (NLS) were introduced downstream of ASN1 and ASN2, encoding asparagine synthetases Asn1p and Asn2p, respectively, in the yeast genome having mCherrry coding sequence downstream of TUB1 encoding alpha-tubulin, a building block of the mitotic spindle. The genomically engineered yeast strains showed co-localization of Asn1p-GFP-NLS (or Asn2p-GFP-NLS) and Tub1p-mCherry in dividing nuclei. In addition, an activity-disrupted mutation was introduced to ASN1 (or ASN2). The yeast mutants still exhibited co-localization between defective asparagine synthetase and mitotic spindle, indicating that the biochemical activity of asparagine synthetase is not required for its association with the mitotic spindle. Furthermore, nocodazole treatment was used to depolymerize the mitotic spindle, resulting in lack of association between the enzyme and the mitotic spindle. Although yeast cell division undergoes closed mitosis, preventing the association of its asparagine synthetase with the mitotic spindle, however, by using yeast constructs with re-localized Asn1/2p have suggested the moonlighting role of asparagine synthetase in cell division of higher eukaryotes.
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Affiliation(s)
- Chalongrat Noree
- Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom, Thailand
| | - Naraporn Sirinonthanawech
- Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom, Thailand
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24
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Simonet JC, Foster MJ, Lynch EM, Kollman JM, Nicholas E, O'Reilly AM, Peterson JR. CTP synthase polymerization in germline cells of the developing Drosophila egg supports egg production. Biol Open 2020; 9:bio050328. [PMID: 32580972 PMCID: PMC7390647 DOI: 10.1242/bio.050328] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Accepted: 06/16/2020] [Indexed: 01/19/2023] Open
Abstract
Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. Here we demonstrate that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, we show that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism.This article has an associated First Person interview with the first author of the paper.
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Affiliation(s)
- Jacqueline C Simonet
- Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
| | - Maya J Foster
- Immersion Science Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
| | - Eric M Lynch
- Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
| | - Justin M Kollman
- Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
| | - Emmanuelle Nicholas
- Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
| | - Alana M O'Reilly
- Molecular Therapeutics Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
| | - Jeffrey R Peterson
- Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
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Nüske E, Marini G, Richter D, Leng W, Bogdanova A, Franzmann TM, Pigino G, Alberti S. Filament formation by the translation factor eIF2B regulates protein synthesis in starved cells. Biol Open 2020; 9:bio046391. [PMID: 32554487 PMCID: PMC7358136 DOI: 10.1242/bio.046391] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Accepted: 05/26/2020] [Indexed: 12/11/2022] Open
Abstract
Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. Many mechanistic details about how starved cells regulate the process of protein synthesis are still unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through filament assembly is an efficient mechanism that allows yeast cells to adapt to fluctuating environments.
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Affiliation(s)
- Elisabeth Nüske
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
| | - Guendalina Marini
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
| | - Doris Richter
- Department of Cellular Biochemistry Biotechnology Center (BIOTEC), Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Tatzberg 47/49, 01307 Dresden, Germany
| | - Weihua Leng
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
| | - Aliona Bogdanova
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
| | - Titus M Franzmann
- Department of Cellular Biochemistry Biotechnology Center (BIOTEC), Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Tatzberg 47/49, 01307 Dresden, Germany
| | - Gaia Pigino
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
| | - Simon Alberti
- Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
- Department of Cellular Biochemistry Biotechnology Center (BIOTEC), Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Tatzberg 47/49, 01307 Dresden, Germany
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26
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Mohammad K, Baratang Junio JA, Tafakori T, Orfanos E, Titorenko VI. Mechanisms that Link Chronological Aging to Cellular Quiescence in Budding Yeast. Int J Mol Sci 2020; 21:ijms21134717. [PMID: 32630624 PMCID: PMC7369985 DOI: 10.3390/ijms21134717] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2020] [Revised: 06/27/2020] [Accepted: 06/30/2020] [Indexed: 12/28/2022] Open
Abstract
After Saccharomyces cerevisiae cells cultured in a medium with glucose consume glucose, the sub-populations of quiescent and non-quiescent cells develop in the budding yeast culture. An age-related chronology of quiescent and non-quiescent yeast cells within this culture is discussed here. We also describe various hallmarks of quiescent and non-quiescent yeast cells. A complex aging-associated program underlies cellular quiescence in budding yeast. This quiescence program includes a cascade of consecutive cellular events orchestrated by an intricate signaling network. We examine here how caloric restriction, a low-calorie diet that extends lifespan and healthspan in yeast and other eukaryotes, influences the cellular quiescence program in S. cerevisiae. One of the main objectives of this review is to stimulate an exploration of the mechanisms that link cellular quiescence to chronological aging of budding yeast. Yeast chronological aging is defined by the length of time during which a yeast cell remains viable after its growth and division are arrested, and it becomes quiescent. We propose a hypothesis on how caloric restriction can slow chronological aging of S. cerevisiae by altering the chronology and properties of quiescent cells. Our hypothesis posits that caloric restriction delays yeast chronological aging by targeting four different processes within quiescent cells.
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27
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Chakraborty A, Lin WC, Lin YT, Huang KJ, Wang PY, Chang IYF, Wang HI, Ma KT, Wang CY, Huang XR, Lee YH, Chen BC, Hsieh YJ, Chien KY, Lin TY, Liu JL, Sung LY, Yu JS, Chang YS, Pai LM. SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network. J Cell Sci 2020; 133:jcs240200. [PMID: 32184263 DOI: 10.1242/jcs.240200] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2019] [Accepted: 03/06/2020] [Indexed: 01/08/2023] Open
Abstract
Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.
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Affiliation(s)
- Archan Chakraborty
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Wei-Cheng Lin
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Yu-Tsun Lin
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Kuang-Jing Huang
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Pei-Yu Wang
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Ian Yi-Feng Chang
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
- Bioinformatics Core Laboratory, Chang Gung University, Taoyuan 33302, Taiwan
| | - Hsiang-Iu Wang
- Bioinformatics Core Laboratory, Chang Gung University, Taoyuan 33302, Taiwan
| | - Kung-Ting Ma
- Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Chun-Yen Wang
- Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Xuan-Rong Huang
- Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Yen-Hsien Lee
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Bi-Chang Chen
- Research Center for Applied Sciences, Academia Sinica, Taipei 11529, Taiwan
| | - Ya-Ju Hsieh
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Kun-Yi Chien
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Clinical Proteomics Core laboratory, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Tzu-Yang Lin
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan
| | - Ji-Long Liu
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Li-Ying Sung
- Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan
- Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan
| | - Jau-Song Yu
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
- Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Yu-Sun Chang
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Li-Mei Pai
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
- Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan
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28
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Abstract
Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, for example, the purinosome, which forms subcellular macrobodies responsible for de novo purine biosynthesis. Here, we construct synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. A synthetic protein phase separation system using component proteins from postsynaptic density in neuronal synapses, GKAP, Shank, and Homer provides the scaffold for assembly. Three sets of guest proteins: a pair of fluorescent proteins (CFP and YFP), three sequential enzymes in menaquinone biosynthesis pathway (MenF, MenD, and MenH), and two enzymes in terpene biosynthesis pathway (Idi and IspA) are assembled via peptide-peptide interactions in the condensate. First, we discover that coassembly of CFP and YFP exhibited a broad distribution of the FRET signal within the condensate. Second, a spontaneous enrichment of the rate-limiting enzyme MenD in the condensate is sufficient to increase the 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate production rate by 70%. Third, coassembly of both Idi and IspA in the protein condensate increases the farnesyl pyrophosphate production rate by more than 50%. Altogether, we show here that phase separation significantly accelerates the efficiency of multienzyme biocatalysis.
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Affiliation(s)
- Miao Liu
- Department of Chemistry, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
| | - Sicong He
- Department of Electronic and Computer Engineering, Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China
| | - Lixin Cheng
- Department of Critical Care Medicine, Shenzhen People's Hospital, First Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518000, China
| | - Jianan Qu
- Department of Electronic and Computer Engineering, Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China
| | - Jiang Xia
- Department of Chemistry, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.,Center for Cell & Developmental Biology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 02522, China
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29
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Noree C, Sirinonthanawech N. Coupled regulations of enzymatic activity and structure formation of aldehyde dehydrogenase Ald4p. Biol Open 2020; 9:bio051110. [PMID: 32295831 PMCID: PMC7197708 DOI: 10.1242/bio.051110] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Accepted: 03/31/2020] [Indexed: 11/20/2022] Open
Abstract
Previously, we have developed an extramitochondrial assembly system, where mitochondrial targeting signal (MTS) can be removed from a given mitochondrial enzyme, which could be used to characterize the regulatory factors involved in enzyme assembly/disassembly in vivo Here, we demonstrate that addition of exogenous acetaldehyde can quickly induce the supramolecular assembly of MTS-deleted aldehyde dehydrogenase Ald4p in yeast cytoplasm. Also, by using PCR-based modification of the yeast genome, cytoplasmically targeted Ald4p cannot polymerize into long filaments when key functional amino acid residues are substituted, as shown by N192D, S269A, E290K and C324A mutations. This study has confirmed that extramitochondrial assembly could be a powerful external system for studying mitochondrial enzyme assembly, and its regulatory factors outside the mitochondria. In addition, we propose that mitochondrial enzyme assembly/disassembly is coupled to the regulation of a given mitochondrial enzyme activity.
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Affiliation(s)
- Chalongrat Noree
- Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand
| | - Naraporn Sirinonthanawech
- Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand
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30
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The structure of helical lipoprotein lipase reveals an unexpected twist in lipase storage. Proc Natl Acad Sci U S A 2020; 117:10254-10264. [PMID: 32332168 DOI: 10.1073/pnas.1916555117] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-Å resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.
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31
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Begovich K, Yelon D, Wilhelm JE. PRPS polymerization influences lens fiber organization in zebrafish. Dev Dyn 2020; 249:1018-1031. [PMID: 32243675 DOI: 10.1002/dvdy.173] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2019] [Revised: 02/28/2020] [Accepted: 03/05/2020] [Indexed: 11/08/2022] Open
Abstract
BACKGROUND The self-assembly of metabolic enzymes into filaments or foci highlights an intriguing mechanism for the regulation of metabolic activity. Recently, we identified the conserved polymerization of phosphoribosyl pyrophosphate synthetase (PRPS), which catalyzes the first step in purine nucleotide synthesis, in yeast and cultured mammalian cells. While previous work has revealed that loss of PRPS activity regulates retinal development in zebrafish, the extent to which PRPS filament formation affects tissue development remains unknown. RESULTS By generating novel alleles in the zebrafish PRPS paralogs, prps1a and prps1b, we gained new insight into the role of PRPS filaments during eye development. We found that mutations in prps1a alone are sufficient to generate abnormally small eyes along with defects in head size, pigmentation, and swim bladder inflation. Furthermore, a loss-of-function mutation that truncates the Prps1a protein resulted in the failure of PRPS filament assembly. Lastly, in mutants that fail to assemble PRPS filaments, we observed disorganization of the actin network in the lens fibers. CONCLUSIONS The truncation of Prps1a blocked PRPS filament formation and resulted in a disorganized lens fiber actin network. Altogether, these findings highlight a potential role for PRPS filaments during lens fiber organization in zebrafish.
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Affiliation(s)
- Kyle Begovich
- Howard Hughes Medical Institute (HHMI) Summer Institute, Marine Biological Laboratory, Woods Hole, Massachusetts, USA.,Division of Biological Sciences, University of California, San Diego, California, USA
| | - Deborah Yelon
- Division of Biological Sciences, University of California, San Diego, California, USA
| | - James E Wilhelm
- Howard Hughes Medical Institute (HHMI) Summer Institute, Marine Biological Laboratory, Woods Hole, Massachusetts, USA.,Division of Biological Sciences, University of California, San Diego, California, USA
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32
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Park CK, Horton NC. Structures, functions, and mechanisms of filament forming enzymes: a renaissance of enzyme filamentation. Biophys Rev 2019; 11:927-994. [PMID: 31734826 PMCID: PMC6874960 DOI: 10.1007/s12551-019-00602-6] [Citation(s) in RCA: 71] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2019] [Accepted: 10/24/2019] [Indexed: 12/19/2022] Open
Abstract
Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI endonuclease system are also highlighted.
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Affiliation(s)
- Chad K. Park
- Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721 USA
| | - Nancy C. Horton
- Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721 USA
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33
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Zhou X, Guo CJ, Hu HH, Zhong J, Sun Q, Liu D, Zhou S, Chang CC, Liu JL. Drosophila CTP synthase can form distinct substrate- and product-bound filaments. J Genet Genomics 2019; 46:537-545. [PMID: 31902586 DOI: 10.1016/j.jgg.2019.11.006] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2019] [Revised: 11/07/2019] [Accepted: 11/10/2019] [Indexed: 01/26/2023]
Abstract
Intracellular compartmentation is a key strategy for the functioning of a cell. In 2010, several studies revealed that the metabolic enzyme CTP synthase (CTPS) can form filamentous structures termed cytoophidia in prokaryotic and eukaryotic cells. However, recent structural studies showed that CTPS only forms inactive product-bound filaments in bacteria while forming active substrate-bound filaments in eukaryotic cells. In this study, using negative staining and cryo-electron microscopy, we demonstrate that Drosophila CTPS, whether in substrate-bound or product-bound form, can form filaments. Our results challenge the previous model and indicate that substrate-bound and product-bound filaments can coexist in the same species. We speculate that the ability to switch between active and inactive cytoophidia in the same cells provides an additional layer of metabolic regulation.
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Affiliation(s)
- Xian Zhou
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Chen-Jun Guo
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Huan-Huan Hu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China; University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Jiale Zhong
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China; University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Qianqian Sun
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; iHuman Institute, ShanghaiTech University, Shanghai, 201210, China
| | - Dandan Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; iHuman Institute, ShanghaiTech University, Shanghai, 201210, China
| | - Shuang Zhou
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China; University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Chia Chun Chang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
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34
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Lin WC, Chakraborty A, Huang SC, Wang PY, Hsieh YJ, Chien KY, Lee YH, Chang CC, Tang HY, Lin YT, Tung CS, Luo JD, Chen TW, Lin TY, Cheng ML, Chen YT, Yeh CT, Liu JL, Sung LY, Shiao MS, Yu JS, Chang YS, Pai LM. Histidine-Dependent Protein Methylation Is Required for Compartmentalization of CTP Synthase. Cell Rep 2019; 24:2733-2745.e7. [PMID: 30184506 DOI: 10.1016/j.celrep.2018.08.007] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2017] [Revised: 05/11/2018] [Accepted: 08/03/2018] [Indexed: 12/13/2022] Open
Abstract
CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. Tetramer conformation-based filament formation restricts CTPS enzymatic activity during nutrient deprivation. CTPS protein levels remain stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth after stress relief. We demonstrate that filament formation is controlled by methylation and that histidine promotes re-methylation of homocysteine by donating one-carbon intermediates to the cytosolic folate cycle. Furthermore, we find that starvation stress and glutamine deficiency activate the GCN2/ATF4/MTHFD2 axis, which coordinates CTPS filament formation. CTPS filament formation induced by histidine-mediated methylation may be a strategy used by cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.
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Affiliation(s)
- Wei-Cheng Lin
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Archan Chakraborty
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Shih-Chia Huang
- Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan
| | - Pei-Yu Wang
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Ya-Ju Hsieh
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Kun-Yi Chien
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Clinical Proteomics Core Laboratory, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Yen-Hsien Lee
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Chia-Chun Chang
- Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan
| | - Hsiang-Yu Tang
- Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Healthy Aging Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Yu-Tsun Lin
- Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
| | - Chang-Shung Tung
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
| | - Ji-Dung Luo
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Bioinformatics Core Laboratory, Chang Gung University, Taoyuan 33302, Taiwan
| | - Ting-Wen Chen
- Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Departments of Otolaryngology-Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Tzu-Yang Lin
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan; Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan
| | - Mei-Ling Cheng
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Healthy Aging Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Clinical Metabolomics Core Laboratory, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Yi-Ting Chen
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Kidney Research Center, Department of Nephrology, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Chau-Ting Yeh
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Ji-Long Liu
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
| | - Li-Ying Sung
- Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan; Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan
| | - Ming-Shi Shiao
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Healthy Aging Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Jau-Song Yu
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan
| | - Yu-Sun Chang
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan
| | - Li-Mei Pai
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan.
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35
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Zhang J, Liu JL. Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe. J Genet Genomics 2019; 46:423-432. [PMID: 31611173 PMCID: PMC6868507 DOI: 10.1016/j.jgg.2019.09.002] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2019] [Revised: 08/29/2019] [Accepted: 09/09/2019] [Indexed: 12/15/2022]
Abstract
The metabolic enzyme CTP synthase (CTPS) is able to compartmentalize into filaments, termed cytoophidia, in a variety of organisms including bacteria, budding yeast, fission yeast, fruit flies and mammals. A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift. Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe. To our surprise, we find that both the length and the occurrence of cytoophidia in S. pombe decrease upon cold shock or heat shock. The temperature-dependent changes of cytoophidia are fast and reversible. Taking advantage of yeast genetics, we demonstrate that heat-shock proteins are required for cytoophidium assembly in S. pombe. Temperature sensitivity of cytoophidia makes S. pombe an attractive model system for future investigations of this novel membraneless organelle.
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Affiliation(s)
- Jing Zhang
- MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom
| | - Ji-Long Liu
- MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom; School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
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36
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Andreadis C, Hulme L, Wensley K, Liu JL. The TOR pathway modulates cytoophidium formation in Schizosaccharomyces pombe. J Biol Chem 2019; 294:14686-14703. [PMID: 31431504 PMCID: PMC6779450 DOI: 10.1074/jbc.ra119.009913] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2019] [Revised: 08/09/2019] [Indexed: 12/30/2022] Open
Abstract
CTP synthase (CTPS) has been demonstrated to form evolutionarily-conserved filamentous structures termed cytoophidia whose exact cellular functions remain unclear, but they may play a role in intracellular compartmentalization. We have previously shown that the mammalian target of rapamycin complex 1 (mTORC1)-S6K1 pathway mediates cytoophidium assembly in mammalian cells. Here, using the fission yeast Schizosaccharomyces pombe as a model of a unicellular eukaryote, we demonstrate that the target of rapamycin (TOR)-signaling pathway regulates cytoophidium formation (from the S. pombe CTPS ortholog Cts1) also in S. pombe Conducting a systematic analysis of all viable single TOR subunit-knockout mutants and of several major downstream effector proteins, we found that Cts1 cytoophidia are significantly shortened and often dissociate when TOR is defective. We also found that the activities of the downstream effector kinases of the TORC1 pathway, Sck1, Sck2, and Psk1 S6, as well as of the S6K/AGC kinase Gad8, the major downstream effector kinase of the TORC2 pathway, are necessary for proper cytoophidium filament formation. Interestingly, we observed that the Crf1 transcriptional corepressor for ribosomal genes is a strong effector of Cts1 filamentation. Our findings connect TOR signaling, a major pathway required for cell growth, with the compartmentalization of the essential nucleotide synthesis enzyme CTPS, and we uncover differences in the regulation of its filamentation among higher multicellular and unicellular eukaryotic systems.
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Affiliation(s)
- Christos Andreadis
- School of Life Sciences and Technology, ShanghaiTech University, 201210 Shanghai, China
| | - Lydia Hulme
- MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom
| | - Katherine Wensley
- MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom
| | - Ji-Long Liu
- School of Life Sciences and Technology, ShanghaiTech University, 201210 Shanghai, China .,MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom
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37
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Hayward D, Kouznetsova VL, Pierson HE, Hasan NM, Guzman ER, Tsigelny IF, Lutsenko S. ANKRD9 is a metabolically-controlled regulator of IMPDH2 abundance and macro-assembly. J Biol Chem 2019; 294:14454-14466. [PMID: 31337707 DOI: 10.1074/jbc.ra119.008231] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 07/10/2019] [Indexed: 12/17/2022] Open
Abstract
Members of a large family of Ankyrin Repeat Domain (ANKRD) proteins regulate numerous cellular processes by binding to specific protein targets and modulating their activity, stability, and other properties. The same ANKRD protein may interact with different targets and regulate distinct cellular pathways. The mechanisms responsible for switches in the ANKRDs' behavior are often unknown. We show that cells' metabolic state can markedly alter interactions of an ANKRD protein with its target and the functional outcomes of this interaction. ANKRD9 facilitates degradation of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in GTP biosynthesis. Under basal conditions ANKRD9 is largely segregated from the cytosolic IMPDH2 in vesicle-like structures. Upon nutrient limitation, ANKRD9 loses its vesicular pattern and assembles with IMPDH2 into rodlike filaments, in which IMPDH2 is stable. Inhibition of IMPDH2 activity with ribavirin favors ANKRD9 binding to IMPDH2 rods. The formation of ANKRD9/IMPDH2 rods is reversed by guanosine, which restores ANKRD9 associations with the vesicle-like structures. The conserved Cys109Cys110 motif in ANKRD9 is required for the vesicle-to-rods transition as well as binding and regulation of IMPDH2. Oppositely to overexpression, ANKRD9 knockdown increases IMPDH2 levels and prevents formation of IMPDH2 rods upon nutrient limitation. Taken together, the results suggest that a guanosine-dependent metabolic switch determines the mode of ANKRD9 action toward IMPDH2.
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Affiliation(s)
- Dawn Hayward
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
| | - Valentina L Kouznetsova
- The Moores Cancer Center, University of California San Diego, La Jolla, California 92093.,San Diego Supercomputer Center University of California San Diego, La Jolla, California 92093
| | - Hannah E Pierson
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
| | - Nesrin M Hasan
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
| | - Estefany R Guzman
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
| | - Igor F Tsigelny
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.,San Diego Supercomputer Center University of California San Diego, La Jolla, California 92093.,Department of Neurosciences, University of California San Diego, La Jolla, California 92093
| | - Svetlana Lutsenko
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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38
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Woo WK, Dzaki N, Thangadurai S, Azzam G. Ectopic miR-975 induces CTP synthase directed cell proliferation and differentiation in Drosophila melanogaster. Sci Rep 2019; 9:6096. [PMID: 30988367 PMCID: PMC6465261 DOI: 10.1038/s41598-019-42369-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2018] [Accepted: 03/25/2019] [Indexed: 11/16/2022] Open
Abstract
CTP synthase (CTPSyn) is an essential metabolic enzyme, synthesizing precursors required for nucleotides and phospholipids production. Previous studies have also shown that CTPSyn is elevated in various cancers. In many organisms, CTPSyn compartmentalizes into filaments called cytoophidia. In Drosophila melanogaster, only its isoform C (CTPSynIsoC) forms cytoophidia. In the fruit fly's testis, cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located, and development is at its most rapid. Here, we report that CTPSynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately 34% of males overexpressing CTPSynIsoC. Immunostaining shows that this bulged phenotype is most likely due to increased numbers of both germline cells and spermatocytes. Through a microRNA (miRNA) overexpression screen, we found that ectopic miR-975 concurrently increases both the expression levels of CTPSyn and the length of its cytoophidia. The bulging testes phenotype was also recovered at a penetration of approximately 20%. However, qPCR assays reveal that CTPSynIsoC and miR-975 overexpression each provokes a differential response in expression of a number of cancer-related genes, indicating that the shared CTPSyn upregulation seen in either case is likely the cause of observed testicular overgrowth. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPSyn in D. melanogaster, and further reaffirms the enzyme's close ties to germline cells overgrowth.
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Affiliation(s)
- Wai Kan Woo
- School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
| | - Najat Dzaki
- School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
| | | | - Ghows Azzam
- School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia.
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39
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Wu Z, Liu JL. Cytoophidia respond to nutrient stress in Drosophila. Exp Cell Res 2019; 376:159-167. [PMID: 30768932 PMCID: PMC6403103 DOI: 10.1016/j.yexcr.2019.02.003] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2018] [Revised: 01/13/2019] [Accepted: 02/09/2019] [Indexed: 01/08/2023]
Abstract
CTP synthase (CTPsyn) is a metabolic enzyme essential for the de novo synthesis of CTP the nucleotide. CTPsyn can be compartmented into filamentous structures named cytoophidia. Cytoophidia are conserved in a wide range of species and are highly abundant in Drosophila ovaries. Here we report that cytoophidia elongate upon nutrient deprivation, CTPsyn overexpression or heat shock in Drosophila ovaries. We also show that the curvature of cytoophidia changes during apoptosis. Moreover, cytoophidia can be transported from nurse cells to the oocyte via ring canals. Our study demonstrates that cytoophidia can respond to stress and are very dynamic in Drosophila ovaries.
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Affiliation(s)
- Zheng Wu
- School of Life Science and Technology, ShanghaiTech University, 230 Haike Road, 201210 Shanghai, China
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, 230 Haike Road, 201210 Shanghai, China; MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, United Kingdom.
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40
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Sun Z, Liu JL. mTOR-S6K1 pathway mediates cytoophidium assembly. J Genet Genomics 2019; 46:65-74. [PMID: 30857853 PMCID: PMC6459811 DOI: 10.1016/j.jgg.2018.11.006] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2018] [Revised: 11/21/2018] [Accepted: 11/30/2018] [Indexed: 01/17/2023]
Abstract
CTP synthase (CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin (mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1 (mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing mTOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells. Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.
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Affiliation(s)
- Zhe Sun
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom.
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41
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Noree C, Sirinonthanawech N, Wilhelm JE. Saccharomyces cerevisiae ASN1 and ASN2 are asparagine synthetase paralogs that have diverged in their ability to polymerize in response to nutrient stress. Sci Rep 2019; 9:278. [PMID: 30670751 PMCID: PMC6342913 DOI: 10.1038/s41598-018-36719-z] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Accepted: 11/26/2018] [Indexed: 11/10/2022] Open
Abstract
Recent work has found that many metabolic enzymes have the ability to polymerize in response to metabolic changes or environmental stress. This ability to polymerize is well conserved for the few metabolic enzyme paralogs that have been studied in yeast. Here we describe the first set of paralogs, Asn1p and Asn2p, that have differential assembly behavior. Asn1p and Asn2p both co-assemble into filaments in response to nutrient limitation. However, the ability of Asn2p to form filaments is strictly dependent on the presence of Asn1p. Using mutations that block enzyme activity but have differential effects on Asn1p polymerization, we have found that Asn1p polymers are unlikely to have acquired a moonlighting function. Together these results provide a novel system for understanding the regulation and evolution of metabolic enzyme polymerization.
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Affiliation(s)
- Chalongrat Noree
- Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom, 73170, Thailand.
| | - Naraporn Sirinonthanawech
- Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhon Pathom, 73170, Thailand
| | - James E Wilhelm
- Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive (MC 0347), La Jolla, CA, 92093-0347, USA.
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42
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Sagot I, Laporte D. The cell biology of quiescent yeast – a diversity of individual scenarios. J Cell Sci 2019; 132:132/1/jcs213025. [DOI: 10.1242/jcs.213025] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
ABSTRACT
Most cells, from unicellular to complex organisms, spend part of their life in quiescence, a temporary non-proliferating state. Although central for a variety of essential processes including tissue homeostasis, development and aging, quiescence is poorly understood. In fact, quiescence encompasses various cellular situations depending on the cell type and the environmental niche. Quiescent cell properties also evolve with time, adding another layer of complexity. Studying quiescence is, above all, limited by the fact that a quiescent cell can be recognized as such only after having proved that it is capable of re-proliferating. Recent cellular biology studies in yeast have reported the relocalization of hundreds of proteins and the reorganization of several cellular machineries upon proliferation cessation. These works have revealed that quiescent cells can display various properties, shedding light on a plethora of individual behaviors. The deciphering of the molecular mechanisms beyond these reorganizations, together with the understanding of their cellular functions, have begun to provide insights into the physiology of quiescent cells. In this Review, we discuss recent findings and emerging concepts in Saccharomyces cerevisiae quiescent cell biology.
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Affiliation(s)
- Isabelle Sagot
- Centre National de la Recherche Scientifique, Université de Bordeaux-Institut de Biochimie et Génétique Cellulaires, UMR5095-33077 Bordeaux cedex, France
| | - Damien Laporte
- Centre National de la Recherche Scientifique, Université de Bordeaux-Institut de Biochimie et Génétique Cellulaires, UMR5095-33077 Bordeaux cedex, France
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43
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Zhang S, Ding K, Shen QJ, Zhao S, Liu JL. Filamentation of asparagine synthetase in Saccharomyces cerevisiae. PLoS Genet 2018; 14:e1007737. [PMID: 30365499 PMCID: PMC6221361 DOI: 10.1371/journal.pgen.1007737] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2018] [Revised: 11/07/2018] [Accepted: 10/03/2018] [Indexed: 11/24/2022] Open
Abstract
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. Asparagine synthetase (ASNS) is an essential enzyme for biosynthesis of asparagine. We have recently shown that ASNS, similar to CTP synthase (CTPS), can assemble into snake-shaped structures termed cytoophidia. In this study, we reveal that the ASNS cytoophidium stays close with the CTPS cytoophidium in most cells. Two ASNS proteins, Asn1 and Asn2, localize in the same structure. The Asn1 protein is important for the formation of the Asn2 filaments. Mutant cells with branching Asn1 cytoophidia grow slower than wild-type cells. Our findings provide a better understanding of the ASNS cytoophidium as well as its relationship with the CTPS cytoophidium.
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Affiliation(s)
- Shanshan Zhang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- Shanghai Institute of Biochemistry and Cell biology, Chinese Academy of Sciences, Shanghai, China
| | - Kang Ding
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- iHuman Institute, ShanghaiTech University, Shanghai, China
| | - Qing-Ji Shen
- MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
| | - Suwen Zhao
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- iHuman Institute, ShanghaiTech University, Shanghai, China
| | - Ji-Long Liu
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
- * E-mail: ,
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44
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McCluskey GD, Bearne SL. Anfractuous assemblies of IMP dehydrogenase and CTP synthase: new twists on regulation? FEBS J 2018; 285:3724-3728. [PMID: 30285320 DOI: 10.1111/febs.14658] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2018] [Accepted: 09/12/2018] [Indexed: 12/16/2022]
Abstract
CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell lines expressing CTPS1 and IMPDH2 fusion proteins. The formation and dismantling of mixed assemblies depends on nucleotide levels, suggesting a co-regulation function.
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Affiliation(s)
- Gregory D McCluskey
- Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Canada
| | - Stephen L Bearne
- Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Canada.,Department of Chemistry, Dalhousie University, Halifax, Canada
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45
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Dzaki N, Wahab W, Azlan A, Azzam G. CTP synthase knockdown during early development distorts the nascent vertebral column and causes fluid retention in multiple tissues in zebrafish. Biochem Biophys Res Commun 2018; 505:106-112. [DOI: 10.1016/j.bbrc.2018.09.074] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2018] [Accepted: 09/12/2018] [Indexed: 01/07/2023]
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46
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Chang C, Keppeke GD, Sung L, Liu J. Interfilament interaction between IMPDH and CTPS cytoophidia. FEBS J 2018; 285:3753-3768. [PMID: 30085408 PMCID: PMC6220823 DOI: 10.1111/febs.14624] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2018] [Revised: 07/03/2018] [Accepted: 08/03/2018] [Indexed: 11/30/2022]
Abstract
Inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS) are two metabolic enzymes that perform rate-limiting steps in the de novo synthesis of purine and pyrimidine nucleotides, respectively. It has been shown that IMPDH and CTPS can comprise a filamentous macrostructure termed the cytoophidium, which may play a role in regulation of their catalytic activity. Although these two proteins may colocalise in the same cytoophidium, how they associate with one another is still elusive. As reported herein, we established a model HeLa cell line coexpressing OFP-tagged IMPDH2 and GFP-tagged CTPS1 and recorded the assembly, disassembly and movement of the cytoophidium in live cells. Moreover, by using super-resolution confocal imaging, we demonstrate how IMPDH- and CTPS-based filaments are aligned or intertwined in the mixed cytoophidium. Collectively, our findings provide a panorama of cytoophidium dynamics and suggest that IMPDH and CTPS cytoophidia may coordinate by interfilament interaction.
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Affiliation(s)
- Chia‐Chun Chang
- Department of Physiology, Anatomy and GeneticsUniversity of OxfordUK
- Institute of BiotechnologyNational Taiwan UniversityTaipeiTaiwan
| | - Gerson D. Keppeke
- Department of Physiology, Anatomy and GeneticsUniversity of OxfordUK
| | - Li‐Ying Sung
- Institute of BiotechnologyNational Taiwan UniversityTaipeiTaiwan
- Agricultural Biotechnology Research CenterAcademia SinicaTaipeiTaiwan
| | - Ji‐Long Liu
- Department of Physiology, Anatomy and GeneticsUniversity of OxfordUK
- School of Life Science and TechnologyShanghaiTech UniversityChina
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47
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Daumann M, Hickl D, Zimmer D, DeTar RA, Kunz HH, Möhlmann T. Characterization of filament-forming CTP synthases from Arabidopsis thaliana. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2018; 96:316-328. [PMID: 30030857 PMCID: PMC6821390 DOI: 10.1111/tpj.14032] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/17/2017] [Revised: 05/14/2018] [Accepted: 05/22/2018] [Indexed: 05/27/2023]
Abstract
Cytidine triphosphate (CTP) is essential for DNA, RNA and phospholipid biosynthesis. De novo synthesis is catalyzed by CTP synthases (CTPS). Arabidopsis encodes five CTPS isoforms that unanimously share conserved motifs found across kingdoms, suggesting all five are functional enzymes. Whereas CTPS1-4 are expressed throughout Arabidopsis tissues, CTPS5 reveals exclusive expression in developing embryos. CTPS activity and substrates affinities were determined for a representative plant enzyme on purified recombinant CTPS3 protein. As demonstrated in model organisms such as yeast, fruit fly and mammals, CTPS show the capacity to assemble into large filaments called cytoophidia. Transient expression of N- and C-terminal YFP-CTPS fusion proteins in Nicotiana benthamiana allowed to monitor such filament formation. Interestingly, CTPS1 and 2 always appeared as soluble proteins, whereas filaments were observed for CTPS3, 4 and 5 independent of the YFP-tag location. However, when similar constructs were expressed in Saccharomyces cerevisiae, no filaments were observed, pointing to a requirement for organism-specific factors in vivo. Indications for filament assembly were also obtained in vitro when recombinant CTPS3 protein was incubated in the presence of CTP. T-DNA-insertion mutants in four CTPS loci revealed no apparent phenotypical alteration. In contrast, CTPS2 T-DNA-insertion mutants did not produce homozygous progenies. An initial characterization of the CTPS protein family members from Arabidopsis is presented. We provide evidence for their involvement in nucleotide de novo synthesis and show that only three of the five CTPS isoforms were able to form filamentous structures in the transient tobacco expression system. This represents a striking difference from previous observations in prokaryotes, yeast, Drosophila and mammalian cells. This finding will be highly valuable to further understand the role of filament formation to regulate CTPS activity.
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Affiliation(s)
- Manuel Daumann
- Pflanzenphysiologie, Fachbereich Biologie, Universität Kaiserslautern, Erwin-Schrodinger-Straße, D-67663, Kaiserslautern, Germany, and
| | - Daniel Hickl
- Pflanzenphysiologie, Fachbereich Biologie, Universität Kaiserslautern, Erwin-Schrodinger-Straße, D-67663, Kaiserslautern, Germany, and
| | - David Zimmer
- Pflanzenphysiologie, Fachbereich Biologie, Universität Kaiserslautern, Erwin-Schrodinger-Straße, D-67663, Kaiserslautern, Germany, and
| | - Rachael A. DeTar
- School of Biological Sciences, Washington State University, PO Box 644236, Pullman, WA, 99164-4236, USA
| | - Hans-Henning Kunz
- School of Biological Sciences, Washington State University, PO Box 644236, Pullman, WA, 99164-4236, USA
| | - Torsten Möhlmann
- Pflanzenphysiologie, Fachbereich Biologie, Universität Kaiserslautern, Erwin-Schrodinger-Straße, D-67663, Kaiserslautern, Germany, and
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48
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McCluskey GD, Bearne SL. "Pinching" the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage. Biochim Biophys Acta Gen Subj 2018; 1862:2714-2727. [PMID: 30251661 DOI: 10.1016/j.bbagen.2018.08.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2018] [Revised: 07/20/2018] [Accepted: 08/06/2018] [Indexed: 01/10/2023]
Abstract
Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5'-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH3 as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH3 passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH3 transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with "pinching" the tunnel, thereby revealing processes that synchronize the transfer of NH3 from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a "bottleneck" effect. The inability of V60F to use exogenous NH3 was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH2OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH3 gate of CTPS to mediate inter-domain NH3 transport.
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Affiliation(s)
- Gregory D McCluskey
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada
| | - Stephen L Bearne
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada; Department of Chemistry, Dalhousie University, Halifax, NS B3H 4R2, Canada.
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Li H, Ye F, Ren JY, Wang PY, Du LL, Liu JL. Active transport of cytoophidia in Schizosaccharomyces pombe. FASEB J 2018; 32:5891-5898. [PMID: 29782206 PMCID: PMC6292696 DOI: 10.1096/fj.201800045rr] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The metabolic enzyme cytidine triphosphate synthase has recently been found to form micrometer-sized filamentous structures termed cytoophidia, which are evolutionarily conserved across prokaryotes and eukaryotes. The cytoophidium represents a novel type of membraneless organelle and behaves dynamically inside the cell. The question of how cytoophidia transport is mediated, however, remains unanswered. For the first time, we detected in this study the active transport of cytoophidia, taking advantage of the fission yeast Schizosaccharomyces pombe as an excellent model for studying membraneless organelles. We demonstrated that actin filaments, not microtubules, are responsible for this transport. Furthermore, we determined that Myo52, a type of myosin V, is required for the active transport of cytoophidia. These results reveal the major players critical to the dynamics of cytoophidia and extend our understanding of intracellular transport of membraneless organelles.—Li, H., Ye, F., Ren, J.-Y., Wang, P.-Y., Du, L.-L., Liu, J.-L. Active transport of cytoophidia in Schizosaccharomyces pombe.
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Affiliation(s)
- Hui Li
- Department of Physiology, Anatomy, and Genetics, Medical Research Council Functional Genomics Unit, University of Oxford, Oxford, United Kingdom.,Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China
| | - Fangfu Ye
- Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.,School of Physical Sciences, University of Chinese Academy of Sciences, Beijing, China
| | - Jing-Yi Ren
- National Institute of Biological Sciences, Beijing, China; and
| | - Peng-Ye Wang
- Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China
| | - Li-Lin Du
- National Institute of Biological Sciences, Beijing, China; and
| | - Ji-Long Liu
- Department of Physiology, Anatomy, and Genetics, Medical Research Council Functional Genomics Unit, University of Oxford, Oxford, United Kingdom.,School of Life Science and Technology, ShanghaiTech University, Shanghai, China
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Extramitochondrial Assembly of Mitochondrial Targeting Signal Disrupted Mitochondrial Enzyme Aldehyde Dehydrogenase. Sci Rep 2018; 8:6186. [PMID: 29670139 PMCID: PMC5906672 DOI: 10.1038/s41598-018-24586-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Accepted: 04/06/2018] [Indexed: 11/17/2022] Open
Abstract
Supramolecular assembly of metabolic enzymes has been studied both in vivo and in vitro for nearly a decade. Experimental evidence has suggested a close relationship between enzymatic activity and enzyme assembly/disassembly. However, most cases were studied with the cytosolic enzymes. Here, I report the evidence for a mitochondrial enzyme with its ability in forming visible intracellular structures. By removing the mitochondrial targeting sequence, yeast mitochondrial enzyme aldehyde dehydrogenase (Ald4p) exhibits reversible supramolecular assembly in the cytoplasm, thus creating a useful system for further characterization of the regulatory factors that modulate the assembly/disassembly of this mitochondrial enzyme.
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