Critical roles of CTP synthase N-terminal in cytoophidium assembly.
Several metabolic enzymes assemble into distinct intracellular structures in prokaryotes and eukaryotes suggesting an important functional role in cell physiology. The CTP-generating enzyme CTP synthase forms long filamentous structures termed cytoophidia in bacteria, yeast, fruit flies and human cells independent of its catalytic activity. However, the amino acid determinants for protein-protein interaction necessary for polymerisation remained unknown. In this study, we systematically analysed the role of the conserved N-terminal of Drosophila CTP synthase in cytoophidium assembly. Our mutational analyses identified three key amino acid residues within this region that play an instructive role in organisation of CTP synthase into a filamentous structure. Co-transfection assays demonstrated formation of heteromeric CTP synthase filaments which is disrupted by protein carrying a mutated N-terminal alanine residue thus revealing a dominant-negative activity. Interestingly, the dominant-negative activity is supressed by the CTP synthase inhibitor DON. Furthermore, we found that the amino acids at the corresponding position in the human protein exhibit similar properties suggesting conservation of their function through evolution. Our data suggest that cytoophidium assembly is a multi-step process involving N-terminal-dependent sequential interactions between correctly folded structural units and provide insights into the assembly of these enigmatic structures.
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom; Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing, China.
Yong
Y
Huang
Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing, China.
Jin-Jun
JJ
Wang
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom. Electronic address: sg805@cam.ac.uk.
Sanjay
S
Ghosh
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. Electronic address: jilong.liu@dpag.ox.ac.uk.
Ji-Long
JL
Liu
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000378:metabolism*; D002999:Clone Cells; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D005799:Genes, Dominant; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D058105:Polymerization; D013329:Structure-Activity Relationship
D000596:Amino Acids; D029721:Drosophila Proteins; D050505:Mutant Proteins; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase
CTP synthase; Cytoophidium; Drosophila; Intracellular filaments; Membrane-less organelle; Metabolic cell biology