Journal article
Characterization of filament‐forming CTP synthases from Arabidopsis thaliana
The Plant journal : for cell and molecular biology, Vol.96(2), pp.316-328
10/2018
Handle:
https://hdl.handle.net/2376/105947
PMCID: PMC6821390
PMID: 30030857
Abstract
Summary
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.
Significance Statement
An initial characterization of the CTP synthase 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 CTP Synthase activity.
Metrics
10 Record Views
Details
- Title
- Characterization of filament‐forming CTP synthases from Arabidopsis thaliana
- Creators
- Manuel Daumann - Universität KaiserslauternDaniel Hickl - Universität KaiserslauternDavid Zimmer - Universität KaiserslauternRachael A DeTar - Washington State UniversityHans‐Henning Kunz - Washington State UniversityTorsten Möhlmann - Universität Kaiserslautern
- Publication Details
- The Plant journal : for cell and molecular biology, Vol.96(2), pp.316-328
- Academic Unit
- Biological Sciences, School of
- Number of pages
- 13
- Grant note
- NIH Biotechnology Training Program NSF Graduate Research Fellowship Program DFG (MO 1032/4‐1; IRTG 1830; IOS‐1553506)
- Identifiers
- 99900546830501842
- Language
- English
- Resource Type
- Journal article