Abstract
Key message
KEG is involved in mediating the proteasome-dependent degradation of FDH, a stress-responsive enzyme. The UPS may function to suppress FDH mediated stress responses under favorable growth conditions.
Abstract
Formate dehydrogenase (FDH) has been studied in bacteria and yeasts for the purpose of industrial application of NADH co-factor regeneration. In plants, FDH is regarded as a universal stress protein involved in responses to various abiotic and biotic stresses. Here we show that FDH abundance is regulated by the ubiquitin proteasome system (UPS). FDH is ubiquitinated in planta and degraded by the 26S proteasome. Interaction assays identified FDH as a potential substrate for the RING-type ubiquitin ligase Keep on Going (KEG). KEG is capable of attaching ubiquitin to FDH in in vitro assays and the turnover of FDH was increased when co-expressed with a functional KEG in planta, suggesting that KEG contributes to FDH degradation. Consistent with a role in regulating FDH abundance, transgenic plants overexpressing KEG were more sensitive to the inhibitory effects of formate. In addition, FDH is a phosphoprotein and dephosphorylation was found to increase the stability of FDH in degradation assays. Based on results from this and previous studies, we propose a model where KEG mediates the ubiquitination and subsequent degradation of phosphorylated FDH and, in response to unfavourable growth conditions, reduction in FDH phosphorylation levels may prohibit turnover allowing the stabilized FDH to facilitate stress responses.
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Acknowledgements
The authors would like to thank Dr. Russ Finley (Wayne State University) and Dr. Sonia Gazzarrini (University of Toronto) for providing the yeast two-hybrid vectors and cDNA library, respectively. This project was supported in part by a Natural Science and Engineering Research Council of Canada (NSERC) Discovery grant to S.L.S.
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DM: data acquisition, analysis of data and wrote the first draft. AS: data acquisition and editing of final draft. SLS conceived and designed the project, data interpretation and revisions of draft initial and subsequent drafts.
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11103_2017_691_MOESM1_ESM.pdf
Supplemental Figure S1. Alignment of FDH amino acid sequences from 12 plant species showing predicted and experimentally determine sites of post-translational modification. Predicted ubiquitination (black outline) and phosphorylation (grey boxes) sites as well as experimentally determined phosphorylation (black arrowheads) sites are indicated. Plant species and associated accession/locus ID are as follows: Arabidopsis, Arabidopsis thaliana (At5g14780); Solanum, Solanum lycopersicum (tomato, NP_001234857); Oryza, Oryza sativa Japonica (rice, XP_015642621.1); Glycine,Glycine max (soybean, NP_001241141.1); Vitis,Vitis vinifera (grape, XP_002278444.1); Brachypodium, Brachypodium distachyon (stiff brome, XP_003563874); Sorghum, Sorghum bicolor (XP_002438408); Populus; Populus trichocarpa (XP_002320501.1); Selaginella, Selaginella moellendorffii (XP_002985142); Hordeum, Hordeum vulgare (barley, BAJ95739.1); Tuberosum, Solanum tuberosum (potato, NP_001274827.1); Capsicum,Capsicum annuum (sweet pepper, NP_001311723). Below the alignment asterisk (*) indicates fully conserved residues; colon (:) indicates amino acids that have highly similar properties; period (.) indicates amino acids that have weakly similar properties. Sequences were aligned using the Clustal Omega protein sequence alignment program available from The European Bioinformatics Institutes (EBI; http://www.ebi.ac.uk/) (McWilliam et al. 2013). (PDF 56 KB)
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McNeilly, D., Schofield, A. & Stone, S.L. Degradation of the stress-responsive enzyme formate dehydrogenase by the RING-type E3 ligase Keep on Going and the ubiquitin 26S proteasome system. Plant Mol Biol 96, 265–278 (2018). https://doi.org/10.1007/s11103-017-0691-8
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DOI: https://doi.org/10.1007/s11103-017-0691-8