Proteins Interacting with S‐Nitrosoglutathione reductase to Regulate Nitric Oxide Homeostasis

Abstract

In plants, nitric oxide (NO) is a crucial signaling molecule involved in many physiological processes and stress pathways. NO can influence plant germination, flowering, and root development1. NO is usually found in an adduct with glutathione, forming S‐nitrosoglutathione (GSNO), which can affect protein S‐nitrosation, an important post‐translational modification that can alter protein activity and stability. The enzyme S‐nitrosoglutathione reductase (GSNOR) catalyzes the irreversible NADH‐dependent reduction of GSNO. GSNOR’s ability to regulate GSNO levels and subsequently affect S‐nitrosation activity makes it a critical enzyme in NO signaling2. The objective of this project is to identify proteins that interact with GSNOR to regulate its activity and stability, thereby impacting NO homeostasis. Based on previous experiments, we hypothesize that thioredoxins and catalase may be recovered as interacting candidates. We are utilizing enzyme‐catalyzed proximity labeling with the biotin ligase, TurboID, coupled to mass spectrometry to identify interacting partners of GSNOR in the plant model Arabidopsis thaliana. Currently, we have shown that a GSNOR‐TurboID fusion protein that has been transformed into a GSNOR null mutant can rescue the mutant phenotype without displaying any adverse effects on plant growth. As controls, A. thaliana lines carrying TurboID fused to YFP and targeted to the nucleus or cytosol have also been characterized. Importantly, upon treatment with biotin, TurboID produces strong biotinylation signals in these lines. These findings indicate that TurboID is suitable for use in A. thalianaand that biotinylated proteins can be recovered for identification by mass spectrometry. These experiments will advance the current understanding of GSNOR’s regulation, leading to an improved model of NO signaling.1. Xu, S., D. Guerra, U. Lee, & E. Vierling. S‐Nitrosoglutathione reductases are low‐copy number, cysteine‐rich proteins in plants that control multiple developmental and defense responses in Arabidopsis. Front. Plant Sci. 4: 1‐13 (2013). doi: 10.3389/fpls.2013.00430. 2. Lee, U., Wie, C., Fernandez, B. O., Feelisch, M. & Vierling, E. Modulation of nitrosative stress by S‐nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis. Plant Cell. 20: 786–802 (2008). doi: 10.1105/tpc/107.052647.