Identification of 14‐3‐3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis

Abstract

SUMMARYThe balance between carbon (C) and nitrogen (N) availability is an important determinant for various phases of plant growth; however, the detailed mechanisms regulating the C/N response are not well understood. We previously described two related ubiquitin ligases, ATL31 and ATL6, that function in the C/N response in Arabidopsis thaliana. Here, we used FLAG tag affinity purification and MS analysis to identify proteins targeted by ATL31, and thus likely to be involved in regulating the phase transition checkpoint based on C/N status. This analysis revealed that 14-3-3 proteins were associated with ATL31, and one of these, 14-3-3v, was selected for detailed characterization. The interaction between ATL31 and 14-3-3v was confirmed by yeast two-hybrid and co-immunoprecipitation analyses. In vitro assays showed that ubiquitination of 14-3-3v is catalyzed by ATL31. Degradation of 14-3-3v in vivo was shown to be correlated with ATL31 activity, and to occur in a proteasomedependent manner. Furthermore, 14-3-3 protein accumulation was induced by a shift to high-C/N stress conditions in Arabidopsis seedlings, and this regulated response required both ATL31 and ATL6. It was also shown that over-expression of 14-3-3v leads to hypersensitivity of Arabidopsis seedlings to C/N stress conditions. These results indicate that ATL31 targets and ubiquitinates 14-3-3 proteins for degradation via the ubiquitin-proteasome system during the response to cellular C/N status.

Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins

“…Each isoform has a different binding affinity to the client proteins (40,41). Previously, we have shown that 14-3-3 belonging to the non-⑀ group is directly associated and ubiquitinated by ATL31 (12). In addition to 14-3-3, immunoprecipitation and MS analysis revealed that several 14-3-3 isoforms are putative targets of ATL31, with the stability of each isoform being different in response to C/N-nutrient conditions (12).…”

“…Previously, we have shown that 14-3-3 belonging to the non-⑀ group is directly associated and ubiquitinated by ATL31 (12). In addition to 14-3-3, immunoprecipitation and MS analysis revealed that several 14-3-3 isoforms are putative targets of ATL31, with the stability of each isoform being different in response to C/N-nutrient conditions (12). To evaluate the specificity of the interaction between ATL31 and each 14-3-3 isoform, we carried out yeast two-hybrid (Y2H) analysis.…”

“…-FLAG) were as described previously (12). Nicotiana benthamiana was grown under conditions of 16 h light/8 h dark at 22°C.…”

“…Portions of the extracts prior to immunoprecipitation were also boiled with SDS sample buffer. The proteins were detected by Western blotting using anti-FLAG M2 antibody (Sigma), anti-Myc tag antibody (MBL), and anti-14-3-3 antibody (12).…”

“…In particular, 14-3-3 proteins have been reported to regulate primary carbon and nitrogen metabolism by directly interacting with key enzymes (16 -18). Further analyses demonstrated that ATL31 directly ubiquitinates 14-3-3 proteins and regulates protein amounts in response to C/N-nutrient conditions, revealing a plant-specific regulatory mechanism of C/N-nutrient signaling via the ubiquitin-proteasome system (12).…”