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ABA and the ubiquitin E3 ligase KEEP ON GOING affect proteolysis of the Arabidopsis thaliana transcription factors ABF1 and ABF3.

Chen YT, Liu H, Stone S, Callis J - Plant J. (2013)

Bottom Line: Loss of ABF1 or ABF3 in the keg background has a phenotypic effect similar to the loss of ABI5, and there is no additional rescue of the keg phenotype in abf1 abf3 abi5 keg seedlings.This result suggests that the abundance of other substrates is altered in keg seedlings, affecting growth.In conclusion, ABF1 and ABF3 abundance is affected by ABA and KEG, and the conserved C4 region serves as a stabilizing element.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, UC-Davis, 1 Shields Ave, Davis, CA 95616, USA.

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KEG ubiquitylates ABF1 and ABF3 and their C4 deletion forms in vitro. In vitro ubiquitylation assays with yeast E1, Arabidopsis His-UBC8 (E2), GST-KEG-RK (E3), ubiquitin (Ub), and His-HA-ABF1, His-HA-ABF3 or their C4 deletion forms (substrates). Substrate proteins visualized by anti-HA immunoblot (top). GST-KEG self-ubiquitylating E3 activity indicated by higher migrating forms in the anti-GST immunoblot (bottom).
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fig09: KEG ubiquitylates ABF1 and ABF3 and their C4 deletion forms in vitro. In vitro ubiquitylation assays with yeast E1, Arabidopsis His-UBC8 (E2), GST-KEG-RK (E3), ubiquitin (Ub), and His-HA-ABF1, His-HA-ABF3 or their C4 deletion forms (substrates). Substrate proteins visualized by anti-HA immunoblot (top). GST-KEG self-ubiquitylating E3 activity indicated by higher migrating forms in the anti-GST immunoblot (bottom).

Mentions: The CHX chase and cell-free degradation assays demonstrated that ABF1 and ABF3 protein degradation is affected by the loss of KEG. To determine whether KEG acts directly through catalyzing ABF ubiquitylation, we performed in vitro ubiquitylation assays using recombinant KEG (GST-KEG), as the E3 ligase E3 and with ABF proteins as substrates. In complete ubiquitylation reactions, higher molecular mass forms of full-length His-HA-ABF1 and His-HA-ABF3, and their ΔC4 forms, were detected by anti-HA antibody (Figure 9, top panel, lanes 1, 7, 12 and 18). GST-KEG activity in these assays can be verified by self-ubiquitylation (Figure 9, lower panels, lanes 1, 6, 7, 12, 17 and 18). Omitting any of the required components abolished higher molecular mass forms, demonstrating that these forms are ubiquitylated by KEG (Figure 9).


ABA and the ubiquitin E3 ligase KEEP ON GOING affect proteolysis of the Arabidopsis thaliana transcription factors ABF1 and ABF3.

Chen YT, Liu H, Stone S, Callis J - Plant J. (2013)

KEG ubiquitylates ABF1 and ABF3 and their C4 deletion forms in vitro. In vitro ubiquitylation assays with yeast E1, Arabidopsis His-UBC8 (E2), GST-KEG-RK (E3), ubiquitin (Ub), and His-HA-ABF1, His-HA-ABF3 or their C4 deletion forms (substrates). Substrate proteins visualized by anti-HA immunoblot (top). GST-KEG self-ubiquitylating E3 activity indicated by higher migrating forms in the anti-GST immunoblot (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3823012&req=5

fig09: KEG ubiquitylates ABF1 and ABF3 and their C4 deletion forms in vitro. In vitro ubiquitylation assays with yeast E1, Arabidopsis His-UBC8 (E2), GST-KEG-RK (E3), ubiquitin (Ub), and His-HA-ABF1, His-HA-ABF3 or their C4 deletion forms (substrates). Substrate proteins visualized by anti-HA immunoblot (top). GST-KEG self-ubiquitylating E3 activity indicated by higher migrating forms in the anti-GST immunoblot (bottom).
Mentions: The CHX chase and cell-free degradation assays demonstrated that ABF1 and ABF3 protein degradation is affected by the loss of KEG. To determine whether KEG acts directly through catalyzing ABF ubiquitylation, we performed in vitro ubiquitylation assays using recombinant KEG (GST-KEG), as the E3 ligase E3 and with ABF proteins as substrates. In complete ubiquitylation reactions, higher molecular mass forms of full-length His-HA-ABF1 and His-HA-ABF3, and their ΔC4 forms, were detected by anti-HA antibody (Figure 9, top panel, lanes 1, 7, 12 and 18). GST-KEG activity in these assays can be verified by self-ubiquitylation (Figure 9, lower panels, lanes 1, 6, 7, 12, 17 and 18). Omitting any of the required components abolished higher molecular mass forms, demonstrating that these forms are ubiquitylated by KEG (Figure 9).

Bottom Line: Loss of ABF1 or ABF3 in the keg background has a phenotypic effect similar to the loss of ABI5, and there is no additional rescue of the keg phenotype in abf1 abf3 abi5 keg seedlings.This result suggests that the abundance of other substrates is altered in keg seedlings, affecting growth.In conclusion, ABF1 and ABF3 abundance is affected by ABA and KEG, and the conserved C4 region serves as a stabilizing element.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, UC-Davis, 1 Shields Ave, Davis, CA 95616, USA.

Show MeSH