ABA and the ubiquitin E3 ligase KEEP ON GOING affect proteolysis of the Arabidopsis thaliana transcription factors ABF1 and ABF3.
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.
Affiliation: Department of Molecular and Cellular Biology, UC-Davis, 1 Shields Ave, Davis, CA 95616, USA.Show MeSH
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).
Affiliation: Department of Molecular and Cellular Biology, UC-Davis, 1 Shields Ave, Davis, CA 95616, USA.