Role of Arabidopsis thaliana RING-Type E3 Ligase XBAT35.2 in Regulating ACD11 Stability

Abstract

Ubiquitination is a post-translational modification that results in a single (monoubiquitination) or multiple (polyubiquitination) ubiquitin molecule(s) being covalently attached to a selected protein substrate. A common consequence of ubiquitination is degradation of the polyubiquitinated protein by the 26S proteasome. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase). E1 activates ubiquitin and E2 receives the activated ubiquitin from E1. The E2-ubiquitin intermediate interacts with the E3, which recruits the substrate. The E2 and E3 then coordinate the attachment of ubiquitin to a lysine residue on the selected protein substrate. My research focuses on characterizing the function of the RING-type E3 ligase, XBAT35.2, via identification of substrates for the E3 ligase. Accelerated Cell Death 11 (ACD11) has been identified as a potential substrate for XBAT35.2. ACD11 is a pathogen-related protein that is an important component of the programmed cell death pathway in plants. acd11, a lethal recessive mutant in Arabidopsis, constitutively expresses cell death and defense related genes. ACD11 is thought to inhibit cell death and activation of defense pathways in the absence of pathogens. The interaction between ACD11 and XBAT35.2 was confirmed using immunoprecipitation assays. Cell free degradation assays were used to determine if ACD11 was a target for the ubiquitin proteasome system and also to demonstrate that XBAT35.2 mediated the turnover of ACD11. My results show that ACD11 is quite stable in a cell free degradation assay, however, when transiently co-expressed with XBAT35.2, ACD11 is rapidly turned over. I also demonstrate that XBAT35.2-mediated turnover of ACD11 is dependent on the function of the 26S proteasome. Unexpectedly, XBAT35.2 is itself unstable and subjected to proteasome dependent degradation. The observed turnover of XBAT35.2 requires its own RING E3 ligase activity as the nonfunctional E3 is not degraded. This suggests that XBAT35.2 undergoes self-ubiquitination and auto-regulation. These results provide evidence that ACD11 is a substrate for XBAT35.2 E3 ligase activity. These results also correlate with previous studies, which show that overexpression of XBAT35.2 is capable of inducing cell death in tobacco cells and promoting plant defense against pathogens in Arabidopsis transgenic plants. A model is proposed where in the absence of pathogen, XBAT35.2 is continually self-ubiquitinated and degraded by the 26S proteasome, allowing ACD11 to accumulate and inhibit cell death and pathogen defense pathways. In the presence of pathogens, XBAT35.2 would be stable and promote the degradation of ACD11, allowing for activation of cell death and defense response pathways.