Abstract
We have reported that a subunit of 19S proteasome, RPT5a, is required for high-boron (B) tolerance in roots of A. thaliana. 19S is a subcomplex of 26S proteasome regulating many cellular processes via active proteolysis. 19S mediates recognition and unfolding of targets. We further established that 19S subunits RPN2a, RPN8a and RPT2a were also required for B tolerance, but RPN2b, RPN8b, RPT2b and RPT5b were not. Considering specific requirement for high-B tolerance among 19S subunits, it is possible that subunit-specific substrates degraded by proteasome in response to high-B are present. In this study, we attempted to screen that kind of substrate using rpt5a mutant. Proteomics of affinity-purified poly-ubiquitinated proteins using iTRAQ and LC-MS/MS found that 28 proteins were induced and were highly accumulated in rpt5a-6 than in the wild type by high-B treatment. 17 of 28 proteins have at least 1 motif associated with the degradation by proteasome, suggesting that our method is effective for substrates screening. We also isolated at least 2 high-B insensitive revertants of rpt5a-6 which are expected to be defective in high-B dependent substrates recognized through RPT5a.
