Proteomic identification of potential target proteins regulated by an ASK1-mediated proteolysis pathway

The ASK1 (ARABIDOPSIS SKP1-LIKE) protein is a critical component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome. To investigate proteins that are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers, we compared the proteomes of the Arabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis (2-DE). Ten protein spots with higher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to further mass spectrometry (MS) analysis. The results showed that they were proteins involved in photomorphogenesis, circadian oscillation, post-translation process, stress-responses and cell expansion or elongation, suggesting that those processes were affected in the ask1 mutant. The transcript levels of these genes were also compared based on the Affymetrix gene chip microarray data. No significant difference was observed for most of the genes, suggesting that the proteins with elevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASK1-mediated proteolysis pathway. These results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes and also demonstrate the importance and necessity of studying protein levels with respect to gene functions.