Identification of HSP90C as a substrate of E3 ligase TaSAP5 through ubiquitylome profiling.

• HSP90s were identified to be substrates of TaSAP5 using two independent methods. • TaSAP5 interacts with HSP90C and facilitates their ubiquitination in vivo. • TaSAP5 promotes degradation of HSP90C protein via the 26S proteasome. Protein ubiquitination is a major post-translational modification important for diverse biological processes. In wheat (Triticum aestivum) and Arabidopsis thaliana , STRESS-ASSOCIATED PROTEIN 5 (SAP5) is involved in drought tolerance, acting as an E3 ubiquitin ligase to target DRIP and MBP-1 for degradation. To identify further target proteins of SAP5, we implemented two independent approaches in this work. We used ubiquitylome capture with a di-Gly-Lys antibody-based peptide enrichment and affinity purification with a polyubiquitin antibody coupled with mass spectrometry to elucidate the SAP5-dependent ubiquitylation of its target proteins in response to osmotic stress. Wild type or TaSAP5 -overexpressing Arabidopsis line, which was more tolerant to osmotic stress according to our previous study, were used here. We identified HSP90C (chloroplast heat shock protein 90) as a substrate of TaSAP5. Further biochemical experiments indicated that TaSAP5 interacts with HSP90C and mediates its degradation by the 26S proteasome. Our work also demonstrates that ubiquitylome profiling is an effective approach to search for substrates of the TaSAP5 E3 ubiquitin ligase when heterologously expressed in Arabidopsis. [ABSTRACT FROM AUTHOR]