State Key Laboratory for Agrobiotechnology and Key Laboratory of Crop Heterosis and Utilization (MOE) and Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China.
Key Laboratory of Integrated Crop Pest Management of Shandong Province, College of Plant Health and Medicine, Qingdao Agricultural University, Qingdao, China.
Plant Sci. 2019 Oct;287:110170. doi: 10.1016/j.plantsci.2019.110170. Epub 2019 Jun 16.
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.
蛋白质泛素化是一种重要的翻译后修饰，对于多种生物过程都非常重要。在小麦（Triticum aestivum）和拟南芥（Arabidopsis thaliana）中，应激相关蛋白 5（SAP5）参与耐旱性，作为 E3 泛素连接酶，靶向 DRIP 和 MBP-1 进行降解。为了进一步鉴定 SAP5 的靶蛋白，我们在这项工作中采用了两种独立的方法。我们使用基于二甘氨酰赖氨酸抗体的肽富集进行泛素组捕获，以及与多聚泛素抗体偶联的亲和纯化，结合质谱法，阐明 SAP5 对其靶蛋白的泛素化依赖，以响应渗透胁迫。野生型或 TaSAP5 过表达的拟南芥系，根据我们之前的研究，对渗透胁迫更耐受，在这里被使用。我们鉴定了 HSP90C（叶绿体热休克蛋白 90）作为 TaSAP5 的底物。进一步的生化实验表明，TaSAP5 与 HSP90C 相互作用，并介导其通过 26S 蛋白酶体降解。我们的工作还表明，当在拟南芥中异源表达时，泛素组谱分析是一种有效的方法，用于寻找 TaSAP5 E3 泛素连接酶的底物。
