Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
Biochem Biophys Res Commun. 2012 Mar 9;419(2):436-40. doi: 10.1016/j.bbrc.2012.02.044. Epub 2012 Feb 14.
Stress associated proteins (SAPs) in plants contain A20-type zinc finger (A20_ZF) domains and are involved with abiotic stress response. A20-type zinc finger domains in animals reportedly recognize ubiquitin as a regulatory signal in cell. However, it remains unclear whether A20_ZF domains in plants perform similar roles. AtSAP5, a SAP from Arabidopsis thaliana, exhibits a unique sequence feature among 10 AtSAPs harboring A20_ZF domains. The highly conserved diaromatic patch is replaced by the dialipathic patch. Here we investigated whether AtSAP5 recognizes ubiquitin and the roles of the dialipathic patch in ubiquitin binding in vitro. GST pulldown assay reveals that AtSAP5 binds polyubiquitin rather than monoubiquitin. AtSAP5 shows preferences for linear and K63-linked polyubiquitin chains to K48-linked one. The A20_ZF domain of AtSAP5 is sufficient for linkage-specific polyubiquitin recognition. The dialipathic patch in AtSAP5 plays an important role in K48-linked polyubiquitin recognition. Taken together, our results suggest that AtSAP5 participates in polyubiquitin recognition in plants and that the dialipathic patch in AtSAP5 is critical in binding K48-linked polyubiquitn chains.
植物中的应激相关蛋白（SAPs）含有 A20 型锌指（A20_ZF）结构域，参与非生物胁迫响应。据报道，动物中的 A20 型锌指结构域可以识别泛素作为细胞中的调节信号。然而，植物中的 A20_ZF 结构域是否发挥类似作用尚不清楚。AtSAP5 是拟南芥中的一种 SAP，在含有 A20_ZF 结构域的 10 种 AtSAP 中表现出独特的序列特征。高度保守的二芳香性补丁被二脂性补丁所取代。在这里，我们研究了 AtSAP5 是否识别泛素以及在体外泛素结合中dialipathic 补丁的作用。GST 下拉测定表明，AtSAP5 结合多泛素而不是单泛素。AtSAP5 对线性和 K63 连接的多泛素链表现出偏好，而不是 K48 连接的多泛素链。AtSAP5 的 A20_ZF 结构域足以特异性识别连接的多泛素。AtSAP5 中的 dialipathic 补丁在 K48 连接的多泛素识别中起着重要作用。总之，我们的研究结果表明，AtSAP5 参与植物中多泛素的识别，并且 AtSAP5 中的 dialipathic 补丁在结合 K48 连接的多泛素链中是至关重要的。
