Development of the substrate identification method against budding yeast SCF complex using site-specific photocross-linking in vivo

调节蛋白的泛素化和随后的蛋白酶体降解控制着各种细胞过程。E3负责识别和招募多泛素化的靶蛋白。由于技术上的困难，E3和底物之间的关系在很大程度上是未知的。在这项研究中，我们在体内进行定点光交联来鉴定SCF复合体的特定底物。Hard3与Hrd1形成复合体，发挥E3的作用。我们在体内利用定点光交联法确定了Hrd3与Hrd1的特异结合区。然而，在这些过程中，我们认识到，由于缺乏结合区域的信息，这种方法不适合检测新的结合蛋白。然后进行酵母双杂交筛选，确定p50为SCFmet30底物的候选者。SCFmet30以细胞周期依赖的方式调节p50的稳定性。我们目前正在研究这种退化的功能意义。

Ubiquitylation and subsequent proteasomal degradation of regulatory proteins control a variety of cellular processes. The E3s are responsible for recognizing and recruiting target proteins for polyubiquitylation. Relationship between E3s and substrates are largely uncharacterized because of the technical difficulty. In this study, we perform site-specific photocross-linking in vivo to identify specific substrates of SCF complex. Hard3 forms complex with Hrd1 and functions as a E3. We identify the specific binding region of Hrd3 with Hrd1 using site-specific photocross-linking in vivo. However, in these processes, we recognize that this method is not suitable for the detection of novel binding protein, because of the lacking of the binding region information. Then we perform yeast two-hybrid screening and identify p50 as a candidate of SCFmet30 substrate. Stability of p50 is regulated by SCFmet30 in the cell cycle dependent manner. We are currently investigating the functional meaning of this degradation.