Functional Analysis of RAD23 Protein

DNA修复蛋白RAD23可以与多种泛素化蛋白和蛋白酶体结合。生化 研究表明，Rad23是一种穿梭因子，可以结合和传递泛素化的底物 到蛋白酶体，并表明它可能促进DNA损伤后的蛋白质降解。RAD23 在核苷酸切除-修复(NER)中扮演两个不同的角色。其中一个功能涉及与 DNA修复蛋白RAD4。值得注意的是，这种相互作用阻止了Ub/蛋白酶体对Rad4的降解 路径。第二个功能涉及RAD23与蛋白酶体的相互作用，但该功能并不相关 到Rad4-稳定，尽管它是NER所必需的。 有令人信服的证据表明，RAD23/蛋白酶体相互作用是其所有不同功能所必需的， 包括(NER)。然而，以前的研究是基于缺乏Ubl结构域的缺失突变体， 它结合了蛋白酶体。由于UBL域有其他生理伙伴，这些研究没有 确定突变蛋白的特定缺陷。RAD23中结合和稳定RAD4的序列 (R4B)。正如预期的那样，该序列的移除阻止了与Rad4的相互作用，并导致 紫外线敏感度。令人惊讶的是，R4B的缺失也取消了RAD23与多泛素化蛋白的相互作用。 有人建议通过产生定义明确的突变来表征UBL和R4B基序。这些 研究对于进一步验证RAD23是穿梭因子的假设将是重要的。尽管广泛 NER蛋白的特征，Rad4的功能尚未描述。我们的研究表明 RAD4可能会影响RAD23的功能，有人建议对此进行研究。 RAD23和蛋白酶体亚基RpnIO的缺失导致严重的生长和蛋白降解缺陷。 STS1作为这些缺陷的基因抑制因子被分离出来。初步研究表明，Sts1可能 促进蛋白酶体的适当亚细胞分布，并可能特异性地调节核- 细胞质运输。这项研究的一个重要目标是确定STS1是如何抑制缺陷的 Rac/23rpnlO突变体。由于RAD23和蛋白酶体对DNA的反应定位于细胞核 这些研究可以提高我们对RAD23在蛋白质降解和蛋白质降解中的功能的理解 DNA修复。 多泛素链被认为足以将底物靶向蛋白酶体。然而， 新出现的证据表明，泛素化蛋白向蛋白酶体的转位需要 监管因素，如RAD23。我们发现RAD23与蛋白酶体结合，类似的发现 现在已经描述了从酵母到人类的其他UBL-UBA蛋白。我们的学习将促进我们的 了解底物递送到蛋白酶体的机制，以及RAD23在NER中的作用。

The DMArepair protein Rad23 can bind multiubiquitinated proteins and the proteasome. Biochemical studies suggested that Rad23functions as a shuttle-factor that can bind and deliver ubiquitinated substrates to the proteasome, and suggested that it might promote protein degradation following DNA damage. Rad23 performs two distinct roles in nucleotide excision-repair (NER). One function involves interaction with the DNA repair protein Rad4. Remarkably,this interaction prevents Rad4 degradation by the Ub/proteasome pathway. A second function involves Rad23 interaction with the proteasome, but this function is not related to Rad4-stabilization, although it is required for NER. There is compelling evidence that Rad23/proteasome interaction is required for all its diverse functions, including (NER). However, previous studies were based on a deletion mutant that lacked the UbL domain, which binds the proteasome. Since the UbL domain has other physiological partners, these studies did not characterize the specific defect of the mutant protein. A sequence in Rad23 that binds and stabilizes Rad4 (R4B) was identified. As expected, removal of this sequence prevented interaction with Rad4, and caused UV sensitivity. Surprisingly, loss of R4B also abolished Rad23 interaction with multiubiquitinated proteins. Studies are proposed to characterize the UbL and R4B motifs, by generating well-defined mutations. These studies will be important for further testing the hypothesis that Rad23 is a shuttle-factor. Despite extensive characterization of NER proteins, a function for Rad4 has not been described. Our studies suggest that Rad4 might influence Rad23 function, and studies are proposed to investigate this idea. The loss of Rad23 and the proteasome subunit RpnIO caused severe growth and proteolytic defects. STS1 was isolated as genetic suppressor of these defects. Preliminary studies indicate that Sts1 might facilitate the proper sub-cellular distribution of proteasomes, and might specifically regulate nucleo- cytoplasmic transport. An important objective of this study is to determine how Sts1 suppressed the defects of the rac/23 rpnlO mutant. Since Rad23 and proteasomes are localized to the nucleus in response to DNA damage, these studies can improve our understanding of the function of Rad23 in protein degradation and DNA repair. Multiubiquitin chains were believed to be sufficient for targeting substrates to the proteasome. However, emerging evidence suggest that the translocation of ubiquitinated proteins to the proteasome requires regulatory factors, such as Rad23. We discovered that Rad23 binds the proteasome, and similar findings have now been described for other UbL-UBA proteins, from yeast to human. Our studies will advance our understanding of the mechanism of substrate delivery to the proteasome, and Rad23 function in NER.