Functional Analysis of RAD23 Protein

DMA修复蛋白Rad 23可以结合多泛素化蛋白和蛋白酶体。生化 研究表明，Rad 23作为一种穿梭因子，可以结合并传递泛素化底物 蛋白酶体，并建议它可能会促进蛋白质降解后的DNA损伤。Rad23 在核苷酸切除修复（NER）中起两种不同的作用。一个功能涉及与 DNA修复蛋白Rad 4。值得注意的是，这种相互作用阻止了Ub/蛋白酶体对Rad 4的降解。 通路第二个功能涉及Rad 23与蛋白酶体的相互作用，但该功能与 Rad 4稳定，虽然它是NER所需的。 有令人信服的证据表明，Rad 23/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， 与蛋白酶体结合由于UbL结构域有其他生理伴侣，这些研究没有 表征突变蛋白的特定缺陷。Rad 23中结合并稳定Rad 4的序列 (R4B)被确认了正如预期的那样，去除该序列阻止了与Rad 4的相互作用，并引起了 紫外线敏感性。令人惊讶的是，R4 B的缺失也消除了Rad 23与多泛素化蛋白的相互作用。 研究提出了UbL和R4 B基序的特点，通过产生明确的突变。这些 研究对于进一步检验Rad 23是穿梭因子的假设是重要的。尽管进行了广泛 在NER蛋白的表征中，Rad 4的功能尚未描述。我们的研究表明， Rad 4可能会影响Rad 23的功能，并提出了研究调查这一想法。 Rad 23和蛋白酶体亚基RpnIO的丢失引起严重的生长和蛋白水解缺陷。 STS 1被分离为这些缺陷的遗传抑制因子。初步研究表明，Sts 1可能 促进蛋白酶体的适当亚细胞分布，并可能特异性调节核- 胞质转运本研究的一个重要目的是确定Sts 1如何抑制缺陷 的外消旋/23 rpnlO突变体。由于Rad 23和蛋白酶体定位于细胞核中以响应DNA 损伤，这些研究可以提高我们对Rad 23在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，在这方面， 新出现的证据表明，泛素化蛋白向蛋白酶体的转运需要 调节因子，如Rad 23。我们发现Rad 23与蛋白酶体结合， 现在已经描述了从酵母到人类的其他UbL-UBA蛋白。我们的研究将促进我们的 了解底物传递到蛋白酶体的机制，以及Rad 23在NER中的功能。

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.