Institute of Biotechnology, Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.
BMC Biol. 2009 Nov 12;7:75. doi: 10.1186/1741-7007-7-75.
Ubiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. A classic role for ubiquitin is to flag proteins for destruction by the proteasome. Recent studies indicate that ubiquitin-binding proteins (e.g. Rad23, Dsk2, Rpn10) play a pivotal role in transferring ubiquitylated proteins to the proteasome. However, the specific role of these ubiquitin receptors remains poorly defined. A key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. Although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging.
Using a genome-wide functional screen, we have identified 11 yeast genes that cause slower growth upon their overexpression in cells lacking two ubiquitin-binding proteins Rad23 and Dsk2. Our results suggest that proper functioning of Rad23 and Dsk2 is required for efficient pheromone response, transcription, amino acid metabolism, and DNA damage response. Two proteins identified by the screen are shown to be proteolytic substrates of Dsk2, validating the large scale synthetic dosage lethality screen as a new strategy for identifying substrates of a specific degradation pathway.
In conclusion, as proof-of-concept, we show that a synthetic dosage lethality screen, which is based on the toxicity induced by gene overexpression, offers an effective, complementary method to elucidating biological functions of proteolytic pathways.
泛素通过共价连接到其底物上来调节无数重要的细胞过程。泛素的一个经典作用是标记蛋白质以供蛋白酶体破坏。最近的研究表明，泛素结合蛋白（例如 Rad23、Dsk2、Rpn10）在将泛素化蛋白质转移到蛋白酶体中起着关键作用。然而，这些泛素受体的具体作用仍未得到明确界定。解开这些泛素受体功能的关键是识别它们涉及的细胞底物和生物回路。尽管已经开发了许多用于分离底物的策略，但证明鉴定蛋白水解途径的生理靶标具有挑战性。
使用全基因组功能筛选，我们已经鉴定出 11 个酵母基因，这些基因在缺乏两种泛素结合蛋白 Rad23 和 Dsk2 的细胞中过表达时会导致生长缓慢。我们的结果表明，Rad23 和 Dsk2 的正常功能对于有效的交配反应、转录、氨基酸代谢和 DNA 损伤反应是必需的。通过筛选鉴定出的两种蛋白质被证明是 Dsk2 的蛋白水解底物，这验证了大规模合成剂量致死性筛选作为鉴定特定降解途径底物的新策略的有效性。
总之，作为概念验证，我们表明，基于基因过表达诱导的毒性的合成剂量致死性筛选提供了一种有效的、互补的方法来阐明蛋白水解途径的生物学功能。
