Ubiquitin ligases (E3s) serve as key regulators for the ubiquitylation-mediated pathway. The identification of the corresponding relationship between E3 and its substrates is challenging but required for understanding the regulatory network of ubiquitylation. The low abundance of ubiquitinated conjugates and high redundancy of E3 substrate regulation made the screening pretty hard. Herein, we combined SILAC-based quantitative proteomics with two contrary genetic methods (overexpression and knockout) in theory for E3 (Hrt3, the F-box subunit of the SCF complex) substrate screening. The knockout method could not overcome the constraint mentioned above, while the overexpression approach turned on the access to the potential substrates of E3. Subsequently, we obtained 77 candidates, which are involved in many critical biological processes and need to be verified in the future. Within these candidates, we confirmed the relationship between one of the candidates Nce103 and Hrt3 and linked Hrt3 with oxygen sensitivity and oxidative stress response in which Nce103 took part as well. This research is also beneficial for understanding the impact of oxygen supply on regulation of yeast growth through the ubiquitination of Nce103.

中文翻译：

---

定量蛋白质组学与两种遗传策略相结合，用于筛选泛素连接酶Hrt3的底物。

泛素连接酶（E3s）作为泛素化介导途径的关键调节剂。鉴定E3与其底物之间的对应关系是具有挑战性的，但是对于理解泛素化的调节网络是必需的。泛素化缀合物的低丰度和E3底物调控的高冗余度使得筛选非常困难。本文中，我们在理论上将基于SILAC的定量蛋白质组学与两种相反的遗传方法（过表达和敲除）相结合，用于E3（Hrt3，SCF复合体的F-box亚基）底物筛选。敲除法不能克服上述限制，而过表达法则开启了进入E3潜在底物的通道。随后，我们获得了77名候选人，其中涉及许多关键的生物过程，并且需要在将来进行验证。在这些候选对象中，我们确认了候选对象Nce103和Hrt3之间的关系，并将Hrt3与Nce103也参与的氧敏感性和氧化应激反应联系起来。这项研究对于了解氧供应通过Nce103泛素化对酵母生长调控的影响也非常有益。