Quantitative Proteomics Combined with Two Genetic Strategies for Screening Substrates of Ubiquitin Ligase Hrt3.

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.