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.