Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in <i>Saccharomyces cerevisiae</i>

Abstract

During Nucleotide Excision Repair (NER) in the yeast S. cerevisiae, ubiquitylation of Rad4 is carried out by the E3 ubiquitin ligase that includes Rad7-Elc1-Cul3 and is critical to optimal NER. Rad7 E3 activity targets Rad4 for degradation by the proteaseome but, in principle, could also trigger other DNA damage responses. We observed increased nuclear ubiquitin foci (Ub-RFP) formation in S. cerevisiae containing a Rad7 E3 ligase mutant (rad7SOCS) in response to DNA damage by benzo[a]pyrenediolepoxide (BPDE) in dividing cells. Immunoblots reveal that ubiquitin conjugates of Rpn10 and Dsk2 accumulate in greater abundance in rad7SOCS compared to RAD7 in dividing cells in response to BPDE which makes Rpn10 and Dsk2 candidates for being the ubiquitylated species observed in our microscopy experiments. Microscopy analysis with strains containing Dsk2-GFP shows that Dsk2-GFP and Dsk2-GFP/Ub-RFP colocalized in nuclear foci form to an increased extent in a rad7SOCS mutant background in dividing cells than in a RAD7 wild-type strain. Further, Dsk2-GFP in the rad7SOCS strain formed more foci at the plasma membrane following BPDE treatment in dividing cells relative to strains containing RAD7 or a rad7Δ deletion mutant. In response to a different agent, UV irradiation, levels of ubiquitylated proteins were increased in rad7SOCS relative to RAD7, and the proteasomal deubiquitylase subunit, Rpn11 was even monoubiquitylated in the absence of damaging agents. Together these data show that Rad7 E3 activity attenuates ubiquitylation of proteins regulating the shuttling of polyubiquitylated proteins to the proteasome (Dsk2 and Rpn10) and removal of ubiquitin chains just prior to degradation (Rpn11). Since Rad7 E3 ligase activity has been shown to increase ubiquitylation of its target proteins, yet our results show increased ubiquitylation in the absence of Rad7 E3, we suggest that Rad7 E3 action regulates ubiquitin ligase and deubiquitylase (DUB) activities that act on Rpn10, Dsk2 and Rpn11.

Integrated Multi-Omics Analysis of Mechanisms Underlying Yeast Ethanol Tolerance

“… 72 On the other hand, Dsk2 overexpression appears to be toxic for cells, causing not only accumulation of ubiquitylated substrates but also cell cycle arrest. By ubiquitylating Dsk2 and proteasome subunit Rpn10, both of which are involved in degradation of cyclins, 70 Rad7 ultimately regulates spindle pole body proteins to fine-tune cell cycle checkpoint activation due to DNA damage. Rendering Dsk2 inactive by the introduction of a STOP codon, observed here in the ethanol-adapted strains, could therefore interfere with this checkpoint.…”

“…Dsk2 is involved in the ubiquitin-proteasome proteolytic pathway through recruiting K48-ubiquitylated proteins for degradation and was previously reported to become essential under salt stress . Its deletion was found to have little effect on protein degradation as Dsk2 is partially functionally redundant with Rad23 .…”