ER-trafficking triggers NRF1 ubiquitination to promote its proteolytic activation.

The transcription factor NRF1 resides in the endoplasmic reticulum (ER) and is constantly transported to the cytosol for proteasomal degradation. However, when the proteasome is defective, NRF1 escapes degradation and undergoes proteolytic cleavage by the protease DDI2, generating a transcriptionally active form that restores proteostasis, including proteasome function. The mechanisms that regulate NRF1 proteolytic activation and transcriptional potential remain poorly understood. This study demonstrates that the ER is a crucial regulator of NRF1 function by orchestrating its ubiquitination through the E3 ubiquitin ligase HRD1. We show that HRD1-mediated NRF1 ubiquitination is necessary for DDI2-mediated processing in cells. Furthermore, we found that deficiency in both RAD23A and RAD23B impaired DDI2-mediated NRF1 processing, indicating that these genes are essential components of the DDI2 proteolytic machinery. Our findings highlight the intricate mechanism by which the ER activates NRF1 to coordinate the transcriptional activity of an adaptation response in cells.