LAUSR

&NA; The topobiological behaviour of Nrf1 dictates its post‐translational modification and its ability to transactivate target genes. Here, we have elucidated that topovectorial mechanisms control the juxtamembrane processing of Nrf1 on the cyto/nucleoplasmic side of endoplasmic reticulum (ER), whereupon it is cleaved and degraded to remove various lengths of its N‐terminal domain (NTD, also refolded into a UBL module) and acidic domain‐1 (AD1) to yield multiple isoforms. Notably, an N‐terminal ˜12.5‐kDa polypeptide of Nrf1 arises from selective cleavage at an NHB2‐adjoining region within NTD, whilst other longer UBL‐containing isoforms may arise from proteolytic processing of the protein within AD1 around PEST1 and Neh2L degrons. The susceptibility of Nrf1 to proteolysis is determined by dynamic repositioning of potential UBL‐adjacent degrons and cleavage sites from the ER lumen through p97‐driven retrotranslocation and ‐independent pathways into the cyto/nucleoplasm. These repositioned degrons and cleavage sites within NTD and AD1 of Nrf1 are coming into their bona fide functionality, thereby enabling it to be selectively processed by cytosolic DDI‐1/2 proteases and also partiality degraded via 26S proteasomes. The resultant proteolytic processing of Nrf1 gives rise to a mature ˜85‐kDa CNC‐bZIP transcription factor, which regulates transcriptional expression of cognate target genes. Furthermore, putative ubiquitination of Nrf1 is not a prerequisite necessary for involvement of p97 in the client processing. Overall, the regulated juxtamembrane proteolysis (RJP) of Nrf1, though occurring in close proximity to the ER, is distinctive from the mechanism that regulates the intramembrane proteolytic (RIP) processing of ATF6 and SREBP1. Graphical abstract Figure. No caption available. HighlightsThe multistep processing of Nrf1 is dictated by its topovectorial mechanisms.The regulated juxtamembrane proteolysis of Nrf1 occurs in close proximity to the ER.Removal of distinct N‐terminal peptides from Nrf1 gives rise to a mature isoform.Cytosolic proteases are required for the selective juxtamembrane proteolysis of Nrf1.