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In rat liver RL-34 cells, endogenous Nrf1 (NF-E2-related factor 1) is localized in the endoplasmic reticulum (ER) where it exists as a glycosylated protein. Electron microscopy has demonstrated that ectopic Nrf1 in COS-1 cells is located in the ER and the nuclear envelope (NE). Subcellular fractionation, together with a membrane proteinase protection assay, revealed that Nrf1 is an integral membrane protein with both luminal and cytoplasmic domains. The N-terminal 65 residues of Nrf1 direct its integration into the ER and NE membranes and tether it to a Triton X-100-resistant membrane microdomain that is associated with lipid rafts. The activity of Nrf1 was increased by the electrophile tert-butyl hydroquinone (tBHQ) probably through an N-terminal domain-dependent process. We found that the NST (Asn/Ser/Thr-rich) domain, along with AD1 (acidic domain 1), contributes positively to the transactivation activity of full-length Nrf1. Further, the NST domain contains seven putative −Asn−X−Ser/Thr− glycosylation sites and when glycation was prevented by substituting all of the seven Asn to either Gln (Nrf1^1-7×N/Q) or Asp (Nrf1^1-7×N/D), the former multiple point mutant possessed less activity than the wild-type factor whereas the latter mutant exhibited substantially greater activity. Lastly, the ER stressors tunicamycin, thapsigargin and brefeldin A were found to inhibit basal Nrf1 activity by ~25%, and almost completely prevented induction of Nrf1-mediated transactivation by tBHQ. Collectively, these results suggest that the activity of Nrf1 critically depends on its topology within the ER, and that this is modulated by redox stressors, as well as its glycosylation status.

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doi:10.1042/BJ20081575 Received 4 August 2008/5 November 2008; Accepted 6 November 2008 Published as Immediate Publication 6 November 2008