The Nrf1 CNC/bZIP protein is a nuclear envelope-bound transcription factor that is activated by t-butyl hydroquinone but not by endoplasmic reticulum stressors

Abstract

In rat liver RL-34 cells, endogenous Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is localized in the ER (endoplasmic reticulum) 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 NE (nuclear envelope). 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 tBHQ (t-butyl hydroquinone) 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. Furthermore, the NST domain contains seven putative -Asn-Xaa-Ser/Thr- glycosylation sites and, when glycosylation was prevented by replacing all of the seven asparagine residues with either glutamine (Nrf1(1-7xN/Q)) or aspartic acid (Nrf1(1-7xN/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 approximately 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 by its glycosylation status.

The Nrf3 Transcription Factor Is a Membrane-bound Glycoprotein Targeted to the Endoplasmic Reticulum through Its N-terminal Homology Box 1 Sequence

“…Both proteins are glycosylated in the ER and are also located in the nuclear envelope. Interestingly, although Nrf1 is also targeted to the ER and located in the nuclear envelope (32), our data suggest that Nrf3 is integrated into membranes in a more complete fashion. Proteinase protection assays have shown that the topology of Nrf3 within the membrane is modulated by NHB2 within the NTD.…”

Transcription Factor Nrf1 Negatively Regulates the Cystine/Glutamate Transporter and Lipid-Metabolizing Enzymes

“…Antiserum raised in rabbits against recombinant mouse Nrf1 protein (i.e., mNrf1␤, residues 293 to 742) has been described previously (25). However, in the present study, this serum was immunopurified prior to use.…”

The Nrf1 CNC-bZIP Protein Is Regulated by the Proteasome and Activated by Hypoxia

“…Nrf1 localizes primarily to the ER (see Figure 1) as well as the nuclear envelope membrane [11]. The ER membrane-resident form of Nrf1 represents a low activity, glycosylated protein with an apparent molecular weight of 120 kDa (p120), while the nuclear form (p95) is an active, non-glycosylated (or deglycosylated) protein [15, see also Figure 1].…”

“…The ER membrane-resident form of Nrf1 represents a low activity, glycosylated protein with an apparent molecular weight of 120 kDa (p120), while the nuclear form (p95) is an active, non-glycosylated (or deglycosylated) protein [15, see also Figure 1]. The ER location of Nrf1 is thought to be suitable for the maintenance of the ER redox homeostasis [11], perhaps by affecting the ER membrane lipid organization via its amphipathic, transmembrane α-helices and participating in membrane-dependent biological events [16]. It has also been proposed that the localization of Nrf1 within the ER determines the activity of this CNC factor and that the ER redox status and Nrf1 glycosylation status could cause Nrf1 to relocate from the ER to the nucleus [11].…”