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

Biochem J. 2009 Mar 1;418(2):293-310. doi: 10.1042/BJ20081575.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Basic-Leucine Zipper Transcription Factors / metabolism
  • Basic-Leucine Zipper Transcription Factors / physiology
  • Brefeldin A / pharmacology
  • COS Cells
  • Cells, Cultured
  • Chlorocebus aethiops
  • Endoplasmic Reticulum / drug effects*
  • Endoplasmic Reticulum / metabolism
  • Glycosylation
  • Hydroquinones / pharmacology*
  • NF-E2-Related Factor 1 / metabolism
  • NF-E2-Related Factor 1 / physiology*
  • Nuclear Envelope / metabolism*
  • Oxidation-Reduction / drug effects
  • Protein Synthesis Inhibitors / pharmacology
  • Rats
  • Stress, Physiological / drug effects
  • Stress, Physiological / physiology*
  • Thapsigargin / pharmacology
  • Transcription Factors / metabolism
  • Transcription Factors / physiology
  • Transcriptional Activation / drug effects*
  • Tunicamycin / pharmacology

Substances

  • Basic-Leucine Zipper Transcription Factors
  • Hydroquinones
  • NF-E2-Related Factor 1
  • Protein Synthesis Inhibitors
  • Transcription Factors
  • Tunicamycin
  • Brefeldin A
  • Thapsigargin
  • 2-tert-butylhydroquinone