The NHB1 (N-terminal homology box 1) sequence in transcription factor Nrf1 is required to anchor it to the endoplasmic reticulum and also to enable its asparagine-glycosylation

Yiguo Zhang, John M. Lucocq, Masayuki Yamamoto, John D. Hayes

Research output: Contribution to journalArticlepeer-review

85 Citations (Scopus)

Abstract

NrfI(nuclear factor-erythroid 2 p45 subunit-related factor 1) is negatively controlled by its NTD (N-terminal domain) that lies between amino acids 1 and 124. This domain contains a leucine-rich sequence, called NHB1 (N-terminal homology box 1; residues 11-30), which tethers NrfI to the ER (endoplasmic reticulum). Electrophoresis resolved NrfI into two major bands of approx. 95 and 120 kDa. The 120-kDa NrfI form represents a glycosylated protein that was present exclusively in the ER and was converted into a substantially smaller polypeptide upon digestion with either peptide:N-glycosidase For endoglycosidase H. By contrast, the 95-kDa NrfI form did not appear to be glycosylated and was present primarily in the nucleus. NHB1 and its adjacent residues conform to the classic tripartite signal peptide sequence, comprising n-, h- and c-regions. The h-region (residues 11-22), but neither the n-region (residues 1-10) nor the c-region (residues 23-30), is required to direct NrfI to the ER. Targeting NrfI to the ER is necessary to generate the 120-kDa glycosylated protein. The n-region and c-region are required for correct membrane orientation of NrfI, as deletion of residues 2-10 or 23-30 greatly increased its association with the ER and the extent to which it was glycosylated. The NHB1 does not contain a signal peptidase cleavage site, indicating that it serves as an ER anchor sequence. Wild-type NrfI is glycosylated through its Asn/Ser/Thr-rich domain, between amino acids 296 and 403, and this modification was not observed in an NrfI(Delta 299-400) mutant. Glycosylation of NrfI was not necessary to retain it in the ER.

Original languageEnglish
Pages (from-to)161-172
Number of pages12
JournalBiochemical Journal
Volume408
Issue number2
DOIs
Publication statusPublished - 1 Dec 2007

Keywords

  • Amino Acid Sequence
  • Animals
  • Asparagine
  • COS Cells
  • Cercopithecus aethiops
  • Endoplasmic Reticulum
  • Glycosylation
  • Membrane Glycoproteins
  • Mice
  • Molecular Sequence Data
  • Nuclear Respiratory Factor 1
  • Peptide Fragments
  • Protein Binding
  • Sequence Homology, Amino Acid

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