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

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    Abstract

    Nrf1 (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 Nrf1 to the ER (endoplasmic reticulum). Electrophoresis resolved Nrf1 into two major bands of approx. 95 and 120 kDa. The 120-kDa Nrf1 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 F or endoglycosidase H. By contrast, the 95-kDa Nrf1 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 Nrf1 to the ER. Targeting Nrf1 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 Nrf1, 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 Nrf1 is glycosylated through its Asn/Ser/Thr-rich domain, between amino acids 296 and 403, and this modification was not observed in an Nrf1(Delta299-400) mutant. Glycosylation of Nrf1 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 - 2007

    Keywords

    • endoplasmic reticulum
    • glycosylation
    • non-alcoholic steatohepatitis (NASH)
    • nuclear factor-erythroid 2-p45 subunit-related factor (Nrf)
    • oxidative stress
    • signal anchor sequence
    • REGULATED INTRAMEMBRANE PROTEOLYSIS
    • ANTIOXIDANT RESPONSE ELEMENT
    • GENE-EXPRESSION
    • MEMBRANE-PROTEINS
    • INDUCIBLE EXPRESSION
    • EMBRYONIC LETHALITY
    • NEGATIVE REGULATION
    • SIGNAL SEQUENCES
    • ALPHA-COP
    • LIVER

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