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
Résumé
Nrf1 (nuclear factor-erythroid 2-p45 subunit-related factor 1) is negatively controlled by its N-terminal domain (NTD) that lies between amino acids 1-124. This domain contains a Leu-rich sequence, called N-terminal homology box 1 (NHB1, residues 11-30), which tethers Nrf1 to the endoplasmic reticulum (ER). Electrophoresis resolved Nrf1 into two major bands of approximately 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 this organelle 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 296-403, and this modification was not observed in an Nrf1 Δ299-400} mutant. Glycosylation of Nrf1 was not necessary to retain it in the ER.
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