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.
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.
Zhang
Yiguo
Y
Biomedical Research Center, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK. y.z.zhang@dundee.ac.uk
Lucocq
John M
JM
Yamamoto
Masayuki
M
Hayes
John D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D051266:Nuclear Respiratory Factor 1 / Q000737:chemistry / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D008562:Membrane Glycoproteins; D051266:Nuclear Respiratory Factor 1; D010446:Peptide Fragments; D001216:Asparagine
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