Identification of topological determinants in the N-terminal domain of transcription factor Nrf1 that control its orientation in the endoplasmic reticulum membrane

Nrf1 [NF-E2 (nuclear factor-erythroid 2)-related factor 1] is a CNC (cap'n'collar) bZIP (basic-region leucine zipper) transcription factor that is tethered to ER (endoplasmic reticulum) and nuclear envelope membranes through its N-terminal signal peptide (residues 1-30). Besides the signal peptide, amino acids 31-90 of Nrf1 also negatively regulate the CNC-bZIP factor. In the present study we have tested the hypothesis that amino acids 31-90 of Nrf1, and the overlapping NHB2 (N-terminal homology box 2; residues 82-106), inhibit Nrf1 because they control its topology within membranes. This region contains three amphipathic alpha-helical regions comprising amino acids 31-50 [called the SAS (signal peptide-associated sequence)], 55-82 [called the CRACs (cholesterol-recognition amino acid consensus sequences)] and 89-106 (part of NHB2). We present experimental data showing that the signal peptide of Nrf1 contains a TM1 (transmembrane 1) region (residues 7-24) that is orientated across the ER membrane in an N-cyt/C-lum fashion with its N-terminus facing the cytoplasm and its C-terminus positioned in the lumen of the ER. Once Nrf1 is anchored to the ER membrane through TM1, the remaining portion of the N-terminal domain (NTD, residues 1-124) is transiently translocated into the ER lumen. Thereafter, Nrf1 adopts a topology in which the SAS is inserted into the membrane, the CRACs are probably repartitioned to the cytoplasmic side of the ER membrane, and NHB2 may serve as an anchor switch, either lying on the luminal surface of the ER or traversing the membrane with an N-cyt/C-lum orientation. Thus Nrf1 can adopt several topologies within membranes that are determined by its NTD.