Figure 3. Post-translational processing of Nrf1 into longer isoforms in response to changes in glucose concentration.

(a) Glycosylation and deglycosylation of Nrf1 in response to glucose supply or glucose starvation. Following transfection with the wild-type Nrf1 expression construct, a group of COS-1 cells (left 6 lanes) was allowed to starve in 1 mM-glucose medium for 16 h, before they were transferred to fresh 25 mM-glucose medium (i.e. glucose supply). Another group of the cells (right 6 lanes) was left to recover for 16 h in 25 mM-glucose medium, before transfer to glucose-free medium (i.e. glucose deprivation). Thereafter, these two groups of cells were disrupted at the indicated times after glucose addition or glucose deprivation, and then examined by western blotting. The left upper panels show two cropped images from the film that was exposed to the immunoblot for different shorter times to eliminate intense bands. The right graph shows the relative abundance of the 120-, 95- and 85-kDa proteins (mean ± S.D, n = 4) that were calculated after normalization to [A]₀ of 120-kDa at t₀ after transfer 25 mM-glucose medium to the starved cells that had been grown in glucose-free medium. (b,c) The vectorial process by which Nrf1 is post-translationally modified was determined by addition to the 1 mM-glucose starved cells of 25 mM-glucose in the presence of DMSO vehicle, TU alone, or TU plus MG132. After transfection, Nrf1-expressing cells were starved in 1 mM- glucose medium for 16 h before transfer to 25 mM-glucose media containing DMSO, TU (1 μg/ml), or plus MG132 (5 μM) for the indicated times. The relative abundance of the 120-, 95- and 85-kDa Nrf1 proteins was determined by western blotting and calculated as described above (right, mean ± S.D, n = 4).