Abstract
The membrane-bound Nrf1 transcription factor regulates critical homeostatic and developmental genes. The conserved N-terminal homology box 1 (NHB1) sequence in Nrf1 targets the cap‘n’collar (CNC) basic basic-region leucine zipper (bZIP) factor to the endoplasmic reticulum (ER), but it is unknown how its activity is controlled topologically within membranes. Herein, we report a hitherto unknown mechanism by which the transactivation activity of Nrf1 is controlled through its membrane-topology. Thus after Nrf1 is anchored within ER membranes, its acidic transactivation domains (TADs), including the Asn/Ser/Thr-rich (NST) glycodomain situated between acidic domain 1 (AD1) and AD2, are transiently translocated into the lumen of the ER, where NST is glycosylated in the presence of glucose to yield an inactive 120-kDa Nrf1 glycoprotein. Subsequently, portions of the TADs partially repartition across membranes into the cyto/nucleoplasmic compartments, whereupon an active 95-kDa form of Nrf1 accumulates, a process that is more obvious in glucose-deprived cells and may involve deglycosylation. The repartitioning of Nrf1 out of membranes is monitored within this protein by its acidic-hydrophobic amphipathic glucose-responsive domains, particularly the Neh5L subdomain within AD1. Therefore, the membrane-topological organization of Nrf1 dictates its post-translational modifications (i.e. glycosylation, the putative deglycosylation and selective proteolysis), which together control its ability to transactivate target genes.
Highlights
Defining the molecular details of membrane protein biogenesis is essential if we are to understand the functions of integral membrane proteins in the cell
The two prototypic membranebound transcription factors ATF6 and SREBP1 are trafficked from the endoplasmic reticulum (ER) into the Golgi apparatus, whereupon both are proteolytically processed through regulated intramembrane proteolysis (RIP) allowing them to be consecutively cleaved by Site-1 and Site-2 proteases [2], in order to allow their active N-terminal portions to be released from membranes prior to nuclear translocation [3,4]
Movement of certain regions of Nrf1 across membranes determines the extent to which its NST domain is glycosylated in the ER lumen and deglycosylated in the extra-luminal cyto/nucleoplasmic compartments, and the extent to which the Nterminal homology box 1 (NHB1)-CNC factor can interact with ARE sequences in target genes and recruit the general transcriptional machinery
Summary
Defining the molecular details of membrane protein biogenesis is essential if we are to understand the functions of integral membrane proteins in the cell. The two prototypic membranebound transcription factors ATF6 and SREBP1 are trafficked from the ER into the Golgi apparatus, whereupon both are proteolytically processed through regulated intramembrane proteolysis (RIP) allowing them to be consecutively cleaved by Site-1 and Site-2 proteases [2], in order to allow their active N-terminal portions to be released from membranes prior to nuclear translocation [3,4]. Other membrane-bound transcription factors, such as certain cap‘n’collar (CNC)-basic basic-region leucine zipper (bZIP) family members [5,6,7], are not processed via RIP and it is unclear how their activity is regulated. We describe how the topology of a membrane-bound CNC transcription factor controls its activity
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