Gamma-secretase complex assembly within the early secretory pathway

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.