γ-secretase complex assembly within the early secretory pathway

Abstract

γ-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 β-secretase-processed β-amyloid precursor protein to liberate the neurotoxic amyloid β-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 γ-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that γ-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 γ-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 γ-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 β-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of β-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/ APH-1/PS) intermediates of γ-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 γ-secretase complex within the ER. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.