Aph-1 contributes to the stabilization and trafficking of the γ-secretase complex through mechanisms involving intermolecular and intramolecular interactions

γ-Secretase cleaves type I transmembrane proteins, including β-amyloid precursor protein and Notch, and requires the formation of a protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2 for its activity. Aph-1 is implicated in the stabilization of this complex, although its precise mechanistic role remains unknown. Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function phenotype in Caenorhabditis elegans. Here, using an untranslated region-targeted RNA interference/rescue strategy in Drosophila Schneider 2 cells, we show that Aph-1 contributes to the assembly of the γ-secretase complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or independent of the conserved glycines. Aph-1 binds to nicastrin forming an early subcomplex independent of the conserved glycines within the endoplasmic reticulum. Certain mutations in the conserved GXXXG motif affect the interaction of the Aph-1-nicastrin subcomplex with presenilin that mediates trafficking of the presenilin-Aph-1-nicastrin tripartite complex to the Golgi. The same mutations decrease the stability of Aph-1 polypeptides themselves, possibly by affecting intramolecular associations through the transmembrane domains. Our data suggest that the proper assembly of the Aph-1-nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the γ-secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex.