A Conserved GXXXG Motif in APH-1 Is Critical for Assembly and Activity of the γ-Secretase Complex

The multipass membrane protein APH-1, found in the γ-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and β-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly¹²³ in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1a^L (G122D) disrupts the physical interaction of APH-1a^L with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced γ-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly¹²², Gly¹²⁶, and Gly ¹³⁰ in the fourth transmembrane region of mammalian APH-1a ^L are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1a^L with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaf-folding role in both the initial assembly and subsequent maturation and maintenance of the active γ-secretase complex.