The multipass membrane protein, APH-1, found in the gamma-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 beta-amyloid precursor protein in mammalian or Drosophila cells. In Caenorhabditis elegans, a mutation of the conserved transmembrane glycine residue 123 in APH-1 (
or28) leads to a notch/glp-1 loss-of-function phenotype. In the present study, we show that the corresponding mutation in mammalian APH-1aL, glycine residue 122 to aspartate (G122D), disrupts the physical interaction of APH-1aL with immature hypoglycosylated nicastrin and presenilin holoprotein, as well as mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduces gamma-secretase activity in intramembrane proteolysis of the membrane-tethered Notch. Moreover, we find that the conserved transmembrane glycine residues 122, 126, and 130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.