The C-terminal PAL motif and transmembrane domain 9 of presenilin 1 are involved in the formation of the catalytic pore of the γ-secretase

γ-Secretase is an unusual membrane-embedded protease, which cleaves the transmembrane domains (TMDs) of type I membrane proteins, including amyloid-β precursor protein and Notch receptor. We have previously shown the existence of a hydrophilic pore formed by TMD6 and TMD7 of presenilin 1 (PS1), the catalytic subunit of γ-secretase, within the membrane by the substituted cysteine accessibility method. Here we analyzed the structure of TMD8, TMD9, and the C terminus of PS1, which encompass the conserved PAL motif and the hydrophobic C-terminal tip, both being critical for the catalytic activity and the formation of the γ-secretase complex. We found that the amino acid residues around the PAL motif and the extracellular/luminal portion of TMD9 are highly water accessible and located in proximity to the catalytic pore. Furthermore, the region starting from the luminal end of TMD9 toward the C terminus forms an amphipathic α-helix-like structure that extends along the interface between the membrane and the extracellular milieu. Competition analysis using γ-secretase inhibitors revealed that the TMD9 is involved in the initial binding of substrates, as well as in the subsequent catalytic process as a subsite. Our results provide mechanistic insights into the role of TMD9 in the formation of the catalytic pore and the substrate entry, crucial to the unusual mode of intramembrane proteolysis by γ-secretase.