The transmembrane domain region of nicastrin mediates direct interactions with APH-1 and the γ-secretase complex

Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the gamma-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other gamma-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the gamma-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfide-bonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human gamma-secretase components, all of the ceNCT/hNCT chimeras interacted with gamma-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the gamma-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.