The Transmembrane Domain Region of Nicastrin Mediates Direct Interactions with APH-1 and the γ-Secretase Complex

• Published in: The Journal of biological chemistry 2003-10, Vol.278 (44), p.43284-43291
• Main Authors: Morais, Vanessa A., Crystal, Adam S., Pijak, Donald S., Carlin, Dan, Costa, Júlia, Lee, Virginia M.-Y., Doms, Robert W.
• Format: Article
• Language: English
• Subjects: Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Line; DNA, Complementary - metabolism; Endopeptidases - chemistry; Endopeptidases - metabolism; HeLa Cells; Homeodomain Proteins - chemistry; Homeodomain Proteins - metabolism; Humans; Immunoblotting; Lipid Bilayers - metabolism; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Microscopy, Confocal; Microscopy, Fluorescence; Precipitin Tests; Protein Binding; Protein Folding; Protein Structure, Tertiary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305685200

Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the γ-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other γ-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 γ-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 γ-secretase components, all of the ceNCT/hNCT chimeras interacted with γ-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 γ-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.