Immature nicastrin stabilizes APH‐1 independent of PEN‐2 and presenilin: identification of nicastrin mutants that selectively interact with APH‐1

γ‐Secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx‐defective‐1 (APH‐1) and presenilin enhancer‐2 (PEN‐2). The complex mediates the intramembraneous proteolysis of β‐secretase cleaved β‐amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease‐associated amyloid β‐peptide (Aβ). In order to dissect functionally important domains of Nct required for γ‐secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH‐1 and PEN‐2 expression as well as by monitoring the accumulation of the APP C‐terminal fragments, the immediate substrates of γ‐secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH‐1 but not of PEN‐2 or PS and thus failed to restore γ‐secretase activity. Interestingly, these immature Nct variants selectively bound to APH‐1, suggesting a stable Nct/APH−1 interaction independent of PS and PEN‐2. Consistent with this finding, expression of APH‐1 remained largely unaffected in the PS double knock‐out and immature Nct co‐immunoprecipitated with APH‐1 in the absence of PS and PEN‐2. Taken together, our findings suggest that immature Nct can stably interact with APH‐1 to form a potential scaffold for binding of PS and PEN‐2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.