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

Gamma-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 beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-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 gamma-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 gamma-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.