Cell-free generation of the notch1 intracellular domain (NICD) and APP-CTfgamma: evidence for distinct intramembranous "gamma-secretase" activities.

PSEN1 and PSEN2 encode polytopic membrane proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2) that play an essential role in intramembranous ("gamma-secretase") proteolysis of selected type I membrane proteins, that include Notch1 and beta-amyloid precursor protein (APP). In order to gain insights into biochemical mechanisms underlying gamma-secretase processing of Notch1 and APP, we have developed a novel in vitro assay in which gamma-secretase-mediated generation of S3/NICD and APP-CTFgamma can be readily detected in isolated membrane fractions derived from immortalized PS1+/- mouse embryonic fibroblasts; production of the APP and Notch1 derivatives are inhibited by a highly selective and potent gamma-secretase inhibitor, L-685,458, with a IC50 of approximately 50 pM. In membranes prepared from PS1-deficient fibroblasts, we detected APP-CTFgamma, albeit at low levels. Unexpectedly, and despite the presence of endogenous PS2 in membranes prepared from PS1-deficient fibroblasts, production of the Notch derivatives, S3/NICD, was nearly undetectable in these reactions. Moreover, S3/NICD production is neither detected in detergent-solubilized membrane preparations from PS1-deficient cells, nor in reactions containing PS1-containing membranes that were co-solubilized with membranes from PS -/- cells expressing a chimeric Notch 1 species. These findings strongly suggest that the factors responsible for intramembranous, gamma-secretase proteolysis of APP and Notch1 are neither equivalent, nor exchangeable.