gamma-Secretase is composed of at least four transmembrane proteins, presenilin (PS) 1/2, nicastrin, anterior pharynx-1 (Aph-1) and presenilin enhancer-2 (Pen-2), and cleaves amyloid precursor protein (APP) to produce amyloid beta peptides (A beta) that is deposited in the brains of Alzheimer disease, However, the mechanism of gamma-secretase-mediated cleavage remains Unclear. To examine the enzymatic properties of gamma-secretase, we established all in vitro assay system Using Saccharomyces cerevisiae, which does not possess homologs of human Ps1/2, nicastrin, Aph-1, or Pen-2. We transformed these Subunits and [tie substrate in pep4 Delta cells with vacuole proteases inactivated, and microsome was isolated for in vitro assay. In the assay, A beta 40, A beta 42, and A beta 43 were produced with an optimal pH of similar to 7.0. We also detected A beta-production by yeast endogenous protease(s), which was abolished by the addition of phosphatidyl choline. This novel system will facilitate the analysis of substrate recognition by gamma-secretase. (C) 2008 Elsevier Inc. All rights reserved