<em>Saccharomyces cerevisiae</em> Models of Alzheimer's Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by γ-Secretase

γ-Secretase is a protease complex embedded in the cell membrane, consisting of the catalytic presenilin subunits (PS1 or PS2) and three additional co-factors: nicastrin, Aph-1, and Pen2. It cleaves the transmembrane domains of type-I transmembrane proteins, such as the amyloid precursor protein (APP) and Notch. The cleavage of APP generates the amyloid β peptides (Aβ), which accumulate in patients with Alzheimer's disease. Despite significant research, the exact mechanism of this unique proteolysis, which occurs within the lipid bilayers, is still not fully understood. To study the enzymatic properties of γ-secretase, we have established a yeast reporter system using artificial γ-secretase substrates containing APP or Notch fragments fused to the transcriptional activator Gal4. The γ-secretase activity was evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane-bound substrates, as assessed by the growth of yeast or β-galactosidase activity. Furthermore, we have developed an in vitro assay to identify the different forms of Aβ produced from yeast microsomes. These yeast models provide a platform to screen mutations, genes, and compounds that affect γ-secretase function. By studying the loss of function properties of PS1 familial Alzheimer's disease (FAD) mutants, it is possible to screen for FAD suppressor mutations and γ-secretase modulators (GSMs), in addition to γ-secretase inhibitors (GSIs). In this report, we describe the genetic and biochemical methods used to study γ-secretase activity in the yeast system with the essential steps of the protocol for the video.