In vitro reconstitution of γ-secretase activity using yeast microsomes

Sosuke Yagishita, Eugene Futai, Shoichi Ishiura

Research output: Contribution to journalArticlepeer-review

32 Citations (Scopus)


γ-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 β peptides (Aβ) that is deposited in the brains of Alzheimer disease. However, the mechanism of γ-secretase-mediated cleavage remains unclear. To examine the enzymatic properties of γ-secretase, we established an 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 the substrate in pep4Δ cells with vacuole proteases inactivated, and microsome was isolated for in vitro assay. In the assay, Aβ40, Aβ42, and Aβ43 were produced with an optimal pH of ∼7.0. We also detected Aβ-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 γ-secretase.

Original languageEnglish
Pages (from-to)141-145
Number of pages5
JournalBiochemical and Biophysical Research Communications
Issue number1
Publication statusPublished - 2008 Dec 5


  • γ-Secretase
  • Amyloid β peptide
  • Presenilin
  • Yeast


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