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γ-Secretase is an intramembrane aspartyl protease that cleaves the C99 fragment of amyloid precursor protein to generate extracellular Aβ peptides.These peptides can oligomerize and aggregate to form amyloid plaques,processes that are widely believed to be causal for Alzheimer’s disease.In spite of this critical function,it remains unknown how γ-secretase recognizes C99 and its other substrates,including Notch.In this study we determined E22-K55 as the minimal C99 fragment that was sufficient and required for initial cleavage.Within this fragment,we identified four determinants:(i) a transferable extracellular determinant that differed between C99 and Notch,and which included negative charge in the case of C99,(ii) the amino acid sequence of the C-terminal half of the transmembrane helix,(iii) an invariant lysine or arginine at the intracellular membrane border,and (iv) a positive charge cluster that included the invariant lysine/arginine.We demonstrated that the charge clusters of C99 and Notch receptors could directly bind phosphatidylinositol 4,5-bisphosphate (PIP2).The PIP2-binding cluster was required for γ-secretase cleavage,and modulation of membrane PIP2 levels strongly affected γ-secretase cleavage levels and the Aβ40/Aβ42 ratio,providing support for the importance of the PIP2 interaction in cells.Together,these studies provide critically needed insight into substrate recognition by γ-secretase.