Notch and the Amyloid Precursor Protein Are Cleaved by Similar γ-Secretase(s)

γ-Secretase is an intramembrane-cleaving protease whose substrates include Notch and the amyloid precursor protein (APP). On the basis of initial genetic and pharmacologic data, the γ-secretase activity responsible for cleavage of both proteins appears to be identical. However, apparent differences in the cleavage site and in sequence specificity raise questions about the degree of similarity between Notch and APP γ-like proteolysis. In an effort to resolve this issue directly, we established an in vitro γ-secretase activity assay that cleaves both APP- and Notch-based substrates, C100Flag and N100Flag. Analysis with specific γ-secretase inhibitors, dominant-negative γ-secretase preparations, and antibody co-immunoprecipitations all demonstrated identical cleavage of these substrates. Most importantly, we found that these substrates prevented cleavage of each other, indicating that the same γ-secretase complex can cleave either protein. Finally, we provide evidence that both substrates are cut at two distinct regions in the transmembrane domain. These data resolve some of the apparent conflicts and strongly indicate that Notch and APP are proteolyzed by the same enzyme(s).