Analysis of transmembrane domain mutants is consistent with sequential cleavage of Notch by γ-secretase

Abstract

γ-Secretase is a lipid-embedded, intramembrane-cleaving aspartyl protease that cleaves its substrates twice within their transmembrane domains (TMD): once near the cytosolic leaflet (at S3/ε) and again in the middle of the TMD (at S4/γ). To address whether this unusual process occurs in two independent or interdependent steps, we investigated how mutations at the S3/ε site in Notch1-based substrates impact proteolysis. We demonstrate that such mutations greatly inhibit not only γ-secretase-mediated cleavage at S3 but also at S4, independent of their impact on NICD stability. These results, together with our previous observationsq2, suggest that hydrolysis at the center of the Notch transmembrane domain (S4/γ) is dependent on the S3/ε cleavage. Notch (and perhaps all γ-secretase substrates) may be cleaved by sequential proteolysis starting at S3. © 2005 International Society for Neurochemistry.