Abstract
Presenilin‐dependent intramembranous proteolysis mediates the dual cleavage of the Notch‐1 protein (S4 and S3) as well as the β amyloid precursor protein (βAPP) (γ40 and ϵ‐site). βAPP has a valine residue just before the γ40 (amyloid β [Aβ] numbering) site and after the ϵ‐site. Both γ40 and ϵ have multiple cleavage sites, and the varieties of γ40 cleavage are associated with Alzheimer's disease (AD). These lines of evidence suggest that valine plays a role in the intramembranous proteolysis. S4 cleavage in the middle of the Notch‐1 transmembrane domain (TMD) corresponds to the γ40 cleavage of βAPP. The cleavage site is in the center of four sequential alanine residues between Ala1731 and Ala1732, neither of which has a valine residue. To investigate the effects of valine on presenilin‐dependent intramembranous proteolysis, we replaced the transmembrane domain residue of Notch‐1 with valine and analyzed the efficiency and precision at S4 and S3. We observed that all valine‐mutated Notch‐1 proteins have a dominant cleavage site (S4) between Ala1731 and Ala1732 with some variations of cleavage precision, suggesting that valine is not indispensable for determining the cleavage site of the Notch‐1 transmembrane domain, but affects the efficiency and precision at S4 cleavage of the Notch‐1 transmembrane domain. © 2006 Wiley‐Liss, Inc.