The intramembrane cleavage site of the amyloid precursor protein depends on the length of its transmembrane domain.

Proteolytic processing of the amyloid precursor protein by beta-secretase generates C99, which subsequently is cleaved by gamma-secretase, yielding the amyloid beta peptide (A beta). This gamma-cleavage occurs within the transmembrane domain (TMD) of C99 and is similar to the intramembrane cleavage of Notch. However, Notch and C99 differ in their site of intramembrane cleavage. The main gamma-cleavage of C99 occurs in the middle of the TMD, whereas the cleavage of Notch occurs close to the C-terminal end of the TMD, making it unclear whether both are cleaved by the same protease. To investigate whether gamma-cleavage always occurs in the middle of the TMD of C99 or may also occur at the end of the TMD, we generated C99-mutants with an altered length of the TMD and analyzed their gamma-cleavage in COS7 cells. The C terminus of A beta and thus the site of gamma-cleavage were determined by using monoclonal antibodies and mass spectrometry. Compared with C99-wild type (wt), most mutants with an altered length of the TMD changed the cleavage site of gamma-secretase, whereas control mutants with mutations outside the TMD did not. Thus, the length of the whole TMD is a major determinant for the cleavage site of gamma-secretase. Moreover, the C99-mutants were not only cleaved at one site but at two sites within their TMD. One cleavage site was located around the middle of the TMD, regardless of its actual length. An additional cleavage occurred within the N-terminal half of their TMD and thus at the opposite side of the Notch cleavage site.