Presenilin (PS1 or PS2) is the catalytic component of the gamma-secretase 
complex, which mediates the final proteolytic processing step leading to the 
Alzheimer's disease (AD)-characterizing amyloid beta-peptide. PS is cleaved 
during complex assembly into its characteristic N- and C-terminal fragments. 
Both fragments are integral components of physiologically active gamma-secretase 
and harbor the two critical aspartyl residues of the active site domain. While 
the minimal subunit composition of gamma-secretase has been defined and numerous 
substrates were identified, the cellular mechanism of the endoproteolytic 
cleavage of PS is still unclear. We addressed this pivotal question by 
investigating whether familial AD (FAD)-associated PS1 mutations affect the 
precision of PS endoproteolysis in a manner similar to the way that such 
mutations shift the intramembrane cleavage of gamma-secretase substrates. We 
demonstrate that all FAD mutations investigated still allow endoproteolysis to 
occur. However, the precision of PS1 endoproteolysis is affected by PS1 
mutations. Comparing the cleavage products generated by a variety of PS1 mutants 
revealed that specifically cleavages at positions 293 and 296 of PS1 are 
selectively affected. Systematic mutagenesis around the cleavage sites revealed 
a stepwise three amino acid spaced cleavage mechanism of PS endoproteolysis 
reminiscent to the epsilon-, zeta-, and gamma-cleavages described for typical 
gamma-secretase substrates, such as the beta-amyloid precursor protein. Our 
findings therefore suggest that intramembranous cleavage by gamma-secretase and 
related intramembrane-cleaving proteases may generally occur via stepwise 
endoproteolysis.