Abstractγ‐secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx‐defective 1 (Aph‐1), and presenilin enhancer 2 (Pen‐2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C‐terminal fragment (PS1C) caused by knockdown of pen‐2 or knockout of NCT or Aph‐1 was recovered by the addition of proteasome inhibitors, indicating that Pen‐2, as well as NCT and Aph‐1α, is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin‐Aph‐1 subcomplex plays not only an important role in γ‐secretase complex assembly but also in recruiting substrate C‐terminal fragment of amyloid precursor protein generated by β‐cleavage. Ablating any one component resulted in the instability of other components of the γ‐secretase complex, and the presence of all three of the other components is required for full maturation of NCT.