In this study, using genetic knockout and siRNA-mediated knockdown cells, we systematically investigated the role of each component of the γ-secretase complex during assembly, maturation, and APP processing activity. Our data revealed several new findings. First, Pen-2 is dispensable for PS endoproteolysis. The data presented here clearly demonstrate that in Pen-2-KD cells, similar to what is observed in NCT-KO and Aph-KO cells, the disappearance of PS1C was prevented by the addition of the proteasome inhibitor MG132 or lactacystin. In particular, our results from the time course experiments undoubtedly show that the accumulated PS1C in Pen-2-KD cells, in the presence of proteasome inhibitors, was newly generated after knockdown of Pen-2. These findings indicate that endoproteolysis of PS occurred in the absence of Pen-2; however, in the absence of MG132, the resulting PS1C was degraded by proteasome. It has been reported that PS1 undergoes endoproteolysis between Thr291 and Ala299 (Podlisny et al. 1997). The PS1C fragment observed in this study was detected by two specific antibodies: one was raised against a peptide corresponding to residues 307–321 of PS1 (Xu et al. 2002) as shown in Figs 1 and 2; the other is specific to the last 20 amino acids of PS1 (from Santa Cruz, data not shown). This result indicates both the N-terminus and the C-terminus of the PS1C fragment observed here remain intact. In addition, the PS1N fragment observed in this study was detected by three PS1N-specific antibodies: anti-PS1N [against residues 27–50 (Zhao et al. 2004)], Figs 1 and 2), N-19 (Santa Cruz, data not shown), and Ab14 [(Luo et al. 2003)], (data not shown). These data strongly indicate that the PS1N and PS1C observed in knockout cells are the same as those detected in wild-type cells, that is, knockout of other components has no effect on the cleavage site of PS1 endoproteolytic processing. Why, in some cases, could full-length PS1 be detected in the Pen-2 knockout cells? In this regard, it is notable that APH-1 and NCT have been shown to form a stable subcomplex that binds to and stabilizes the PS1 holoprotein (LaVoie et al. 2003; Takasugi et al. 2003). Therefore, it is very possible that, in the Pen-2 knockdown cells, the residual unprocessed full-length PS1 resulting from inefficient processing was protected from degradation by the NCT-Aph-1 subcomplex and can be detected under certain experimental conditions. However, in the NCT-KO or Aph-1-KO cells, the knockout of NCT or Aph-1 abolishes the formation of the NCT-Aph-1 subcomplex, resulting in the degradation of the unprotected full-length PS1. Thus, in conclusion, though it cannot be ruled out that the presence of all of other components may accelerate the endoproteolytic processing of PS, the observations that depletion of other components of γ-secretase did not abolish the production of PS1N and PS1C strongly indicate that none of these γ-secretase components is absolutely required for endoproteolysis of PS, but they all are required to stabilize the processing product.
Second, formation of the NCT-Aph-1 subcomplex is not required for NCT and Aph-1 to interact with PS, but it is required for them to interact with Pen-2. Using two-dimensional PAGE and differential detergent dissociation approaches, it was revealed that NCT-Aph-1 forms an intermediate subcomplex (LaVoie et al. 2003; Fraering et al. 2004). However, the role of this subcomplex in γ-secretase assembly remains elusive. In this study, using knockout cells and siRNA approaches, our data confirmed that NCT and Aph-1 can form a complex in the absence of both PS1 and PS2. In investigating the role of this subcomplex in γ-secretase assembly, our co-immunoprecipitation data demonstrated that PS1 can be co-immunoprecipitated with Aph-1 in the absence of NCT and vice versa. Our data also demonstrated that Pen-2 can be co-immunoprecipitated with PS1 in the absence of NCT or Aph-1. These data indicate that the NCT-Aph-1 subcomplex is not required for PS to interact with NCT, Aph-1, or Pen-2. Our data further demonstrate that in wild-type cells both NCT and Aph-1 can be co-immunoprecipitated with Pen-2, but neither NCT nor Aph-1 can be co-immunoprecipitated with Pen-2 in the absence of PS protein, indicating this subcomplex is not sufficient to interact with Pen-2, and its interaction with Pen-2 may be mediated by PS protein. However, our data demonstrated that knockout of NCT abolished the interaction of Aph-1 with Pen-2, and only a trace amount of Pen-2 was co-immunoprecipitated with NCT in the absence of Aph-1, indicating that formation of the NCT-Aph-1 subcomplex is required for NCT and Aph-1 to efficiently interact with Pen-2 in the presence of PS1.
Third, our data suggest that formation of the NCT-Aph-1 subcomplex plays not only an important role in γ-secretase complex assembly but also in recruiting substrate CTFβ to the complex. It is interesting to note that our data clearly demonstrated that both NCT and Aph-1 can be co-immunoprecipitated with CTFβ in the PS1 and PS2 double knockout cells and also in Pen-2-KD cells, indicating that formation of the NCT-Aph-1 subcomplex is sufficient for interaction with CTFβ, the initial substrate of γ-secretase. Moreover, no CTFβ can be co-immunoprecipitated with PS1 in the NCT-KO or Aph-KO cells, indicating that the interaction between PS and CTFβ is mediated by the formation of the NCT-Aph subcomplex, that is, NCT-Aph subcomplex formation is necessary for γ-secretase to interact with its initial substrate CTFβ. It was also notable that more CTFβ was co-immunoprecipitated with NCT than with other γ-secretase components, suggesting that NCT has the highest affinity for CTFβ and plays an important role in recruiting substrates to γ-secretase. This finding is consistent with a previous observation that suggests NCT may function as a substrate receptor (Shah et al. 2005). On the other hand, Aph-1α may contribute to recruiting CTFβ by interacting with CTFβ through the GxxxG motif (Mao et al. 2009).