# Specific Mutations in Aph1 Cause \(\gamma\)-Secretase Activation
## 2 Results
### Aph1 Mutations Activate \(\gamma\)-Secretase in Mouse Fibroblasts  
We analyzed the Aph1 mutations in embryonic fibroblasts obtained from Aph1-a/Aph1-b/Aph1-c triple knockout mice (Aph1 TKO cells). We introduced C99 and Aph1 aL genes into Aph1-KO MEF cells using retroviral vectors. The amount of Aph1 L30F/T164A was lower than the amount of wild-type Aph1, but similar amounts of PS1 NTF were detected (Figure 6a, lanes 2 and 3), indicating that Aph1 mutants formed mature \(\gamma\)-secretase complexes. Next, we analyzed the effect of the L30F/T164A mutation on A\(\beta\) production. Western blotting showed that the amount of A\(\beta\) produced by the L30F/T164A mutant was approximately twice the amount produced by the WT (Figure 6b). We used ELISA to quantify A\(\beta\)40 and A\(\beta\)42 production by the L30F/T164A mutant; we found that the L30F/T164A mutation resulted in approximately twice as much A\(\beta\)40 and A\(\beta\)42, compared with the amounts produced by the WT (Figure 6c). The ratio of long A\(\beta\)42 with A\(\beta\)40 was similar between the WT and mutant (Figure 6d). Figure 3: \(\beta\)-Galactosidase activity of Aph1 active mutants in the \(\gamma\)-secretase complex. (**a**) \(\beta\)-Galactosidase activity was assayed in cells expressing the Aph1 wild-type (WT) or active mutants, PS1, FLAG-Pen2, and nicastrin. The experimental conditions were identical to Figure 2. (**b**) Expression of WT or Aph1 mutants and PS1 in lysates were analyzed by immunoblotting using specific antibodies. Endoproteolysis of PS1 was detected by the production of mature fragment PS1 CTF. Asterisks on each bar indicate \(p<0.01\) (**) or \(p<0.001\) (***) (**a**). Figure 4: Aph1 mutations activate \(\gamma\)-secretase in yeast microsomes. Microsomes were prepared from yeast transformants expressing Aph1 WT or Aph1 X30F/T164A mutants, PS1, NCT, FLAG-Pen2, and the C55 fragment of APP; they were then subjected to \(\gamma\)-secretase assays. CHAPSO- solubilized microsomes (40 \(\upmu\)g protein) were incubated at 37 \({}^{\circ}\)C for 24 h in the presence or absence of phosphatidylcholine (PC) (0.1%) and \(\gamma\)-secretase-specific inhibitor (L685, 458). (**a,b**) Total Aß production was analyzed by immunoblotting using the antibody 82E1. (**c,d**) Aß species were separated by urea/SDS-PAGE and analyzed by immunoblotting with 82E1. Synthetic Aß38, Aß40, Aß42, and Aß43 (30 pg) were loaded as controls in the left-most lanes (**a,c**). Amount of Aß (**b,d**) and the ratio of Aß species (**e**) were quantified from three independent assays; data are shown with standard deviations. Statistical analyses were performed using one-way ANOVA followed by [PERSON]’s multiple comparison test. Asterisks on each bar indicate \(p<\) 0.01 (**) or \(p<\) 0.05 (**) with respect to Aph1 WT (**b,d,e**). Next, we analyzed complex formation by co-immunoprecipitation. The \(\gamma\)-secretase complex was isolated from the total membrane fraction using a PS1 antibody; the amount of Aph1 was examined. The amounts of PS1 and Aph1 aL in the complex were similar between the WT and L30F/T164A mutant (Figure 6e, lanes 1 and 2), suggesting that the L30F/T164A mutation does not affect complex formation. Figure 5: Aph1 aL L30F/T164A mutant forms \(\gamma\)-secretase complexes in yeast. Microsomes were prepared from yeast transformants as in Figure 4 and solubilized with buffer containing 1% CHAPSO. The \(\gamma\)-secretase complex was purified from extracts using an antibody against the PS1-loop region (G1L3) or rabbit pre-immune serum, as indicated. Immunoprecipitants and the input fraction were analyzed by immunoblotting using specific antibodies. The input represents 25% of the microsomal extract. Figure 6: Production of A\(\beta\) by Aph1 mutants in mouse embryonic fibroblasts. Using the retroviral system, Aph1 knockout cells were transfected with Aph1 WT or Aph1 L30F/T164A. Cells were harvested; extracts were then analyzed by immunoblotting with the indicated antibodies (**a**).