# Specific Mutations in Aph1 Cause \(\gamma\)-Secretase Activation
## 2 Results  
### Identification of Aph1 Mutations That Activate \(\gamma\)-Secretase in the Absence of NCT  
We used a yeast reconstitution system expressing human \(\gamma\)-secretase subunits and an APP-based substrate, APP\({}_{\text{C}55}\)-Gal4p, to screen Aph1 aL mutations. Recombinant plasmids were introduced into the yeast strain PJ69-4A, which possesses _HIS3_, _ADE2_, and _lacZ_ markers under Gal4p control. When \(\gamma\)-secretase cleaves the substrate, Gal4p is released from membrane-bound APP\({}_{\text{C}55}\)-Gal4p and activates the expression of _HIS3_, _ADE2_, and _lacZ_. Therefore, the activity of \(\gamma\)-secretase can be assessed by cell growth in media lackinghistidine and adenine, or by \(\beta\)-galactosidase activity [29; 31]. In the media, cells could grow with four subunit genes introduced, but not when any single subunit was absent.  
We previously identified PS1 mutants active in the absence of NCT [29]. These mutants possessed two missense mutations in PS1: S438P at the ninth transmembrane domain together with one mutation distributed throughout the molecule. These PS1 mutations enhanced and modulated the \(\gamma\)-secretase activity and decreased the ratio of long A\(\beta\)s [32]. We then isolated Aph1 or Pen2 mutants able to grow in the absence of NCT. After we had screened cells with Pen2 or Aph1 mutations and the PS1 S438P mutation, we found 13 Aph1 mutants that grew in the absence of NCT (Table 1 and Figure 1). Six mutants possessed two mutations and seven mutants possessed one mutation. These Aph1 mutants did not grow with the wild-type PS1 (Table 1), indicating that the PS1 S438P mutation is necessary for the activation of growth without NCT. No Pen2 mutants were active without NCT. These results indicate that PS1 and Aph1 mutations act synergistically to activate \(\gamma\)-secretase in the absence of NCT.  
\(\beta\)-galactosidase activity determined the cleavage of APP\({}_{\text{C55}}\)-Gal4p by Aph1 mutants. Aph1 L30F/T164A, L47I/T164A, L47F/V51G, S103P/P216S, V131A/T164A, and I135V/P216S possessed high activity (Figure 2a-f), compared to cells with each replacement. L30F, L47I, S103P, V131A, I135V, T164A, and P216S showed low but significant activities (Figure 2a-f). L47F and V51G showed diminished activities (Figure 2c), suggesting that two mutations are required for full activation. Including mutations regarded as single mutants (Figure 2g), we identified 10 primary mutations that activate \(\gamma\)-secretase (10% to 40% of wild-type Aph1 with NCT), and two secondary mutations that act only in combination (Figure 1).  
Figure 1: Aph1 mutants active in the absence of NCT map to transmembrane domains. Mutations in double or single mutants are indicated in the model with the seven transmembrane domains (TMDs) of Aph1. Mutations active as single mutants are shown as primary mutations; mutations active only in combination are shown as secondary mutations. The Aph1 mutations are in close proximity to PS1 TMD1 or TMD9 connected to the C-terminal loop, as indicated by arrows. L30F/T164A mutations, which activated \(\gamma\)-secretase, are shown in bold.  
\begin{table}
\begin{tabular}{c c c c} \hline
**Aph1 Mutants** & **Growth with PS15438P** & **Growth with PS1 WT** & **No. of Clones** \\ \hline L30F/T164A & +++ & - & 1 \\ L47I & +++ & - & 4 \\ L47I/T164A & +++ & - & 1 \\ L47F/V51G & +++ & - & 1 \\ A102V & +++ & - & 1 \\ S103P/P216S & +++ & - & 1 \\ V131A/T164A & +++ & - & 1 \\ I135V/P216S & +++ & - & 1 \\ I135T & ++ & - & 1 \\ T164A & +++ & - & 3 \\ S201L & ++ & - & 1 \\ L215P & +++ & - & 2 \\ P216S & +++ & - & 1 \\ \hline \end{tabular}
\end{table}
Table 1: Screening for Aph1 mutants active without NCT. The growth of cells with APP-Gal4, PS1 (WT or S438P), and Pen2 were analyzed on SD-LWHUAc medium after 3 days at 30 \({}^{\circ}\)C. “++” represent full growth, cells formed colonies (>1 mm); “++” represent partial growth with colonies (>0.5 mm); and “-” represent no growth. 1.1 \(\times\) 10\({}^{6}\) Cells were screened.  
Figure 2: \(Cont\).