Nicastrin is dispensable for gamma-secretase protease activity in the presence of specific presenilin mutations

Abstract

gamma-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial gamma-secretase substrates containing amyloid precursor protein or Notch fragments. The gamma-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or beta-galactosidase assay. We screened and evaluated gamma-secretase mutants using this reconstitution system in yeast, which does not possess endogenous gamma-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the beta-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid beta peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of gamma-secretase.

FIGURE 1.

Reconstitution of PS1 FAD mutants in yeast. A and B, γ-secretase subunits were introduced into yeast with C1–55-Gal4p. Gal4p cleaved from C1–55-Gal4p activates the reporter genes, HIS3 and ADE2, and the release was assessed by the growth on the selection medium, lacking histidine and adenine. Cells expressing PS1 wild type (WT)(A) or FAD mutants (B), other γ-secretase subunits, and C1–55-Gal4p were examined for growth after 3 days at 30 °C on selection (SD-LWHUAde) or nonselection (SD-LWU) media, as indicated. Three independent clones were tested for each strain. C, β-galactosidase activity was measured for each yeast strain with PS1 wild type or FAD mutants, as indicated. One unit of β-galactosidase activity corresponds to 1 nmol of o-nitrophenyl β-d-galactopyranoside hydrolyzed per min, and activity was calculated as unit/(min × mg of protein in lysate). The activity was normalized by the subtraction with the activity in the absence of NCT, 38.7 unit/(min × mg of protein). Representative results from three independent assays are shown with the standard deviations. Statistical analyses were performed by one-way analysis of variance followed by Dunnett's multiple comparison test. Asterisks indicate p < 0.01 with respect to PS1 wild type. D, expression of wild type and PS1 mutants in lysates were analyzed by immunoblotting using antibody against PS1 (G1L3). Arrows indicate full-length PS1 and CTF (carboxyl-terminal fragment). ΔExon9 mutant migrates faster than the wild type. ^** indicates nonspecific bands.

FIGURE 2.

PS1 mutant, F411Y/S438P, active in the absence of NCT. A and B, cells expressing PS1 wild type, F411Y/S438P (A), F411Y, or S438 (B) mutants, otherγ-secretase subunits, and C1–55-Gal4p were examined for growth after 3 days at 30 °C on selection (SD-LWHUAde) or nonselection (SD-LWU) media, as indicated. C, β-galactosidase activity was measured for each yeast strain containing wild type or mutant PS1, as indicated. The experimental conditions are as in Fig. 1C. Data represent means ± S.D.;n=3. Asterisks indicate p < 0.01 with respect to PS1 wild type. D, cells expressing D385A/F411Y/S438P, the catalytic site mutant, were examined for growth as indicated.

FIGURE 3.

PS1 mutants active in the absence of NCT. A and B, mutations in double mutants (A) or a triple mutant (B) are indicated in the model with nine transmembrane domains of PS1. All double mutants contained S438P as a primary mutation with a variable secondary mutation. Conserved residues are in boldface. Catalytic residues in TM6 and TM7 (Asp²⁵⁷ and Asp³⁸⁵) are also indicated.

FIGURE 4.

Endoproteolysis of F411Y/S438P mutant in yeast cells. A, microsomes were prepared from yeast transformants expressing PS1 wild type or PS1 F411Y/S438P mutant, NCT, Aph-1-HA, and FLAG-Pen2, as indicated. These subunits were analyzed by immunoblotting using specific antibodies. The endoproteolysis of PS1 was detected by the production of fragments, PS1 NTF and PS1 CTF. B, microsomes expressing γ-secretase and C1–99 fragment of APP were subjected to the γ-secretase assay. CHAPSO-solubilized microsomes were incubated at 37 °C for 0, 8, or 24 h. Aβ and AICD production was analyzed by immunoblotting using specific antibodies. Synthetic Aβ40 (30 pg) was loaded as a marker in the leftmost lanes. The asterisks indicate nonspecific bands.

FIGURE 5.

Endoproteolysis, Aβ, and NICD production by F411Y/S438P mutant in mouse embryonic fibroblasts. A, PS double knock-out cells were transiently transfected with C1–99pcDNA, PS1pcDNA3.1/Zeo, or F411Y/S438PpcDNA3. 1/Zeo, and siRNA for NCT or control siRNA (cont. siRNA), as indicated. Cells were recovered and analyzed by immunoblotting with specific antibodies. The endoproteolysis of PS1 was detected by the production of PS1 CTF. B, NCT knock-out (KO) cells or wild type (WT) fibroblast cells were transiently transfected with C1–99pcDNA, PS1pcDNA3.1/Zeo, or F411Y/S438PpcDNA3.1/Zeo as indicated. Cells and media were recovered and analyzed by immunoblotting with specific antibodies (human PS1-specific antibody (PS1L) was used for PS1). Aβ was detected in media. Synthetic Aβ40 (30 pg) was loaded as indicated. C, NCT knock-out cells (KO) or wild type (WT) fibroblast cells were transiently transfected with pCS2+mN^ΔEmyc, PS1pcDNA3.1/Zeo, or F411Y/S438PpcDNA3.1/Zeo as indicated. Membrane-bound mNotch-1 (N^ΔE) was expressed from the pCS2+mN^ΔEmyc vector. 48 h after transfection, cells were incubated with 10 μm lactacystin for 4 h, recovered, and analyzed by immunoblotting with specific antibodies. The asterisks indicate nonspecific bands.

FIGURE 6.

F411Y/S438P mutant forms γ-secretase complexes in yeast. A, microsomes from yeast transformants with PS1 wild type, NCT, Aph-1-HA, and FLAG-Pen2 were solubilized with buffer containing 1% CHAPSO. γ-Secretase complex was purified from the extracts using the antibody against PS1-loop region (G1L3) or rabbit preimmune serum, as indicated. Immunoprecipitates and the input fraction were analyzed by immunoblotting using specific antibodies. The input represents 25% of the microsomal extract. B, microsomes were prepared from yeast transformants expressing PS1 wild type or F411Y/S438P mutant, NCT, Aph-1-HA, and FLAG-Pen2 as indicated. IP, immunoprecipitation. γ-Secretase complexes were immunopurified by G1L3 antibody and analyzed by immunoblotting as A.