Co-expression of Nicastrin and Presenilin Rescues a Loss of Function Mutant of APH-1*

γ-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of β-amyloid precursor protein to liberate the neurotoxic amyloid-β peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute γ-secretase activity in yeast. Although PS seems to contribute the catalytic core of the γ-secretase complex, no distinct function could be attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, we now found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, we expressed APH-1a G122D in HEK293 cells. As reported previously, overexpressed APH-1a G122D was not incorporated into the γ-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allowed the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitated the formation of a fully active γ-secretase complex. Under these conditions, APH-1a G122D supported the production of normal amounts of Aβ. We conclude that cooperative effects may stabilize a trim-eric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for γ-secretase activity.

of the transmembrane domain (2). The aspartyl protease ␤-site APP-cleaving enzyme has been identified as the sole ␤-secretase in humans (3). Several lines of evidence have suggested that the polytopic membrane protein presenilin (PS) functions as ␥-secretase. In the absence of both mammalian presenilin homologues PS1 and PS2, no A␤ is produced (4,5). Moreover, mutations in the PSs associated with familial Alzheimer's disease shift the ratio from the predominant 40-amino-acid A␤ species to the more aggregation-prone 42-amino-acid variant (2). Furthermore, active site ␥-secretase inhibitors can be crosslinked to PSs (6), and mutagenesis of two highly conserved aspartyl residues suggests that PSs belong to a novel class of intramembrane-cleaving aspartyl proteases (7). Indeed, the Cterminal aspartyl residue is part of a conserved GXGD motif that is also contained in other polytopic aspartyl proteases including the signal peptide peptidase and its relatives (8,9).
Biochemical purification and genetic studies identified nicastrin (NCT), APH-1, and PEN-2 as additional integral membrane proteins crucial for ␥-secretase activity (10 -13). As for PSs, two homologues of APH-1 have been identified in mammals, APH-1a and APH-1b. PS forms a complex together with NCT, APH-1, and PEN-2 (14 -16) that is sufficient to reconstitute ␥-secretase activity in Saccharomyces cerevisiae, which lacks endogenous ␥-secretase (17). Furthermore, reconstitution of active ␥-secretase in yeast was associated with endoproteolysis of PS1 into an N-terminal fragment and a C-terminal fragment, which together are believed to represent the principal cellular form of PS (18). So far, little is known about the distinct functions of the recently identified complex components NCT, APH-1, and PEN-2. Extensive cross-regulation of the expression level of the four complex components severely complicates a separate analysis of the individual components. Both genetic knock-out and RNA interference-mediated downregulation of PS1, NCT, APH-1, and PEN-2 typically decrease the levels of the other components and prevent maturation of NCT (19 -23). Only immature NCT and APH-1 seem to be rather stable even in the absence of other components (20 -25). This might be explained by a stable "subcomplex" containing only immature NCT and APH-1 (25)(26)(27)(28), although its functional role during physiological ␥-secretase assembly has not been demonstrated. During complex assembly, NCT undergoes a conformational change that renders it resistant to trypsin digestion (29). PEN-2 assembly has been implicated in initiating PS endoproteolysis because removal of PEN-2 leads to accumulation of unprocessed PS holoprotein, which is not observed upon knockdown of any other complex component (14,24,30). Furthermore, PEN-2 is required to stabilize the Nterminal fragment/C-terminal fragment heterodimer within the ␥-secretase complex (24).
A loss of function allele of APH-1 in Caenorhabditis elegans has drawn the attention to a highly conserved GXXXG motif in the fourth transmembrane domain (12,13,31). Such motifs are known to mediate helix-helix interactions within transmembrane domains (32). A point mutation of the first glycine residue of the GXXXG motif in APH-1 was lethal in C. elegans (12) and disrupted ␥-secretase complex formation of the human homologue APH-1a (31), demonstrating the general importance of this motif. We now reconstituted ␥-secretase in yeast to further dissect the role of the conserved 122 GXXXG 126 in human APH-1a for ␥-secretase complex assembly and activity. Surprisingly, we found that the mutation causing a loss of function in C. elegans was fully active when ␥-secretase was reconstituted in yeast. Moreover, by overexpressing PS1 and NCT together with APH-1a G122D in human cells, we were able to fully rescue the loss of function APH-1 allele.
cDNA Constructs-The 265-amino-acid-long splice form (13) of human APH-1a with a C-terminal HA tag was cloned in pcDNA3.1/Hygro(ϩ). For expression in yeast, the constructs were as described in Ref. 17. Mutations were introduced by PCR mutagenesis and confirmed by DNA sequencing.
Cell Lines and Cell Culture-Human embryonic kidney 293 cells (HEK293) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and as appropriate, 200 g/ml G418 (to select for APP expression), 150 g/ml hygromycin (for APH-1a-HA), 5 g/ml blasticidin (for NCT), and 200 g/ml zeocin (for PS1 selection). HEK293 cells stably expressing ␥-secretase components were generated by transfection of HEK293/Sw cells stably expressing APP 695 containing the Swedish mutation as in Ref. 20. For APH-1a, only low expressing clones were analyzed further to avoid aggregation of APH-1a. Moreover, clones expressing high amounts of APH-1a showed growth deficits after several passages. For transient transfection of cells stably expressing APH-1a-HA G122D, Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's instruction.
Yeast Strains and Methods-Standard methods (35) were used for yeast culture, transformation with plasmids, protein extraction, and ␤-galactosidase assay. A yeast strain identical to the previously described DEY1 but lacking the mutation V717I in the APP domain of C 1-55 -GAL4 was used to express PS1, NCT, APH-1a-HA (WT, G122A, or G122D), and FLAG-PEN-2 as described (17).
To investigate this surprising finding on a biochemical level, we stably expressed HA-tagged APH-1a WT and G122D in HEK293 cells. We performed co-immunoprecipitation studies with antibody 3027 against the PS1-C-terminal fragment and an anti-HA antibody against HA epitope-tagged APH-1a. The PS1 antibody co-immunoprecipitated APH-1a HA WT, together with endogenous PS1 N-terminal fragment, NCT, and PEN-2 (Fig. 1B). In contrast, overexpressed APH-1a G122D was not co-immunoprecipitated with PS1 (Fig. 1B). Similarly, anti-HA immunoprecipitation of APH-1a WT did co-precipitate endogenous PS1, NCT, and PEN-2, whereas APH-1a G122D did not interact with endogenous ␥-secretase components (Fig. 1B) as reported previously (21,31). This suggests that APH1a G122D cannot compete with endogenous APH-1a for the other complex components present in HEK293 cells at endogenous levels. Thus, the GXXXG motif in APH-1 may be required for ␥-secretase complex assembly by mediating the interaction with one or more other ␥-secretase components, as suggested previously (31).
In yeast, all ␥-secretase components were expressed at fairly high levels, which might overcome a weaker binding affinity of APH-1a G122D to other complex components. We therefore expressed APH-1a G122D in all double and triple combinations with PS1, NCT, and PEN-2 in HEK293 cells to analyze whether excess amounts of other ␥-secretase components would facilitate the incorporation of APH-1a G122D into a functional ␥-secretase complex as observed when expressed in yeast. In addition, this approach might also reveal which protein-protein interaction requires the GXXXG motif. We transiently transfected the cell line stably expressing APH-1a-HA G122D additionally with PS1, NCT, and PEN-2 and combinations thereof (Fig. 2). By anti-HA immunoprecipitation, we analyzed whether APH-1a-HA G122D formed a stable complex with PS1, NCT, and PEN-2 (Fig. 2). Indeed, excess amounts of PS1 holoprotein (Fig. 2, lanes 2 and 9), immature NCT (lanes 3  and 10), and PEN-2 (lanes 4 and 11) allowed the interaction with APH-1a G122D. Thus, GXXXG-independent interactions can occur between APH-1a and PS1, NCT, and PEN-2. How-

FIG. 2. Overexpression of PS1 and NCT restores assembly of mutant APH-1 into the ␥-secretase complex in HEK293 cells.
HEK293/Sw cells stably expressing APH-1a-HA G122D were transiently transfected with combinations of PS1, NCT, and PEN-2 as indicated and subjected to co-immunoprecipitation (IP) using anti-HA antibody H6908 as in Fig. 1B. NCT, PS1, APH-1a-HA, and PEN-2 were detected by immunoblotting as described in the legend for Fig. 1B. Note that the minor amounts of mature NCT (NCT mat ) bound to APH-1a G122D without co-transfection of PS1 most likely represent premature export from the endoplasmic reticulum due to transient overexpression. The asterisk denotes the IgG heavy chain. NCT immat , immature NCT; NTF, N-terminal fragment. ever, overexpression of APH-1a G122D with only one additional component did not facilitate the full assembly of a ␥-secretase complex containing APH-1a G122D with the remaining ␥-secretase components present at endogenous levels. In accordance with that finding, APH-1a G122D almost exclusively bound to unprocessed PS1 holoprotein and immature NCT, respectively (lanes 9 -11). In contrast, when overexpressed together with PS1 and NCT, the mutant APH-1a G122D associated with large amounts of PS1 endoproteolytic fragments and mature NCT and also with endogenous PEN-2 (Fig. 2, lanes 5 and 12). Therefore, excess amounts of PS1 and NCT fully rescued the complex assembly defect in APH-1a G122D. In contrast, overexpression of APH-1a G122D in the other triple combinations (Fig. 2, compare lanes 6 and 13 and  lanes 7 and 14, respectively) was not sufficient to rescue complex assembly of APH-1a G122D. This may support a model of ordered assembly of ␥-secretase components. Interestingly, such a trimeric complex of PS1, NCT, and APH-1a has been proposed as the final intermediate during ␥-secretase complex assembly before PEN-2 binding initiates endoproteolysis of PS1 (14). The fact that excess PS1 and NCT are necessary to allow the complex incorporation of APH-1a G122D indicates that the GXXXG motif is required for stabilizing such a putative trimeric complex of PS1, NCT, and APH-1a. Most likely, the direct interaction of PS1 and NCT is able to stabilize such a trimer as long as PS1 and NCT are in excess. Upon full assembly through the binding of PEN-2, the complex containing APH-1a G122D may be further stabilized through additional interactions via cooperative binding among the components.
Reconstitution of ␥-secretase activity in yeast with APH-1a G122D suggests that the GXXXG motif is not required for proteolytic activity once APH-1a is incorporated into the ␥-secretase complex. To recapitulate this in mammalian cells, we analyzed ␥-secretase function in a cell-free assay using recombinant C100-His 6 substrate (17). We used transient transfection to overexpress all combinations of PS1, NCT, and PEN-2 together with APH-1a G122D (as in Fig. 2). As expected, FIG. 3. APH-1a G122D allows normal A␤ production upon overexpression of PS1 and NCT. A, HEK293/Sw cells stably expressing APH-1a-HA G122D were transiently transfected with PS1, NCT, and PEN-2 as in Fig. 2 and subjected to co-immunoprecipitation (IP) analysis (as in Fig. 1B but using HA-7 agarose). ␥-Secretase activity associated with APH-1a-HA G122D was assayed using recombinant C100-His 6 substrate. A␤ generation was detected by immunoblotting with antibody 6E10. Note that the recombinant substrate contains an N-terminal methionine, giving rise to A␤ species carrying an N-terminal methionine (Met-A␤). B, APH-1a-HA WT or G122D was immunoprecipitated from cells stably expressing APH-1a, PS1, and NCT as indicated and assayed for ␥-secretase activity using recombinant C100-His 6 substrate (as in panel A). Immunoblotting with antibody 6E10 was used to analyze A␤ production. The ␥-secretase inhibitor L-685,458 was used at 1 M to verify that A␤ is generated by genuine ␥-secretase activity (6). C, A␤ was generated as in panel B using cells stably expressing APH-1a WT or G122D together with PS1 and NCT. Met-A␤ 40 and Met-A␤ 42 species were separated on a Tris-Bicine-urea gel system (36) by immunoprecipitation with antibody 3926 and immunodetection with antibody 6E10. APH-1a G122D immunoprecipitated from HEK293 cells stably overexpressing mutant APH-1a alone is not associated with ␥-secretase activity (Fig. 3A). Only upon co-transfection of both PS1 and NCT was the defective A␤ production of APH-1a G122D restored. In contrast, all other combinations of ␥-secretase components failed to recruit APH-1a G122D into an active ␥-secretase complex. Apparently, only the combined overexpression of PS1 and NCT overcomes the deficient complex assembly of APH-1a G122D and restores ␥-secretase activity. For further analysis, we generated stable HEK293 cell lines co-expressing PS1 and NCT with either APH-1a WT or G122D. Without additional overexpression of PS1 and NCT, only APH-1a WT was associated with ␥-secretase activity (Fig. 3B). In contrast, when stably overexpressed together with PS1 and NCT, both APH-1a WT and G122D interacted with PS1, NCT, and PEN-2 (compare Fig. 2) and generated similar amounts of A␤ from the recombinant substrate. This is genuine ␥-secretase activity since A␤ production was fully blocked by L-685,458, a ␥-secretase inhibitor known to directly interact with PSs (6). The increased A␤ production in triple-transfected cells results from increased levels of APH-1a and enhanced amounts of mature ␥-secretase complexes (data not shown) (14 -16, 27).
We next used a Tris-Bicine-urea gel system (36) that allows the specific identification of A␤ 40 and A␤ 42 to analyze the A␤ species produced in vitro from active ␥-secretase containing APH-1a G122D. The ratio of A␤ 40 and A␤ 42 was largely unaffected in the APH-1a mutant (Fig. 3C). This shows that the mature ␥-secretase complex no longer vitally depends on the GXXXG motif for its stabilization. Once the assembly defect of APH-1a G122D is overcome, it allows A␤ production in amounts very similar to APH-1 WT. The mutation G122D does not affect the ratio of A␤ 40 /A␤ 42 in vitro, suggesting that glycine 122 is only required during ␥-secretase complex assembly but is not directly required for the proteolytic function of ␥-secretase.
Taken together, we demonstrate that the combined overexpression of PS1 and NCT with the loss of function APH-1a G122D mutant fully restores ␥-secretase complex assembly and activity. A similar rescue of inactive APH-1 G122D was not observed upon transfection of other combinations of PS1, NCT, and PEN-2, supporting a putative trimeric complex containing APH-1, PS1, and NCT as an intermediate during ordered complex assembly (14,26). Most likely, the GXXXG motif in APH-1 facilitates the interaction of APH-1 with PS1 or NCT or both PS1 and NCT. Strikingly, PS1 contains a well conserved 378 GXXXG 382 motif, which may mediate the interaction with APH-1, although most GXXXG motifs analyzed so far mediate homodimerization (32,37). Interestingly, the signal peptide peptidase, which does not require additional components (9), shares the GXGD active site motif with PSs but lacks this GXXXG motif (9). Up to now, APH-1a G122D is the only loss of function allele in a ␥-secretase component that can be rescued by overexpression of other ␥-secretase components.