FIGURE 1.

Purification of γ-secretase complexes. A, schematic representation of the subunit composition and structural heterogeneity of the γ-secretase complex. A color code, indicated on top, is applied to the PSEN and APH1 subunits to show local homology between PSEN1 and PSEN2 or APH1A and APH1B subunits, respectively (based on ClustalW using Blosum matrix). PSEN1 and PSEN2 show 65% homology, whereas APH1A and APH1B show 56% homology at the amino acid level. Stars on presenilins TMD6 and TMD7 denote catalytic aspartate residues Asp-257 and Asp-385, respectively (46). Stars on the ectodomain of NCT indicate complex glycosylation. B, schematic representation of the purification methodology. Baculovirus expressed γ-secretase complex is purified using the GFP tag linked to the cytoplasmic domain of NCT. The complex is eluted with Prescission protease. C, Coomassie staining of purified γ-secretase complexes. Next to the γ-secretase subunits, the Prescission protease remains present in all purified samples. An unidentified band is also visible in all purifications (asterisk). D, Western blot of purified γ-secretase complexes. PEN-2 subunit immunoreactivity was used to estimate and normalize γ-secretase complex levels used in further in vitro activity assays.