title: Comparison of presenilin 1 and presenilin 2 γ-secretase activities using a yeast reconstitution system.
abstract: γ-Secretase is composed of at least four proteins, presenilin (PS), nicastrin (NCT), Aph1, and Pen2. PS is the catalytic subunit of the γ-secretase complex, having aspartic protease activity. PS has two homologs, namely, PS1 and PS2. To compare the activity of these complexes containing different PSs, we reconstituted them in yeast, which lacks γ-secretase homologs. Yeast cells were transformed with PS1 or PS2, NCT, Pen2, Aph1, and artificial substrate C55-Gal4p. After substrate cleavage, Gal4p translocates to the nucleus and activates transcription of the reporter genes ADE2, HIS3, and lacZ. γ-Secretase activity was measured based on yeast growth on selective media and β-galactosidase activity. PS1 γ-secretase was ∼24-fold more active than PS2 γ-secretase in the β-galactosidase assay. Using yeast microsomes containing γ-secretase and C55, we compared the concentration of Aβ generated by PS1 or PS2 γ-secretase. PS1 γ-secretase produced ∼24-fold more Aβ than PS2 γ-secretase. We found the optimal pH of Aβ production by PS2 to be 7.0, as for PS1, and that the PS2 complex included immature NCT, unlike the PS1 complex, which included mature NCT. In this study, we compared the activity of PS1 or PS2 per one γ-secretase complex. Co-immunoprecipitation experiments using yeast microsomes showed that PS1 concentrations in the γ-secretase complex were ∼28 times higher than that of PS2. Our data suggest that the PS1 complex is only marginally less active than the PS2 complex in Aβ production.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan.
authors.forename: Yoji
authors.initials: Y
authors.lastname: Yonemura
authors.forename: Eugene
authors.initials: E
authors.lastname: Futai
authors.forename: Sosuke
authors.initials: S
authors.lastname: Yagishita
authors.forename: Satoshi
authors.initials: S
authors.lastname: Suo
authors.forename: Taisuke
authors.initials: T
authors.lastname: Tomita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Iwatsubo
authors.forename: Shoichi
authors.initials: S
authors.lastname: Ishiura
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D053764:Presenilin-1 / Q000235:genetics / Q000378:metabolism*; D053766:Presenilin-2 / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C508853:PSEN1 protein, human; C508861:PSEN2 protein, human; D053764:Presenilin-1; D053766:Presenilin-2; C413604:nicastrin protein; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases
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