The question of how proteins such as the β-amyloid precursor protein (β-APP), Notch-1 and others can be cleaved in the plane of the membrane has challenged cell biologists and researchers into Alzheimer’s disease for some time. Processing of β-APP by γ-secretase is considered an important therapeutic target, as it constitutes the final step in the release of the amyloid β-peptide (Aβ), the principal constituent of the amyloid plaques in the brains of Alhzeimer’s patients. Recently, γ-secretase activity was linked to two proteins, presenilin-1 (PS1) and presenilin-2 (PS2;refs 1,2). Presenilins are strongly hydrophobic proteins embedded in intracellular membranes, and missense mutations in their genes cause a rare, aggressive form of Alzheimer’s disease³. It has been proposed that two aspartate residues in the transmembrane domains of presenilins constitute the catalytic site of γ-secretase². One argument against this hypothesis is the important residual γ-secretase activity observed in cells derived from PS1- or PS2-deficient mice^1,4. The question is therefore whether cells that are completely devoid of PS1 and PS2 maintain this γ-secretase activity or not.

mNotchΔE is another substrate for PS1-dependent γ-secretase^7,8. Processing of this construct mimics that of full-length Notch-1, and results in release of the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates, together with transcription factors of the CBF-1 family, several genes, includingHES-1 (ref. 9). We therefore co-transfected the embryonic stem cells with plasmids encoding mNotchΔE and luciferase under the control of theHES-1 promoter (kindly provided by R. Kopan and A. Israel), and measured induction of luciferase activity¹⁰. As a control, we also transfected embryonic stem cells with plasmids encoding the NICD. Induction ofHES-1 activity with mNotchΔE was observed inPS1^+/+PS2^–/– cells, but not inPS1^–/–PS2^–/– cells (Fig. 1d). In contrast, NICD strongly induced luciferase activity, implying that the Notch signalling pathway downstream of the proteolytic processing of mNotchΔE was essentially intact inPS1^–/–PS2^–/– cells. This was further confirmed by the observation that signalling inPS1^–/– PS2^–/– cells was also restored after co-transfection with PS1 complementary DNA (data not shown). These observations extend to the molecular level our previous conclusion that the phenotype ofPS1^–/–PS2^–/– embryos is similar to that of Notch-deficient embryos^4,11. We further confirmed that proteolysis of mNotchΔE inPS1^–/–PS2^–/– cells was completely inhibited using a biochemical assay, as before⁷ (Fig. 1e). Quantification of the obtained signals by phosphorimaging indicated that NICD production inPS1^–/–PS2^–/– cells was <1% of that observed inPS1^+/+PS2^–/– cells. InPS1^+/+PS2^–/– cells, a weak NICD signal was already observed after 30 min of pulse labelling (Fig. 1f), whereas it was never observed inPS1^–/–PS2^–/– cells.

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