γ-Secretase is an intramembrane-cleaving aspartyl protease required for the normal development of metazoans because it processes Notch within cellular membranes to release its signaling domain. More than two dozen additional substrates of diverse functions have been reported, including the Notch ligands Delta and Jagged, N- and E-cadherins, and a sodium channel subunit. The protease is causally implicated in Alzheimer’s disease because it releases the neurotoxic amyloid β-peptide (Aβ) from its precursor, APP. γ-Secretase occurs as a large complex containing presenilin (bearing the active site aspartates), nicastrin, Aph-1, and Pen-2. Because the complex contains at least 18 transmembrane domains, crystallographic approaches to its structure are difficult and remote. We recently purified the human complex essentially to homogeneity from stably expressing mammalian cells. Here, we use EM and single-particle image analysis on the purified enzyme, which produces physiological ratios of Aβ40 and Aβ42, to obtain structural information on an intramembrane protease. The 3D EM structure revealed a large, cylindrical interior chamber of ∼20–40 Å in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer. Lectin tagging of the nicastrin ectodomain enabled proper orientation of the globular, ∼120-Å-long complex within the membrane and revealed ∼20-Å pores at the top and bottom that provide potential exit ports for cleavage products to the extra- and intracellular compartments. Our reconstructed 3D map provides a physical basis for hydrolysis of transmembrane substrates within a lipid bilayer and release of the products into distinct subcellular compartments.

Lazarov, V. K., Fraering, P. C., Ye, W., Wolfe, M. S., Selkoe, D. J., & Li, H. (2006). Electron microscopic structure of purified, active γ-secretase reveals an aqueous intramembrane chamber and two pores. Proceedings of the National Academy of Sciences, 103(18), 6889–6894.