Direct and Potent Regulation of -Secretase by Its Lipid Microenvironment*

-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP andNotch. Several reports suggest that cleavage of APP to produce the A peptide is regulated in part by lipids. As -secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate -activity. To determine the direct contribution of the lipid microenvironment to -secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional -activity assay was modified to include a detergentremoval step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased -secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APPand Notch-like substrates in a dose-dependentmanner. Reconstitution of -secretase in complex lipid mixtures revealed that a lipid raft-like composition supported thehighest level of activity compared with othermembrane compositions. Taken together, these results demonstrate thatmembrane lipid composition is a direct and potentmodulator of -secretase and that cholesterol, in particular, plays a major regulatory role.