Partitioning of γ-Secretase and its Substrates in Lipid Microdomains

Abstract

γ-Secretase is a multiprotein complex that catalyzes intramembranous cleavage of mutiple transmembrane I proteins. The sequential proteolysis of the amyloid precursor protein (APP) by γ-Secretase generates amyloid beta peptides that form plaques, one of the main pathological hallmarks of Alzheimer's disease. γ-Secretase also cleaves Notch the abnormal signaling of which can lead to cancer. The lipid environment regulates the location and activity of many transmembrane proteins and defines membrane micro or nanodomains, which serve as spatio-temporal platforms for proteins to function properly, and therefore have a modulatory impact on the intramembrane proteolysis. Mechanistically, the lipids may affect substrate recognition by binding to γ-secretase and substrates and by excluding or enriching both partners within a microdomain. Despite substantial advances in elucidating how this enzyme complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Here, we characterize the partitioning of APP and Notch1 substrate and γ-secretase using solid supported membranes to determine whether the function and activity of γ-Secretase and its substrates r is influenced by the membrane lateral organization. As a membrane model we chose a canonical raft mixture containing phosphatidylcholine, sphingomyelin and cholesterol. Atomic force microscopy (AFM) data revealed membrane heterogeneities, ordered (lo) and disordered (ld) domains, and the selective localization and clustering of membrane proteins. Notch substrate is uniformly distributed in the ld phase while APP substrate clusters prefer the boundary between ld and lo domains in agreement with fluorescence microscopy measurements carried out on proteolipobeads, suggesting that proteolysis and specificity of these substrates are modulated by membrane microenvironement. In addition, AFM time-course measurements suggests that γ-secretase recruits specific membrane components and thus creates a favorable lipidic environment for its assembly and activity.