Inhibition by KMI-574 Leads to Dislocalization of BACE1 From Lipid Rafts

BACE1 initiates processing of the amyloid precursor protein (APP) in the production of amyloid beta (A beta) peptide. After P-cleavage by BACE1, the C-terminal stub of the APP fragment is further processed by the gamma-secretase complex to produce A beta. Because APP, A beta, the -ysecretase complex, and BACE1 are found in lipid raft membranes, A beta production is widely accepted to occur in lipid rafts. However, whether BACE1 is activated within the rafts is unclear. To analyze the relationship between the activity and the localization of BACE1, we used a new BACE1 inhibitor, KMI-574, and separated raft membranes on sucrose density gradients. In the presence of KMI-574, the localization of BACE1 shifted from the rafts to nonraft membranes in HEK293 cells. We also analyzed the proteolytically inactive mutants, D93A, D289A, and D93A/D289A, of BACE1. These mutants also moved from rafts to nonrafts, and the D93A/D289A double-mutant localized exclusively to nonraft membranes. The mutants were defective in maturation by glynosylation and formed hyperoligomers, suggesting that the BACE1 oligomers could not exit from the ER and be transported to the Golgi apparatus. Our findings suggest that the activated conformation of BACE1 is important for protein transport and localization to lipid rafts. (c) 2008 Wiley-Liss, Inc.