γ-Secretase Activity Is Present in Rafts but Is Not Cholesterol-Dependent

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Abstract
Cholesterol has been claimed to be involved in the generation and/or accumulation of amyloid β protein (Aβ). However, the underlying molecular mechanisms have not been fully elucidated yet. Here, we have investigated the effect of membrane cholesterol content on γ-secretase activity using Chinese hamster ovary cells stably expressing β-amyloid precursor protein (APP) and either wild-type or N141I mutant-type presenilin 2. Cholesterol was acutely depleted from the isolated membrane by methyl-β-cyclodextrin, and Aβ production was assessed in a cell-free assay system. Reduced cholesterol did not significantly alter the amounts of Aβ produced by either total cell membranes or cholesterol-rich low-density membrane domains. Even its extremely low levels in the latter domains did not affect Aβ production. This indicates that the membrane cholesterol content does not directly modulate the activity of γ-secretase. To ascertain that γ-secretase resides in cholesterol-rich membrane domains, low-density membrane domains were further fractionated with BCθ (biotinylated θ-toxin nicked with subtilisin Carlsberg protease), which has recently been shown to bind selectively to rafts of intact cells. The membrane domains purified with BCθ did indeed produce Aβ. These observations indicate that the γ-cleavage required for generating Aβ occurs in rafts, but its activity is virtually cholesterol-independent.