The transmembrane domain of the amyloid precursor protein in microsomal membranes is on both sides shorter than predicted

Abstract

The amyloid precursor protein is cleaved within its ectodomain by β-amyloid-converting enzyme (BACE) yielding C99, which is further cleaved by γ-secretase within its putative transmembrane domain (TMD). Because it is difficult to envisage how a protease may cleave within the membrane, alternative mechanisms have been proposed for γ-cleavage in which the TMD is shorter than predicted or positioned such that the γ-cleavage site is accessible to cytosolic proteases. Here, we have biochemically determined the length of the TMD of C99 in microsomal membranes. Using a single cysteine mutagenesis scan of C99 combined with cysteine modification with a membrane-impermeable labeling reagent, we identified which residues are accessible to modification and thus located outside of the membrane. We find that in endoplasmic reticulum-derived microsomes the TMD of C99 consists of 12 residues that span from residues 37 to 48, which is N- and C-terminally shorter than predicted. Thus, the γ-cleavage sites are positioned around the middle of the lipid bilayer and are unlikely to be accessible to cytosolic proteases. Moreover, the center of the TMD is positioned at the γ-cleavage site at residue 42. Our data are consistent with a model in which γ-secretase is a membrane protein that cleaves at the center of the membrane.