beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms

We have analyzed the cellular processing pathways which produce the 4-kDa amyloid beta-peptide (A beta) and a 3-kDa derivative (p3) of the beta-amyloid precursor protein (beta APP) found in conditioned media of tissue culture cells and in cerebrospinal fluid. Pulse-chase experiments reveal that both peptides are secreted in parallel with soluble beta APP (APPs); no precursor-product relation between A beta and p3 was found. The protease inhibitor leupeptin did not influence the production of either peptide. In contrast, the weak base ammonium chloride (NH4Cl) showed a dose-dependent inhibition of A beta production with less decrease in p3. A similar effect was observed using the monovalent ionophore monensin. Brefeldin A completely inhibited the generation of both peptides, indicating that proteases located in the endoplasmic reticulum or early Golgi are not sufficient for the production of the small peptides. Deletion of the beta APP cytoplasmic domain, which removes a consensus sequence that probably mediates reinternalization, caused an increase in secretion of both APPs and p3 and did not abolish A beta production. These observations suggest that completely mature beta APP within the late Golgi and/or at the cell surface is a prerequisite for A beta production but processing within the lysosome might not be directly required. p3 appears to derive from the 10-kDa C-terminal stub of beta APP following secretion of APPs.