GENERATION OF ALZHEIMER BETA-AMYLOID PROTEIN IN THE TRANS-GOLGI NETWORK IN THE APPARENT ABSENCE OF VESICLE FORMATION

beta-amyloid protein (AP) formation was reconstituted in permeabilized neuroblastoma cells expressing human Alzheimer beta-amyloid precursor protein (beta APP) harboring the Swedish double mutation associated w ith familial early-onset Alzheimer disease. Permeabilized cells were p repared following metabolic labeling and incubation at 20 degrees C, a temperature that allows beta APP to accumulate in the trans-Golgi net work (TGN) without concomitant A beta formation. Subsequent incubation at 37 degrees C led to the generation of A beta. A beta production in the TGN persisted even under conditions in which formation of nascent post-TGN vesicles was inhibited by addition of guanosine 5'-O-(3-thio triphosphate), a nonhydrolyzable GTP analogue, or by omission of cytos ol. These and other results indicate that vesicle budding and traffick ing may not be required for proteolytic metabolism of beta APP to A be ta, a process that includes ''gamma-secretase'' cleavage within the be ta APP transmembrane domain.