gamma-Secretase cleavage site specificity differs for intracellular and secretory amyloid beta

The final step in A beta generation is the cleavage of the C-terminal 99 amino acid residues of the amyloid precursor protein by gamma-secretase. gamma-Secretase activity is closely linked to the multi-transmembrane-spanning proteins presenilin 1 and presenilin 2. To elucidate whether the cleavage site specificities of gamma-secretase leading to the formation of secreted and intracellular A beta are identical, we made use of point mutations close to the gamma-cleavage site, known to have a dramatic effect on the 42/40 ratio of secreted A beta. We found that the selected point mutations only marginally influenced the 42/40 ratio of intracellular A beta, suggesting differences in the gamma-secretase cleavage site specificity for the generation of secreted and intracellular A beta. The analysis of the subcellular compartments involved in the generation of intracellular A beta revealed that A beta is not generated in the early secretory pathway in the human SH-SY5Y neuroblastoma cell line. In this study we identified late Golgi compartments to be involved in the generation of intracellular A beta. Moreover, we demonstrate that the presence of processed PS1 is not sufficient to obtain gamma-secretase processing of the truncated amyloid precursor protein construct C99, proposing the existence of an additional factor downstream of the endoplasmic reticulum and early Golgi required for the formation of an active gamma-secretase complex.