An unusual intramembranous cleavage of the β-amyloid precursor protein (APP) by γ-secretase is the final step in the generation of amyloid β-peptide (Aβ). Two conserved aspartates in transmembrane (TM) domains 6 and 7 of presenilin (PS) 1 are required for Aβ production by γ-secretase. Here we report that the APP C-terminal fragments, C83 and C99, which are the direct substrates of γ-secretase, can be coimmunoprecipitated with both PS1 and PS2. PS/C83 complexes were detected in cells expressing endogenous levels of PS. The complexes accumulate when γ-secretase is inactivated either pharmacologically or by mutating the PS aspartates. PS1/C83 and PS1/C99 complexes were detected in Golgi-rich and trans-Golgi network-rich vesicle fractions. In contrast, complexes of PS1 with APP holoprotein, which is not the immediate substrate of γ-secretase, occurred earlier in endoplasmic reticulum-rich vesicles. The major portion of intracellular Aβ at steady state was found in the same Golgi/trans-Golgi network-rich vesicles, and Aβ levels in these fractions were markedly reduced when either PS1 TM aspartate was mutated to alanine. Furthermore, de novo generation of Aβ in a cell-free microsomal reaction occurred specifically in these same vesicle fractions and was markedly inhibited by mutating either TM aspartate. Thus, PSs are complexed with the γ-secretase substrates C83 and C99 in the subcellular locations where Aβ is generated, indicating that PSs are directly involved in the pathogenically critical intramembranous proteolysis of APP.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 1 Aug 2000|