Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid beta-protein generation

An unusual intramembranous cleavage of the beta-amyloid precursor protein ( APP) by gamma-secretase is the final step in the generation of amyloid beta -peptide (A beta). Two conserved aspartates in transmembrane (TM) domains 6 and 7 of presenilin (PS) 1 are required for A beta production by gamma-sec retase. Here we report that the APP C-terminal fragments, C83 and C99, whic h are the direct substrates of gamma-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 gamma-secretase is i nactivated either pharmacologically or by mutating the PS aspartates, PS1/C 83 and PS1/C99 complexes were detected in Golgi-rich and trans-Golgi networ k rich vesicle fractions. In contrast, complexes of PS1 with APP holoprotei n, which is not the immediate substrate of gamma-secretase, occurred earlie r in endoplasmic reticulum-rich vesicles. The major portion of intracellula r A beta at steady state was found in the same Golgi/trans-Golgi network-ri ch vesicles, and A beta levels in these fractions were markedly reduced whe n either PS1 TM aspartate was mutated to alanine, Furthermore, de novo gene ration of A beta in a cell-free microsomal reaction occurred specifically i n these same vesicle fractions and was markedly inhibited by mutating eithe r TM aspartate, Thus, PSs are complexed with the gamma-secretase substrates C83 and C99 in the subcellular locations where A beta is generated, indica ting that PSs are directly involved in the pathogenically critical intramem branous proteolysis of APP.