Retention of the Alzheimer's Amyloid Precursor Fragment C99 in the Endoplasmic Reticulum Prevents Formation of Amyloid β-Peptide

Abstract

γ-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's β-amyloid precursor protein (APP), resulting in the release of amyloid β-peptide (Aβ). The molecular identity of γ-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that γ-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of AJ3. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the Aβ 42/Aβ40 ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of γ-secretase cleavage. Biogenesis of both Aβ40 and Aβ42 was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for γ-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as γ-secretase. The results suggest that presenilins may acquire the characteristics of γ-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.