gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.

Amyloid ␤-peptide (A␤) is generated by the consecutive cuts of two membrane-bound proteases. ␤-Secretase cuts at the N terminus of the A␤ domain, whereas ␥-secretase mediates the Cterminal cut. Recent evidence suggests that the presenilin (PS) proteins, PS1 and PS2, may be ␥-secretases. Because PSs principally exist as high molecular weight protein complexes, biologically active ␥-secretases likely require other cofactors such as nicastrin (Nct) for their activities. Here we show that preferentially mature Nct forms a stable complex with PSs. Furthermore, we have down-regulated Nct levels by using a highly specific and efficient RNA interference approach. Very similar to a loss of PS function, down-regulation of Nct levels leads to a massive accumulation of the C-terminal fragments of the ␤-amyloid precursor protein. In addition, A␤ production was markedly reduced. Strikingly, downregulation of Nct destabilized PS and strongly lowered levels of the high molecular weight PS1 complex. Interestingly, absence of the PS1 complex in PS1 ؊/؊ cells was associated with a strong downregulation of the levels of mature Nct, suggesting that binding to PS is required for trafficking of Nct through the secretory pathway. Based on these findings we conclude that Nct and PS regulate each other and determine ␥-secretase function via complex formation.

The Alzheimer disease-associated presenilin (PS) proteins apparently provide the active site of ␥-secretase, an unusual intramembrane-cleaving aspartyl protease. PSs principally occur as high molecular weight protein complexes that contain nicastrin (Nct) and additional so far unidentified components. Recently, PEN-2 has been implicated in ␥-secretase function. Here we identify PEN-2 as a critical component of PS1/␥-secretase and PS2/␥-secretase complexes. Strikingly, in the absence of PS1 and PS1/PS2, PEN-2 levels are strongly reduced. Similarly, PEN-2 levels are reduced upon RNA interference-mediated down-regulation of Nct. On the other side, down-regulation of PEN-2 by RNA interference is associated with reduced PS levels, impaired Nct maturation, and deficient ␥-secretase complex formation. We conclude that PEN-2 is an integral ␥-secretase complex component and that ␥-secretase complex components are expressed in a coordinated manner.The Alzheimer disease-associated polytopic membrane proteins presenilin 1 (PS1) 1 and presenilin 2 (PS2) are required for the intramembranous ␥-secretase cleavage of the ␤-amyloid precursor protein (APP) (1). Following an initial cleavage by ␤-secretase within the APP ectodomain, ␥-secretase cleavage releases the 40 -42-amino acid amyloid ␤-peptide (A␤) from the membrane (1). The majority of familial Alzheimer disease cases are associated with mutations in the PS1 gene (1). Apparently all PS1 mutations investigated cause an increased generation of the highly amyloidogenic A␤42 (1). Absence of PS1 reduces ␥-secretase activity (2, 3) and absence of PS1/PS2 eliminates ␥-secretase function completely (4, 5). Mounting evidence suggests that PSs are unusual aspartyl proteases with ␥-secretase activity (6). All PSs contain two aspartates within transmembrane domains 6 and 7 that are critically required for ␥-secretase activity (7,8). Moreover, ␥-secretase inhibitors designed to mimic the transition state of the catalytic mechanism of aspartyl proteases can be covalently cross-linked to PSs (9, 10). Finally, PSs are apparently members of a group of polytopic membrane-bound aspartyl proteases that are all characterized by a GXGD (X ϭ variable amino acid) signature motif in which the C-terminal active site aspartate is embedded (11). Besides the PSs, the bacterial type 4 prepilin peptidases (11, 12) and signal peptide peptidase and its related proteins carry this signature motif (13,14).The PSs reside in high molecular weight complexes (15)(16)(17)(18)). An integral component of these high molecular weight complexes is the membrane glycoprotein nicastrin (Nct) (18 -20). Down-regulation of Nct in cultured mammalian or Drosophila cells inhibits ␥-secretase cleavage of APP (18, 21) and site 3 (S3) cleavage of Notch (21) and reduces PS levels (18,21,22). On the other side, absence of PS1 and PS1/PS2 causes a strong inhibition of Nct maturation (18, 23).PSs are not only required for the ␥-secretase-mediated processing of APP but also for intramembrane proteolysis of several other type I transm...

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