Gamma-secretase catalyzes the proteolytic processing of a number of integral 
membrane proteins, including amyloid precursor protein (APP) and Notch. The 
native gamma-secretase is a heterogeneous population of large membrane protein 
complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, 
nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase 
complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, 
nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and 
the Notch-like substrate N160 and displays the characteristic features of 
gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor, 
upregulation of Abeta42 production by a familial Alzheimer's disease (FAD) 
mutation in the APP gene, and downregulation of Notch processing by PS1 FAD 
mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted 
gamma-secretase is significantly higher than that of the native enzyme from 293 
cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in 
Abeta42 production, PS1 FAD missense mutations in the reconstitution system 
alter the cleavage sites in the C99 substrate without changing the 
Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in 
both C99 and N160 processing. Reconstitution of gamma-secretase provides a 
homogeneous system for studying the individual gamma-secretase complexes and 
their roles in Abeta production, Notch processing and AD pathogenesis. These 
studies may provide important insight into the development of a new generation 
of selective gamma-secretase inhibitors with an improved side effect profile.