Gamma-secretase is a multiprotein, intramembrane-cleaving protease with a 
growing list of protein substrates, including the Notch receptors and the 
amyloid precursor protein. The four components of gamma-secretase 
complex--presenilin (PS), nicastrin (NCT), Pen2, and Aph1--are all thought to be 
essential for activity. The catalytic domain resides within PS proteins, NCT has 
been suggested to be critical for substrate recognition, and the contributions 
of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by 
the observation that a critical residue (E332) in NCT, which had been thought to 
be essential for gamma-secretase activity, is instead involved in complex 
maturation. Here, we report that NCT is dispensable for gamma-secretase 
activity. NCT-independent gamma-secretase activity can be detected in two 
independent NCT-deficient mouse embryonic fibroblast lines and blocked by the 
gamma-secretase inhibitors 
N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and 
L-685,458. This catalytic activity requires prior ectodomain shedding of the 
substrate and can cleave ligand-activated endogenous Notch receptors, indicating 
presence of this activity at the plasma membrane. Small interfering RNA 
knockdown experiments demonstrated that NCT-independent gamma-secretase activity 
requires the presence of PS1, Pen2, and Aph1a but can tolerate knockdown of PS2 
or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active 
enzyme, displaying biochemical properties similar to those of gamma-secretase 
and roughly 50% of its activity when normalized to PS1 N-terminal fragment 
levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts 
to stabilize gamma-secretase but is not required for substrate recognition.