γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β 
protein precursor (AβPP) and of many other important physiological substrates. 
γ-secretase is a multiproteic complex made of four main core components, namely 
presenilin 1 or 2, APH-1, PEN-2, and Nicastrin. Since APH-1 exists as different 
variants, combinations of these proteins can theoretically yield distinct 
γ-secretase complexes. Whether γ-secretase complexes trafficking and targeting 
to either similar or distinct subcellular compartments depend upon their 
molecular composition remains unknown. A differential complex-specific 
distribution may drive a narrow specificity for a subset of substrates that 
would traffic within the same cellular compartments. Here, we generated bigenic 
expression vectors to co-express untagged nicastrin or presenilin 1 together 
with either PEN-2 or distinct variants of APH-1 (aL, aS and b) tagged with 
complementary fragments of the fluorescent protein Venus. We show that these 
constructs allow the formation of functional γ-secretase complexes and their 
visualization with bimolecular fluorescence complementation (BiFC). BiFC can be 
detected at the plasma membrane as well as in endosomes/lysosomes in addition to 
the endoplasmic reticulum (ER) of COS-7 cells transfected with the different 
variants of APH-1. However, the majority of cells co-transfected with APH-1b 
presented BiFC signal only in the ER, suggesting enhanced retention/retrieval of 
APH-1b-containing γ-secretase complexes. Therefore, the new tools described here 
should be helpful to decipher the precise subcellular trafficking of γ-secretase 
complexes and to delineate the distinct variant-linked pathways in various 
cellular systems.