γ-Secretase complexes achieve the production of amyloid peptides playing a key 
role in Alzheimer disease. These proteases have many substrates involved in 
important physiological functions. They are composed of two constant subunits, 
nicastrin and PEN2, and two variable ones, presenilin (PS1 or PS2) and APH1 
(APH1aL, APH1aS, or APH1b). Whether the composition of a given γ-secretase 
complex determines a specific cellular targeting remains unsolved. Here we 
combined a bidirectional inducible promoter and 2A peptide technology to 
generate constructs for the temporary, stoichiometric co-expression of six 
different combinations of the four γ-secretase subunits including EGFP-tagged 
nicastrin. These plasmids allow for the formation of functional γ-secretase 
complexes displaying specific activities and maturations. We show that 
PS1-containing γ-secretase complexes were targeted to the plasma membrane, 
whereas PS2-containing ones were addressed to the trans-Golgi network, to 
recycling endosomes, and, depending on the APH1-variant, to late endocytic 
compartments. Overall, these novel constructs unravel a presenilin-dependent 
subcellular targeting of γ-secretase complexes. These tools should prove useful 
to determine whether the cellular distribution of γ-secretase complexes 
contributes to substrate selectivity and to delineate regulations of their 
trafficking.