Visualization of specific γ-secretase complexes using bimolecular fluorescence complementation.

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.