Affiliations: Université de Nice Sophia-Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, Equipe labellisée Fondation pour la Recherche Médicale et Laboratoire d'Excellence Distalz, Sophia-Antipolis, Valbonne, France
Correspondence:
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Correspondence to: Frédéric Checler, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR7275, 660 route des lucioles, 06560 Valbonne, France. Tel.: +33 4 93 95 34 60; E-mail: checler@ipmc.cnrs.fr.
Abstract: 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.
Keywords: APH-1, fluorescence microscopy, γ-secretase, nicastrin, PEN-2, presenilin, transmembrane protein transport
