title: Visualization of specific γ-secretase complexes using bimolecular fluorescence complementation.
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.
authors.affiliation: 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.
authors.forename: Xavier
authors.initials: X
authors.lastname: Meckler
authors.affiliation: 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.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Checler
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D053764:Presenilin-1 / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009842:Oligopeptides; C449186:PS1 antigen; C469349:PSENEN protein, human; D053764:Presenilin-1; D021122:Protein Subunits; C413604:nicastrin protein; D049452:Green Fluorescent Proteins; D053829:Amyloid Precursor Protein Secretases
keywords: APH-1; PEN-2; fluorescence microscopy; nicastrin; presenilin; transmembrane protein transport; γ-secretase