title: Oligomerization is required for normal endocytosis/transcytosis of a GPI-anchored protein in polarized hepatic cells.
abstract: Targeting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) in polarized epithelial cells depends on their association with detergent-resistant membrane microdomains called rafts. In MDCK cells, GPI-APs associate with rafts in the trans-Golgi network and are directly delivered to the apical membrane. It has been shown that oligomerization is required for their stabilization in rafts and their apical targeting. In hepatocytes, GPI-APs are first delivered to the basolateral membrane and secondarily reach the apical membrane by transcytosis. We investigated whether oligomerization is required for raft association and apical sorting of GPI-APs in polarized HepG2 cells, and at which step of the pathway oligomerization occurs. Model proteins were wild-type GFP-GPI and a double cysteine GFP-GPI mutant, in which GFP dimerization was impaired. Unlike wild-type GFP-GPI, which was efficiently endocytosed and transcytosed to the apical surface, the double cysteine mutant was basolaterally internalized, but massively accumulated in early endosomes, and reached the bile canaliculi with delayed kinetics. The double cysteine mutant was less resistant to Triton X-100 extraction, and formed fewer high molecular weight complexes. We conclude from these results that, in hepatocytes, oligomerization plays a key role in targeting GPI-APs to the apical membrane, by increasing their affinity for rafts and allowing their transcytosis.
authors.affiliation: INSERM, UMR_S938, Centre de Recherche Saint-Antoine, Paris, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Galmes
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Delaunay
authors.forename: Michèle
authors.initials: M
authors.lastname: Maurice
authors.forename: Tounsia
authors.initials: T
authors.lastname: Aït-Slimane
mesh_terms: D048708:Cell Growth Processes / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology; D003545:Cysteine; D004705:Endocytosis / Q000502:physiology; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D021381:Protein Transport; D057900:Transcytosis
chemical_list: D058851:GPI-Linked Proteins; D003545:Cysteine
keywords: Hepatocyte; Rafts; Transcytosis