Lipid Rafts and Clathrin Cooperate in the Internalization of PrP.sup.C in Epithelial FRT Cells

• Published in: PloS one Vol. 4; no. 6; p. e5829
• Main Authors: Sarnataro, Daniela, Caputo, Anna, Casanova, Philippe, Puri, Claudia, Paladino, Simona, Tivodar, Simona S, Campana, Vincenza, Tacchetti, Carlo, Zurzolo, Chiara
• Format: Journal Article
• Language: English
• Published: Public Library of Science 08.06.2009
• Subjects: Bovine spongiform encephalopathy; Clathrin; Prions (Proteins)
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0005829

The cellular prion protein (PrP.sup.C) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP.sup.Sc). Although endocytosis appears to be required for this conversion, the mechanism of PrP.sup.C internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. We have investigated the mechanism of PrP.sup.C endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP.sup.C internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP.sup.C endocytosis. PrP.sup.C internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP.sup.C co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP.sup.C can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. These findings are of particular interest if we consider that the internalization route/s undertaken by PrP.sup.C can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.