Lipid rafts and clathrin cooperate in the internalization of PrP in epithelial FRT cells.
BACKGROUND
The cellular prion protein (PrP(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(Sc)). Although endocytosis appears to be required for this conversion, the mechanism of PrP(C) internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved.


RESULTS
We have investigated the mechanism of PrP(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(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(C) endocytosis. PrP(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(C) co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP(C) can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization.


CONCLUSIONS
These findings are of particular interest if we consider that the internalization route/s undertaken by PrP(C) can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.
Sarnataro
Daniela
D
Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, Napoli, Italy.
Caputo
Anna
A
Casanova
Philippe
P
Puri
Claudia
C
Paladino
Simona
S
Tivodar
Simona S
SS
Campana
Vincenza
V
Tacchetti
Carlo
C
Zurzolo
Chiara
C
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis; D005799:Genes, Dominant; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry*; D011328:Prions / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D013961:Thyroid Gland / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
D002135:Calcium-Binding Proteins; D051242:Caveolin 1; D002966:Clathrin; C526966:Eps15 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D011328:Prions; D002784:Cholesterol; D020764:cdc42 GTP-Binding Protein; D034285:Dynamin II