Doppel and PrPC co-immunoprecipitate in detergent-resistant membrane domains of epithelial FRT cells.
Dpl (doppel) is a paralogue of the PrPC (cellular prion protein), whose misfolded conformer (the scrapie prion protein, PrPSc) is responsible for the onset of TSEs (transmissible spongiform encephalopathies) or prion diseases. It has been shown that the ectopic expression of Dpl in the brains of some lines of PrP-knockout mice provokes cerebellar ataxia, which can be rescued by the reintroduction of the PrP gene, suggesting a functional interaction between the two proteins. It is, however, still unclear where, and under which conditions, this event may occur. In the present study we addressed this issue by analysing the intracellular localization and the interaction between Dpl and PrPC in FRT (Fischer rat thyroid) cells stably expressing the two proteins separately or together. We show that both proteins localize prevalently on the basolateral surface of FRT cells, in both singly and doubly transfected clones. Interestingly we found that they associate with DRMs (detergent-resistant membranes) or lipid rafts, from where they can be co-immunoprecipitated in a cholesterol-dependent fashion. Although the interaction between Dpl and PrPC has been suggested before, our results provide the first clear evidence that this interaction occurs in rafts and is dependent on the integrity of these membrane microdomains. Furthermore, both Dpl and PrPC could be immunoprecipitated with flotillin-2, a raft protein involved in endocytosis and cell signalling events, suggesting that they share the same lipid environment.
Caputo
Anna
A
Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli 'Federico II', Napoli 80131, Italy.
Sarnataro
Daniela
D
Campana
Vincenza
V
Costanzo
Maddalena
M
Negro
Alessandro
A
Sorgato
M Catia
MC
Zurzolo
Chiara
C
D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism; D058851:GPI-Linked Proteins; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000378:metabolism; D018621:PrPC Proteins / Q000032:analysis / Q000378:metabolism*; D011328:Prions / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011916:Rats, Inbred F344; D013961:Thyroid Gland / Q000166:cytology
D058851:GPI-Linked Proteins; D008565:Membrane Proteins; D018621:PrPC Proteins; D011328:Prions; C122036:Prnd protein, mouse; C106354:flotillins