Clathrin-independent internalization of normal cellular prion protein in neuroblastoma cells is associated with the Arf6 pathway

To understand the role of clathrin-mediated endocytosis in the internalization of normal cellular prion protein (PrP^c) in neuronal cells, N2a cells were depleted of clathrin by RNA interference. PrP^c internalization via the constitutive endocytic pathway in the absence of Cu²⁺ and the stimulated pathway in the presence of Cu²⁺ were measured in both control and clathrin-depleted cells. Depletion of clathrin had almost no effect on the internalization of PrP^c either in the presence or absence of Cu²⁺, in contrast to the marked reduction observed in transferrin uptake. By contrast, the internalization of PrP^c was inhibited by the raft-disrupting drugs filipin and nystatin, and by the dominant-negative dynamin-1 mutant dynamin-1 K44A, both in the presence and absence of Cu²⁺. The internalized PrP^c was found to colocalize with cargo that traffic in the Arf6 pathway and in large vacuoles in cells expressing the Arf6 dominant-active mutant. These results show that PrP^c is internalized in a clathrin-independent pathway that is associated with Arf6.