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 Cu(2+) and the stimulated pathway in the presence of Cu(2+) 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 Cu(2+), 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 Cu(2+). 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.