Clathrin and clathrin adaptors on clathrin-coated pits exchange with cytosolic clathrin and clathrin adaptors in vivo. This exchange might require the molecular chaperone Hsc70 and J-domain-protein auxilin, which, with ATP, uncoat clathrin-coated vesicles both in vivo and in vitro. We find that, although Hsc70 and ATP alone could not uncoat clathrin-coated pits, further addition of auxilin caused rapid uncoating of clathrin but not AP2 and epsin. By contrast, cytosol uncoats clathrin, AP2 and epsin from pits in permeabilized cells, and, concomitantly, these proteins in the cytosol rebind to the same pits, establishing that, like in vivo, these proteins exchange in permeabilized cells. Dissociation and exchange of clathrin in permeabilized cells can be prevented by inhibiting Hsc70 activity. The presence of clathrin-exchange in the permeabilized system substantiates our in vivo observations, and is consistent with the view that Hsc70 and auxilin are involved in the clathrin-exchange that occurs as clathrin-coated pits invaginate in vivo.
To understand the role of clathrin-mediated endocytosis in the internalization of normal cellular prion protein (PrPc) in neuronal cells, N2a cells were depleted of clathrin by RNA interference. PrPc 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 PrPc either in the presence or absence of Cu2+, in contrast to the marked reduction observed in transferrin uptake. By contrast, the internalization of PrPc 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 PrPc 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 PrPc is internalized in a clathrin-independent pathway that is associated with Arf6.
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