Exchange of clathrin, AP2 and epsin on clathrin-coated pits in permeabilized tissue culture cells

Abstract

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.

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…Full-length GAK was found to bind most tightly to monophosphorylated PIs, notably PI4P Massol et al 2006), which prompted the hypothesis that conversion of PIP2 to PI4P by a PI5 0 phosphatase might act as the trigger recruiting GAK to newly scissioned vesicles (Massol et al 2006). Indeed it was already known that the 5 0 -PI phosphatase synaptojanin is required for CCV uncoating (Verstreken et al 2003) and knockout in mice of auxilin1 þ 2 (Yim et al 2005;Lee et al 2008) or synaptojanin (Cremona et al 1999) similarly led to accumulation of synaptic CCVs. Loss of PIP2 from the newly scissioned vesicle via synaptojanin-mediated PIP2 dephosphorylation would explain why CCVs also lose their AP2 shell because binding of AP2 to the membrane is mediated by PIP2 (Honing et al 2005).…”

Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors

“…Transfected GFP-clathrin on the plasma membrane was imaged using TIR-FM in mock-depleted (e) and GAK-depleted (f) cells. both during clathrin uncoating and clathrin exchange Umeda et al, 2000;Yim et al, 2005).…”

Cellular prion protein (PrPC) protects neuronal cells from the effect of huntingtin aggregation

“…Cells were maintained in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum, 2 mM glutamine, and 1% penicillin-streptomycin in 75 cm 2 culture bottles in a 5% CO 2 atmosphere at 37°C. CHO cells were cultured and seeded as described previously (Yim et al, 2005).…”