Clathrin-mediated endocytosis in AP-2–depleted cells

TLDR

RNA interference was used to deplete the AP‑2 μ2 subunit and clathrin heavy chain in HeLaM cells. AP‑2 depletion reduces clathrin‑coated pit abundance by 12‑fold and blocks transferrin receptor endocytosis, whereas EGF and LDL receptor uptake remain normal, indicating AP‑2 is not required for clathrin‑coated vesicle formation but is essential for certain cargo, with alternative adaptors compensating for EGF and LDL receptors.

Abstract

We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.