To assess the contribution of individual endocytic
proteins to the assembly of
clathrin coated pits, we depleted the
clathrin heavy chain and the
alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with
clathrin heavy chain-specific
short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual
clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of
adaptors, clathrin assembly lymphoid
myeloid leukemia protein (CALM),
epsin,
dynamin, and Eps15 was only moderately affected by the knockdown and all
proteins still displayed a punctate staining distribution.
Clathrin depletion inhibited the uptake of
transferrin but not that of the
epidermal growth factor. However, efficient sorting of the
epidermal growth factor into
hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of
alpha-adaptin abolished almost completely the plasma membrane association of
clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of
transferrin was efficiently blocked in
alpha-adaptin knockdown cells, the internalization and sorting of the
epidermal growth factor was not significantly impaired. Since neither
clathrin nor AP-2 is essential for the internalization of
EGF, we conclude that it is taken up by an alternative mechanism.