Abstract
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.
Highlights
To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the ␣-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%
Epsin and clathrin assembly lymphoid myeloid leukemia protein (CALM)/ AP180 can associate through their ENTH domain with the rare membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which might define the sites of coat assembly [8, 9]
To quantitate the reduction in clathrin heavy chain expression the transfected cells were lysed in SDS-sample buffer and analyzed by immunoblotting with antibodies directed against the clathrin heavy chain, clathrin light chain and actin as a control, respectively (Fig. 1A)
Summary
Antibodies and Reagents—Mouse monoclonal antibodies used for immunofluorescence were as follows: X22, anti-clathrin heavy chain [45]; AP., anti-␣-adaptin [46]; monoclonal antibody 100/3, anti-␥-adaptin, [47]; anti-EEA1 (BD Biosciences, catalog number 610456). Rabbit polyconal antibodies for immunofluorescence and Western blotting were as follows: affinity-purified R461 directed against clathrin light chains [47]. 24 h before transfection with siRNA the cells were trypsindigested and resuspended at a density of 0.5– 0.8 ϫ 105 cells/ml in fresh medium containing 10% fetal calf serum but no pyruvate or antibiotics. Control cells were transfected with firefly luciferase siRNA. For some experiments cells transfected with clathrin or ␣-adaptin siRNA were trypsin-digested after 24 h and mixed with approximately the same number of control cells and replated on glass cover slips. This allowed viewing and photographing control and knockdown cells next to each other within the same field. This allowed viewing and photographing control and knockdown cells next to each other within the same field. 48 h after
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