Effect of Clathrin Heavy Chain- and α-Adaptin-specific Small Inhibitory RNAs on Endocytic Accessory Proteins and Receptor Trafficking in HeLa Cells
To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the α-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, th...
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Veröffentlicht in: | The Journal of biological chemistry 2003-11, Vol.278 (46), p.45160 |
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Sprache: | eng |
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Zusammenfassung: | To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin
heavy chain and the α-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 α-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 α-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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M307290200 |