Effect of clathrin heavy chain- and alpha-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 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.
Hinrichsen
Lars
L
Department of Cell Biology in the Center of Anatomy, Hannover Medical School, D-30623 Hannover, Germany.
Harborth
Jens
J
Andrees
Lars
L
Weber
Klaus
K
Ungewickell
Ernst J
EJ
D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics*; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000235:genetics*; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D033965:Adaptor Protein Complex alpha Subunits; D002966:Clathrin; D034741:RNA, Small Interfering; D014168:Transferrin; D033922:Clathrin Heavy Chains; D004815:Epidermal Growth Factor