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, 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. 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. Clathrin-coated vesicles move cell surface receptors to endosomes and return some of them through recycling endosomes back to the plasma membrane (1Brodsky F. M. Chen C. Y. Knuehl C. Towler M. C. Wakeham D. E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (538) Google Scholar, 2van Dam E. M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Crossref PubMed Scopus (178) Google Scholar). Lysosomal enzymes are transported by clathrin coated vesicles from the trans-Golgi network (TGN) 1The abbreviations used are: TGN, trans-Golgi network; BSA, bovine serum albumin; CALM, clathrin assembly lymphoid myeloid leukemia protein; EEA1, early endosomal antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EH domain, Eps15 homology domain; ENTH, epsin N-terminal homology domain; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; MVE, multivesicular endosomes; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; PIP2, phosphatidylinositol 4, 5-bisphosphate; RNAi, RNA interference; siRNA, short interfering RNA; HC, heavy chain; LC, light chain. to the endosomal compartment (1Brodsky F. M. Chen C. Y. Knuehl C. Towler M. C. Wakeham D. E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (538) Google Scholar). Moreover, clathrin coats are also involved in the sorting of mono-ubiquitinated proteins into internal vesicles of multivesicular endosomes (MVE) (3Stahl, P. D. , and Barbieri, M. A. (2002) Science's STKE http: /stke. sciencemag. org/cgi/content/full/OCₛigtrans;2002/141/pe32Google Scholar). MVE play an essential role in the down-regulation of signaling receptors such as the activated epidermal growth factor receptor (EGFR) (4Katzmann D. J. Babst M. Emr S. D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar). Principal structural components of plasma membrane-derived clathrin-coated vesicles are clathrin that consists of three heavy and three light chains and the heterotetrameric adaptor protein AP-2 composed of α, β2, μ2, and σ2 subunits. The structurally related AP-1 adaptor complex is found mainly in the TGN (5Stoorvogel W. Oorschot V. Geuze H. J. J. Cell Biol. 1996; 132: 21-33Crossref PubMed Scopus (320) Google Scholar). The clathrin coat on MVE does not include any of the conventional adaptor complexes (AP-1, M. Oorschot V. J. Mol. Biol. Cell. 2002; 13: PubMed Scopus Google but for the clathrin protein growth factor-regulated tyrosine kinase C. A. J. 2001; PubMed Scopus Google Scholar). to a of proteins are involved in clathrin coat assembly on the plasma include Eps15 and the proteins epsin, CALM, and and through with the membrane phosphatidylinositol the of coat assembly D. J. A. 2001; PubMed Scopus Google Scholar, V. Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that the association of the epsin with the membrane an increase in that the P. W. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). also with the clathrin heavy chain P. PubMed Scopus Google Scholar, Chen P. J. Biol. Full Text Full Text PDF PubMed Scopus Google with the of AP-2 P. PubMed Scopus Google Scholar, Chen P. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Cell. Full Text Full Text PDF PubMed Scopus Google and with of AP-2 D. J. J. PubMed Google Scholar). with clathrin and AP-2 P. PubMed Scopus Google Scholar, Chen P. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. J. PubMed Google Scholar, J. PubMed Scopus Google Scholar, W. E. M. J. PubMed Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). short the of the endocytic proteins with clathrin and Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the more CALM with AP-2 is not as A. Mol. Biol. Cell. PubMed Scopus Google Scholar). from the domain, the chain of both, epsin and are C. J. C. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the efficient of AP-2 and clathrin to of coat it was that the proteins and clathrin are to clathrin-coated on D. J. A. 2001; PubMed Scopus Google Scholar). The endocytic protein Eps15 with the of α-adaptin A. A. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google but not with clathrin A. M. A. J. Cell PubMed Google Scholar). it was on the of plasma membrane clathrin-coated pits A. M. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google and on the membrane of early endosomes Mol. Biol. Cell. PubMed Scopus Google it is to with C. A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The of Eps15 on endosomes is not but transfection with Eps15 that it is essential for the of AP-2 to the plasma membrane A. C. A. J. Cell Biol. PubMed Scopus Google Scholar). are endocytic proteins not that are involved in the of and Cell Biol. 2001; 13: PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google into coated pits and of the and P. Chen C. P. PubMed Scopus Google Scholar, Google Scholar, E. M. J. Cell Biol. PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). the of coated vesicles into the the clathrin coat is by the of and A. P. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, W. PubMed Scopus Google Scholar, A. A. J. Cell Biol. PubMed Scopus Google Scholar, J. Cell Biol. 2001; PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). endocytic proteins such as and are to the in Rev. 2002; PubMed Scopus Google Scholar). The of endocytic proteins in the by homology was by on receptor internalization as an of the protein in it the in the endocytic a protein is Clathrin coat to by a complex of that and of almost any of the endocytic in to the of more alternative that the of components of the for the of proteins is RNA was to the transfection of RNA with in the of and in the depletion of the protein J. W. A. 2001; PubMed Scopus Google Scholar). we in to the of the clathrin heavy chain and that of the AP-2 α-adaptin The were by cell and that proteins were efficiently depleted from the 48 The knockdown of clathrin heavy chain was by the of clathrin light Residual clathrin on the plasma membrane was mainly found in shallow coated The membrane association of the endocytic proteins CALM, and epsin was only uptake was strongly inhibited in the knockdown cells, but the uptake of to However, of the was impaired. Moreover, the of the early endosomal EEA1, of Hrs, and that of the receptor was clathrin depletion a on the membrane association of clathrin and also inhibited transferrin uptake but on and used for were as heavy chain D. J. F. M. A. PubMed Scopus Google F. M. J. Cell Biol. PubMed Scopus Google A. J. PubMed Scopus Google used for were as A. J. PubMed Scopus Google heavy chain and from for and were as clathrin light chains A. J. PubMed Scopus Google Scholar). CALM were the and Eps15 were the To an epsin, the was used as and were by was on by the serum bovine in the were using the to The were with The the receptor was from was from M. and the serum epsin that was used for staining epsin in was from J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and were from The were from transferrin was by to and transferrin were from was from clathrin heavy chain and was from The α-adaptin was from and a α-adaptin was from was from were to the to that only the was was as J. W. A. 2001; PubMed Scopus Google Scholar). Cell and were and in with serum and were to h transfection with the were and a of in serum but the were in was used for the of into the of J. J. Cell 2001; PubMed Google Scholar). of RNA were were with some the was we that the transfection was the for any The of the transfection was were 48 h after transfection in the to the The of was by of cell To for any in cell the of the protein was related to that of in some the from the protein was related to protein as by protein of three transfection in were in the The was with the were with and in were by using J. Biol. Full Text PDF PubMed Google and membrane were using the from of the membrane with the membranes were for in a were and on were in some with clathrin α-adaptin were after h and with the of and on and and knockdown to the 48 h after transfection were for in by in The was used in the were three with and with in for the were in and with the for h were with The were the with and The was also with in and the was h were with in for on the were in were with a with were with a using were with and and h after transfection the were serum for with of in with of transferrin in with were to on and for h on uptake of transferrin were used of a that was by with serum and was to in for of the were as uptake was by a in by a with the A. J. Biol. Full Text PDF PubMed Google Scholar). with were in with and in a transferrin was by using to were by Cell were in for 48 h and with and with The were in of and by The was for in a and were in and to and and in were with and for with in the were for with of for h in a and in an of and The were from the surface of the with to a with a of and and for were into a of and and for was by an for in The was more and to for h and by h were using an were on and with and The were with a of Clathrin Depletion on the of the clathrin heavy we an that the of the heavy chain were with a with the transfection the of the not the To the in clathrin heavy chain the were in and by with the clathrin heavy clathrin light chain and as a was with taken the transfection and the after transfection both, the from the clathrin heavy and light chains were strongly the only of the by and to the that the of the clathrin heavy chain to on with The of was on a of the heavy chain is to the growth of The in heavy chain is by that of the light chains as with a the of the light chains and that light chains are not with heavy also that the heavy chain knockdown affected the of only were after the of the the cell was the but 48 h after transfection still only of that were with The not neither in using and staining for the of the of W. V. P. M. J. PubMed Scopus Google any of in knockdown not the cell after of heavy chain is the of we after The of the of the clathrin heavy chain a of h for clathrin in is in with the of clathrin in of clathrin heavy chain using a A. 2002; PubMed Scopus Google Scholar). However, in a of h was for clathrin in a cell F. M. J. Cell Biol. PubMed Scopus Google Scholar). not the for by is to Dev. 2001; PubMed Scopus Google Scholar). and the The clathrin that was used to the clathrin heavy chain in a in the clathrin in from the heavy chain as an for clathrin siRNA, it to the of the clathrin heavy chain that the staining of clathrin heavy and light chains was in the as from the the of of clathrin and were h after and the of and the clathrin heavy chain knockdown were with the chain we that was more than the is the clathrin heavy chain. is to the of more than a of the light chain by the serum than to a heavy association of light chains with membranes and the chain serum as a for the clathrin heavy chain knockdown in of the that after the clathrin was but it not in the the of clathrin coated is the some staining was still and with a RNA that the of the clathrin N-terminal and we the of the adaptor complexes AP-2 and AP-1 endocytic proteins were affected by the of the clathrin heavy chain to AP-2 a punctate staining from coated on the plasma after clathrin heavy chain depletion and the of the AP-2 were only and by a the AP-1 adaptor to the but it is also (1Brodsky F. M. Chen C. Y. Knuehl C. Towler M. C. Wakeham D. E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (538) Google Scholar, D. J. Emr S. D. Rev. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). clathrin depletion to more to the and of endocytic proteins to the assembly of clathrin-coated pits on the plasma are the proteins CALM and epsin, and and epsin with clathrin and also with the D. J. A. 2001; PubMed Scopus Google Scholar, P. Chen J. A. 2002; PubMed Scopus Google Scholar, A. 2001; PubMed Scopus Google Scholar). To the association of proteins with the plasma membrane is clathrin heavy chain we in CALM with plasma membrane clathrin coated pits as A. Mol. Biol. Cell. PubMed Scopus Google and clathrin knockdown the of CALM was not significantly and The protein we was were with association with the clathrin heavy chain was the staining of Eps15 significantly and by an of Eps15 for the by an membrane association in the of clathrin. to the for the and staining of epsin was not significantly affected by the clathrin knockdown and that the endocytic proteins CALM, epsin, and Eps15 not to on clathrin for association with the plasma Clathrin the receptor from TGN to the endosomal compartment and back from to the TGN C. J. P. P. J. PubMed Scopus Google Scholar). clathrin depletion the receptor more the and Clathrin is also on endosomal membranes it in receptor sorting (5Stoorvogel W. Oorschot V. Geuze H. J. J. Cell Biol. 1996; 132: 21-33Crossref PubMed Scopus (320) Google Scholar). The early endosomal was used to by endosomes were also affected by the clathrin are the in However, of clathrin the endosomes the and was by E. M. Towler M. C. F. M. Mol. Biol. Cell. 2001; PubMed Scopus Google that of clathrin of it was that the hepatocyte growth factor-regulated tyrosine kinase clathrin to early endosomes C. D. J. Cell Biol. 2002; PubMed Scopus Google Scholar). with that of in C. A. D. J. A. J. Cell 2001; PubMed Google Scholar). in after clathrin knockdown was also found on and that and of Clathrin the of clathrin heavy chain depletion on the membrane association of adaptors, and endocytic from and clathrin knockdown were and by into a membrane and a that the of the coat of coated vesicles but coated A. PubMed Scopus Google it also of coat components not The of by and that the membrane association of clathrin was from to and the of the AP-1 was after clathrin the of and CALM, was that of epsin and not any of the of any of the adaptor proteins depletion of the clathrin heavy chain was also for dynamin, is with the A. PubMed Scopus Google of a increase in a of clathrin by RNA of Clathrin of and clathrin knockdown cells, 48 h after were by a in clathrin-coated membranes by The were from three and on the of cell taken from was also that the of shallow coated pits by a than to from in to in clathrin knockdown that clathrin depletion The of shallow coated in knockdown a related of Depletion on Clathrin and AP-2 adaptor is to a of clathrin coats on the plasma membrane it with and clathrin. However, endocytic proteins such as are also of clathrin to and D. J. A. 2001; PubMed Scopus Google Scholar). was of to knockdown the α-adaptin subunit of The of the was the to of The efficiently the of α-adaptin by h and that the α-adaptin a 48 h depletion of α-adaptin in a in cell with the clathrin depletion that to the of α-adaptin than to that of the clathrin heavy chain. The knockdown of the α-adaptin subunit of AP-2 affected also the of the and the was to and the to The in the of the the and is by the that the used for the is of and the related is a of the AP-1 adaptor complex A. J. PubMed Scopus Google Scholar). the subunit is to in the of the of α-adaptin was also in with was a that is and after h that the α-adaptin was strongly in was by a of the clathrin light chain and the on the of α-adaptin to we an almost of membrane not that the AP-2 adaptor is essential for the of clathrin to the plasma was also of to the of α-adaptin depletion on in it was that Eps15 is with AP-2 and for the of AP-2 to the plasma membrane A. M. A. J. Cell PubMed Google Scholar, A. J. A. J. Cell Biol. PubMed Scopus Google Scholar). we in α-adaptin that the association of Eps15 with the plasma membrane to significantly but than that of α-adaptin and The of CALM and and epsin and were in the α-adaptin knockdown Cell that the α-adaptin depletion the of clathrin by that of Eps15 by and that of CALM by The of α-adaptin by that of epsin association of coat proteins α-adaptin and were in and by into a membrane and a The were to and by using The were by that only the clathrin heavy chains and Eps15 were to a into the α-adaptin in the knockdown was that for the of clathrin in coated plasma membrane the α-adaptin subunit of AP-2 is of it also the plasma membrane association of of Clathrin and on and of clathrin and α-adaptin on the of transferrin and were by and uptake The clathrin knockdown strongly inhibited the uptake of transferrin with α-adaptin efficiently the uptake of transferrin and were by uptake transferrin and and more complex were for the uptake of in and that to multivesicular endosomes (3Stahl, P. D. , and Barbieri, M. A. (2002) Science's STKE http: /stke. sciencemag. org/cgi/content/full/OCₛigtrans;2002/141/pe32Google Scholar). clathrin the staining of was of in endosomal the was in of and and To that was taken up by the cells, we the uptake of in and in clathrin only in the uptake of transferrin and clathrin heavy chain and α-adaptin 48 h clathrin and were and with transferrin for h on by an for the The were and by was used to the The is in the as in the to was used for with and depleted of the clathrin heavy chain were with for h on by an for the The were and the was in a are of three that clathrin depletion on the uptake of we the internalization of α-adaptin we to that a AP-2 adaptor is not for the internalization of and Since AP-2 is strongly inhibited after we also the uptake of on after transfection and and that the of is not to a of an alternative uptake for that clathrin and AP-2 are for efficient transferrin the of EGF, clathrin not essential for the uptake but to for of in multivesicular the knockdown of mainly in in on To the of individual endocytic proteins to the of clathrin-coated we used to the clathrin heavy chain and the subunit of the AP-2 adaptor in by transfection with heavy chain-specific the clathrin to than The of clathrin in to only is with the of clathrin in the heavy chain was by a clathrin the of a A. 2002; PubMed Scopus Google Scholar). a of h for the clathrin heavy chain was by in the cell F. M. J. Cell Biol. PubMed Scopus Google Scholar). we not a with clathrin the of clathrin is a of cell with the depletion of the heavy chain we a in light chain that light chains are not in Clathrin depletion on the of adaptors, epsin, and it was that of clathrin RNA in the of of RNA A. PubMed Scopus Google Scholar). to increase in the of an of clathrin-coated pits on the plasma membrane and on endosomes was noted. was the that of the coated pits were to the plasma membrane by Moreover, a early of transferrin uptake the clathrin was by only A. PubMed Scopus Google Scholar). we also a clathrin increase in shallow coated pits the of coated were were not also not an increase in the of clathrin depletion and that of A. PubMed Scopus Google by the of cell The of shallow pits after clathrin depletion in is with in after that clathrin the and J. Cell Biol. 2001; PubMed Scopus Google Scholar). was that the of the clathrin the J. Cell Biol. 2001; PubMed Scopus Google Scholar). a of clathrin to in an of shallow coated is also by the of cell that the clathrin of knockdown was also a that was the of the clathrin knockdown were by and by Whereas after transfection the a depletion of the clathrin heavy chain by coated membranes to by only Depletion of clathrin only a on the membrane association of and CALM and not a on epsin and the of Eps15 in However, we not that an increase in the membrane association of Eps15 clathrin is of the membrane not a from the of Eps15 to the membrane is an of Eps15 for clathrin is not the of the clathrin all endocytic proteins that were punctate staining that are still on the membrane in were with the AP-2 and of clathrin to with into the punctate of AP-2 A. A. J. Cell Biol. PubMed Scopus Google Scholar). AP-1 staining was also not affected in and J. Cell 2001; PubMed Google membrane association of was also clathrin was in A. 2002; PubMed Scopus Google and in that clathrin heavy chain A. PubMed Scopus Google Scholar). Clathrin depletion a in the of and endosomal to the we a of the receptor to are with by clathrin that N-terminal and in cells, a of the endosomal compartment E. M. Towler M. C. F. M. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). Depletion of α-adaptin from not only efficiently but also h more than of the α-adaptin was a for The knockdown also a on the membrane association of clathrin and we only clathrin coated were still in the not the of AP-2 for the membrane association of clathrin was but not for protein was to essential for the of AP-2 to the plasma a Eps15 that of three EH to to membranes and the membrane association of AP-2 and clathrin A. M. A. J. Cell PubMed Google Scholar). a with were with and by for the of Whereas clathrin and AP-2 were completely by Eps15 still a punctate staining an association with the membrane that is of AP-2 and clathrin C. W. J. Cell Biol. PubMed Scopus Google Scholar). strongly a for Eps15 in AP-2 to the plasma However, that Eps15 also AP-2 for to the membrane and that only a complex of AP-2 and Eps15 membrane Eps15 by in than coat but does not the of to the membrane in The of the membrane association of the protein CALM was also after depletion of is to the depletion of AP-2 than that of depletion of clathrin not membrane association of CALM epsin to with the plasma membrane in the of AP-2 and by of for the membrane D. J. A. 2001; PubMed Scopus Google Scholar). To the of the clathrin and α-adaptin we the uptake of transferrin and uptake was blocked clathrin and AP-2 was not it is that the transferrin receptor through clathrin-coated pits M. C. Cell. Full Text PDF PubMed Scopus Google Scholar). uptake by was not significantly affected by clathrin depletion as by the uptake of However, the of from that in we in and are to to is that activated signaling receptors are into the membrane of the vesicles for by However, after clathrin the not in but in that are not from the cell α-adaptin depletion not the of in that in the internalization of does not on the but it is essential for sorting to clathrin coat found on the membrane of MVE in the sorting of receptors into the internal vesicles C. D. J. Cell Biol. 2002; PubMed Scopus Google Scholar). it is that clathrin depletion with a of an of AP-2 in the uptake of A. J. A. J. Cell Biol. PubMed Scopus Google Scholar, C. W. J. Cell Biol. PubMed Scopus Google Scholar, A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. W. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, W. A. Biol. Full Text Full Text PDF PubMed Scopus Google are three that are with that the AP-2 were in with as the receptor A. A. PubMed Scopus Google Scholar). Moreover, it that a subunit of AP-2 the uptake of transferrin but not that of A. A. J. PubMed Scopus Google Scholar). it was that of a of efficiently transferrin not with the uptake of J. J. J. Cell 2002; PubMed Google Scholar). for an alternative of into from we with from membranes and is to the of clathrin coated to an of shallow coated pits A. A. PubMed Scopus Google Scholar). Whereas transferrin was we only of with pits not an alternative uptake for is that the activated membrane that in the of V. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, C. Biol. Full Text Full Text PDF PubMed Google Scholar, J. J. J. Cell 2002; PubMed Google Scholar). the than the clathrin and it is to affected by a clathrin that in uptake clathrin-coated pits only a role but clathrin is essential for in and To the of the clathrin heavy chain siRNA, we with a RNA a the of the heavy chain the of the heavy chain to a was into was the in three a RNA was in the of α-adaptin in cells, and as on the of in and the α-adaptin in clathrin depletion the growth of cells, were of to the depletion of of clathrin Moreover, growth with the depletion of more than of α-adaptin of the a of the of individual endocytic proteins by RNAi, but it also more such as internalization and of the coated in C. and for A. for and M. for essential and and A. for on the