title: Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin.
abstract: Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.
authors.affiliation: Laboratory of Cell Biology, NHLBI and Experimental Diabetes, Metabolism and Nutrition Section, NIDDK, NIH, Bethesda, MD, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zhao
authors.forename: T
authors.initials: T
authors.lastname: Greener
authors.forename: H
authors.initials: H
authors.lastname: Al-Hasani
authors.forename: S W
authors.initials: SW
authors.lastname: Cushman
authors.forename: E
authors.initials: E
authors.lastname: Eisenberg
authors.forename: L E
authors.initials: LE
authors.lastname: Greene
mesh_terms: D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000502:physiology* / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology*; D051275:Glucose Transporter Type 4; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D034041:Monomeric Clathrin Assembly Proteins; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D000070876:Tensins; D014162:Transfection; D014168:Transferrin / Q000378:metabolism*
chemical_list: D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D051275:Glucose Transporter Type 4; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D008840:Microfilament Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C495692:SLC2A4 protein, human; D000070876:Tensins; D014168:Transferrin; C439023:clathrin assembly protein AP180