INTRODUCTION During receptor-mediated endocytosis, clathrin triskelions polymerize and form clathrin-coated pits on the plasma membrane that then invaginate into the cell to form clathrin- coated vesicles (Keen, 1990; Pearse and Robinson, 1990). Similar clathrin-coated pits also form on the trans-Golgi network. In addition to clathrin and receptors, these pits contain assembly proteins (APs) that catalyze the polymerization of clathrin triskelions and in some cases bind the receptors localized in the clathrin-coated pits. A number of different APs have been described. AP1, AP2, AP3 and AP4 are multimeric subunit complexes of about 270 kDa (Keen, 1990; Robinson and Kreis, 1992; Simpson et al., 1997; Dell’Angelica et al., 1999; Hirst et al., 1999); AP1 occurs on the trans-Golgi network, AP2 on the plasma membrane, AP3 on both the trans-Golgi membrane and endosomes, and AP4 on perinuclear structures. AP180 (Ungewickell and Oestergaard, 1989) and auxilin (Ahle and Ungewickell, 1990) are neuronal specific APs that consist of single subunits of 92 kDa and 100 kDa, respectively; CALM and GAK, which have recently been described, are the non-neuronal homologs of AP180 (Dreyling et al., 1996) and auxilin (Kanaoka et al., 1997; Greener et al., 2000), respectively. Finally β-arrestin is specifically involved in recruiting β-adrenergic receptors to clathrin-coated pits but unlike APs it does not induce clathrin polymerization (Goodman et al., 1996; Goodman et al., 1997). In addition to APs a number of other proteins have been discovered that are involved in the formation, invagination, and pinching off of clathrin coated vesicles including dynamin, amphiphysin, epsin, eps15, endophilin, syndapin I and the small GTPase protein, Rab5-GDI (Van der Bliek et al., 1993; Takei et al., 1995; David et al., 1996; Chen et al., 1998; Tebar et al., 1996; McLauchlan et al., 1998; Ringstad et al., 1999; Schmidt et al., 1999; Qualmann et al., 1999). In addition, rho, rac, phospholipids, and actin are involved in clathrin coat assembly and receptor recruitment (Rapoport et al., 1997; Takei et al., 1998; Lamaze et al., 1996; Lamaze et al., 1997; Munn et al., 1995; Gaidarov et al., 1999) as is dephosphorylation of many of the proteins involved in formation of clathrin-coated pits (Wilde and Brodsky, 1996; Slepnev et al., 1998). Finally, after they pinch off, the clathrin- coated vesicles are uncoated in an ATP dependent process by Hsc70 and its partner proteins, auxilin or cyclin G-associated kinase (GAK). These partner proteins not only assemble clathrin but also have J-domains that enable them to interact with Hsc70 (Prasad et al., 1993; Ungewickell et al., 1995; Jiang et al., 1997; Greener et al., 2000). Recently, Cremona et al. 353 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. Key words: Auxilin, AP180, Endocytosis SUMMARY Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin Xiaohong Zhao 1 , Tsvika Greener 1 , Hadi Al-Hasani 2 , Samuel W Cushman 2 , Evan Eisenberg 1 and Lois E. Greene 1, * 1 Laboratory of Cell Biology, NHLBI and 2 Experimental Diabetes, Metabolism and Nutrition Section, NIDDK, NIH, Bethesda, MD, USA *Author for correspondence (e-mail: greenel@helix.nih.gov) Accepted 27 October 2000 Journal of Cell Science 114, 353-365 © The Company of Biologists Ltd RESEARCH ARTICLE