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Expression of Auxilin Or AP180 Inhibits Endocytosis by Mislocalizing Clathrin: Evidence for Formation of Nascent Pits Containing AP1 Or AP2 but Not Clathrin

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Expression of Auxilin Or AP180 Inhibits Endocytosis by Mislocalizing Clathrin: Evidence for Formation of Nascent Pits Containing AP1 Or AP2 but Not Clathrin RESEARCH ARTICLE 353 Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin Xiaohong Zhao1, Tsvika Greener1, Hadi Al-Hasani2, Samuel W Cushman2, Evan Eisenberg1 and Lois E. Greene1,* 1Laboratory of Cell Biology, NHLBI and 2Experimental Diabetes, Metabolism and Nutrition Section, NIDDK, NIH, Bethesda, MD, USA *Author for correspondence (e-mail: [email protected]) Accepted 27 October 2000 Journal of Cell Science 114, 353-365 © The Company of Biologists Ltd SUMMARY Although uncoating of clathrin-coated vesicles is a key provided us with an opportunity to determine whether the event in clathrin-mediated endocytosis it is unclear what absence of clathrin from clathrin-coated pits affected the prevents uncoating of clathrin-coated pits before they pinch distribution of the clathrin assembly proteins AP1 and off to become clathrin-coated vesicles. We have shown that AP2. Surprisingly we found almost no change in the the J-domain proteins auxilin and GAK are required for association of AP2 and AP1 with the plasma membrane uncoating by Hsc70 in vitro. In the present study, we and the trans-Golgi network, respectively. This was expressed auxilin in cultured cells to determine if this particularly obvious when auxilin or GAK was expressed would block endocytosis by causing premature uncoating with functional J-domains since, in these cases, almost all of clathrin-coated pits. We found that expression of auxilin of the clathrin was sequestered in granules that also indeed inhibited endocytosis. However, expression of contained Hsc70 and auxilin or GAK. We conclude that auxilin with its J-domain mutated so that it no longer expression of clathrin-binding proteins inhibits clathrin- interacted with Hsc70 also inhibited endocytosis as did mediated endocytosis by sequestering clathrin so that it is expression of the clathrin-assembly protein, AP180, or its no longer available to bind to nascent pits but that assembly clathrin-binding domain. Accompanying this inhibition, we proteins bind to these pits independently of clathrin. observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which Key words: Auxilin, AP180, Endocytosis INTRODUCTION in recruiting β-adrenergic receptors to clathrin-coated pits but unlike APs it does not induce clathrin polymerization During receptor-mediated endocytosis, clathrin triskelions (Goodman et al., 1996; Goodman et al., 1997). polymerize and form clathrin-coated pits on the plasma In addition to APs a number of other proteins have been membrane that then invaginate into the cell to form clathrin- discovered that are involved in the formation, invagination, and coated vesicles (Keen, 1990; Pearse and Robinson, 1990). pinching off of clathrin coated vesicles including dynamin, Similar clathrin-coated pits also form on the trans-Golgi amphiphysin, epsin, eps15, endophilin, syndapin I and the network. In addition to clathrin and receptors, these pits small GTPase protein, Rab5-GDI (Van der Bliek et al., 1993; contain assembly proteins (APs) that catalyze the Takei et al., 1995; David et al., 1996; Chen et al., 1998; Tebar polymerization of clathrin triskelions and in some cases bind et al., 1996; McLauchlan et al., 1998; Ringstad et al., 1999; the receptors localized in the clathrin-coated pits. A number of Schmidt et al., 1999; Qualmann et al., 1999). In addition, rho, different APs have been described. AP1, AP2, AP3 and AP4 rac, phospholipids, and actin are involved in clathrin coat are multimeric subunit complexes of about 270 kDa (Keen, assembly and receptor recruitment (Rapoport et al., 1997; 1990; Robinson and Kreis, 1992; Simpson et al., 1997; Takei et al., 1998; Lamaze et al., 1996; Lamaze et al., 1997; Dell’Angelica et al., 1999; Hirst et al., 1999); AP1 occurs on Munn et al., 1995; Gaidarov et al., 1999) as is the trans-Golgi network, AP2 on the plasma membrane, AP3 dephosphorylation of many of the proteins involved in on both the trans-Golgi membrane and endosomes, and AP4 on formation of clathrin-coated pits (Wilde and Brodsky, 1996; perinuclear structures. AP180 (Ungewickell and Oestergaard, Slepnev et al., 1998). Finally, after they pinch off, the clathrin- 1989) and auxilin (Ahle and Ungewickell, 1990) are neuronal coated vesicles are uncoated in an ATP dependent process by specific APs that consist of single subunits of 92 kDa and 100 Hsc70 and its partner proteins, auxilin or cyclin G-associated kDa, respectively; CALM and GAK, which have recently been kinase (GAK). These partner proteins not only assemble described, are the non-neuronal homologs of AP180 (Dreyling clathrin but also have J-domains that enable them to interact et al., 1996) and auxilin (Kanaoka et al., 1997; Greener et al., with Hsc70 (Prasad et al., 1993; Ungewickell et al., 1995; Jiang 2000), respectively. Finally β-arrestin is specifically involved et al., 1997; Greener et al., 2000). Recently, Cremona et al. 354 JOURNAL OF CELL SCIENCE 114 (2) (Cremona et al., 1999) have shown that hydrolysis of PIP2 by USA). Monoclonal anti-HA antibody (HA. 11) was purchased from synaptojanin is also important for uncoating in vivo. Berkeley Antibody Co. (Richmond, CA, USA). Rabbit antibody An important question regarding regulation of uncoating by against Golgi β coatomer (β-COP), monoclonal anti-clathrin heavy Hsc70 is why premature uncoating of clathrin-coated pits does chain (X22), monoclonal anti-α-adaptin (AP.6) are from Affinity BioReagents, Inc. (Golden, CO, USA). Monoclonal antibody against not occur before they pinch-off to form clathrin-coated γ vesicles. Since the J-domain proteins auxilin and GAK are -adaptin of AP1 (100/3) was obtained from Sigma (St Louis, MO, USA). Monoclonal mouse anti-human transferrin receptor antibody critical for uncoating it seemed possible that the level of auxilin was purchased from Biomeda Corp. (Foster City, CA, USA). A rabbit or GAK present in the cell would not only affect the rate and antiserum to human transferrin was obtained from Boehringer extent of uncoating of clathrin-coated vesicles but also whether Mannhein (Indianapolis, IN, USA). Monoclonal and polyclonal or not clathrin-coated pits were uncoated by Hsc70; if excess antibodies against Hsc70 were purchased from Stressgen auxilin or GAK caused premature uncoating of clathrin-coated Biotechnologies Corp. (Victoria, BC, Canada). Fluorescence- pits before they pinched off to form clathrin-coated vesicles, it conjugated secondary antibodies were from Jackson might markedly inhibit clathrin-mediated endocytosis. On the ImmunoResearch laboratories, Inc. (West Grove, PA, USA). 125I- other hand, quantitative western blot analysis showed that the sheep anti-mouse antiserum was from Amersham Pharmacia Biotech level of auxilin present in neuronal cells is almost 10 times the (Piscataway, NJ, USA). level of GAK present in non-neuronal cells (Greener et al., Plasmid construction 2000), probably because recycling of synaptic vesicles requires Auxilin and AP180 and their truncated mutants were prepared as Flag clathrin-mediated endocytosis to occur much more rapidly in fusion proteins (Fig. 1A-B) using a pFlag-CMV-2 expression vector neuronal cells than in non-neuronal cells. Therefore, markedly from Kodak Scientific Imaging System (Rochester, NY, USA). increasing the level of auxilin or GAK present in cultured cells Auxilin and AP180 cDNA were subcloned to give pTG176 and might actually increase the rate of uncoating of clathrin-coated pTG135 expressing wild-type AP180 and wild-type auxilin, vesicles and thereby increase the rate of clathrin-mediated respectively. This wild-type construct of auxilin was later used to endocytosis rather than inhibit it. make pTG177 expressing mutated auxilin contains a non-active J- In the present study, we found that expression of auxilin or domain, where the HPDK conserved motif was changed to AAAK. GAK markedly decreased clathrin-mediated endocytosis in N- and C-terminal fragments of auxilin were made as follows. The HeLa and Cos cells and, at the same time, in many of the cells first 1,224 base pairs of auxilin cDNA were subcloned to give pTG168 expressing the 45 kDa N-terminal fragment of auxilin which contains led to the formation of clathrin-Hsc70-auxilin granules in the the tensin domain. The last 1,521 base pairs of auxilin cDNA were cytosol and a decrease in clathrin associated with clathrin- subcloned to give pTG197 expressing the 56 kDa C-terminal fragment coated pits on the plasma membrane and the trans-Golgi of auxilin which contains the clathrin binding and J-domains. N- and network. However, clathrin-mediated endocytosis was also C-terminal fragments of AP180 were made as follows. The first 1,674 inhibited by auxilin with its J-domain mutated so that it no base pairs of AP180 cDNA were subcloned to give pTG190 longer supported uncoating by Hsc70 in vitro although in this expressing the 61 kDa N-terminal domain of AP180 containing the case the clathrin in the cytosol did not form granules but domain which interacts with phospholipase D. The last 1,791 base appeared to become aggregated in the cytosol. A similar effect pairs of AP180 cDNA were subcloned to give pTG192 expressing the occurred when AP180 or its clathrin-binding domain was 65 kDa C-terminal domain of AP180 containing its clathrin binding expressed. Surprisingly, however, in none of these cases was domain (Lee et al., 1997). GAK was subcloned into GFP vector (Kioka et al., 1999) with the epitope tag at the N-terminal of GAK. localization of AP1 or AP2 affected despite the mislocalization of clathrin suggesting first, that expression of clathrin-binding Endocytosis assays proteins inhibits endocytosis by causing mislocalization of For immunofluorescence microscopy studies, cells were grown on clathrin away from nascent pits, and second, that the binding glass coverslips and transfected 24-48 hours before the assay. Cells of APs to these pits occurs independently of clathrin.
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