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. were washed three times with PBS and incubated with DMEM containing 0. 5% BSA for 30 minutes at 37°C. Human transferrin was then added to the media at the final concentration of 30 µg/ml. Incubation was continued at 37°C for 5 to 10 minutes. To measure MATERIALS AND METHODS bulk fluid-phase endocytosis, 1 mg/ml lysine-fixable FITC-dextran (70,000) from Molecular Probes was added to the cells in DMEM Cell culture and transfection containing 0.5% BSA for 1 hour at 37°C. Cells were washed quickly HeLa and Cos cells were purchased form ATCC. Mouse with PBS, fixed in 2% formaldehyde and processed for neuroblastoma N2A cells were a gift from Dr Y. Peng Loh (NICHD, immunofluorescence microscopy. NIH). Cells were maintained in DMEM supplemented with 10% To measured transferrin internalization biochemically, a modified fetal bovine serum, 2 mM glutamine, penicillin (100 unit/ml), and biotinylated transferrin uptake assay (Smythe et al., 1992) was used. streptomycin (100 unit/ml) in a humidified incubator with 5% CO2 at Briefly, Cos cells grown in 6-well plates were first depleted of 37°C. All media and supplements were obtained from Biofluids, Inc. endogenous transferrin. Biotinylated transferrin was then added and (Rockville, MD, USA). For the purpose of immunofluorescence incubated with cells for minutes. After removing free biotinylated studies calcium phosphate precipitation was used to transfect cell. In transferrin by washing, avidin was added to the plates to mask surface- order to get higher transfection efficiency in biochemical assays, bound biotinylated transferrin. Internalized biotin activity in cell SuperFect (QIAGEN, Valencia, CA, USA) was used as transfection lysates were then assayed quantitatively using streptavidin- reagent. horseradish peroxidase in an ELISA plate coated with a rabbit antibody against transferrin. Experiments were performed in Antibodies triplicate. Monoclonal antibody M5 against Flag was obtained from Kodak Scientific Imaging System (Rochester, NY USA). A rabbit antiserum Immunofluorescence microscopy to Flag was from ZYMED Laboratories, Inc. (San Francisco, CA, Cells were treated as indicated in each experiment, fixed in 2% Expression of auxilin or AP180 inhibits endocytosis 355

Fig. 1. Auxilin and AP180 DNA constructs used in the study. (A) Auxilin; (B) AP180. All constructs are N-terminal Flag-tagged. formaldehyde at room temperature for 15 minutes. After washing the cells three time with PBS containing 10% FBS, cells were incubated with primary antibodies for 1 hour at room temperature. Cells were washed again three times and incubated with fluorescence-conjugated secondary antibodies. GLUT4 glucose transporter endocytosis Rat adipose cells from male rats were prepared and GLUT4 endocytosis assays were conducted as previously described (Al- Hasani et al., 1998). Briefly, cells were transfected by electroporation with HA- GLUT4 alone or cotransfected with HA- GLUT4 and Flag-AP180, auxilin or their mutants as indicated. Cell surface GLUT4 in absence or presence of insulin (1×104 microunits/ml) were measured by the binding of monoclonal anti-HA antibody to cell surface HA-GLUT4 followed by the addition of 125I-sheep anti-mouse antibody. Cell surface associated radioactivity was counted in a γ-counter. Unless stated otherwise, the values obtained from transfected cells were subtracted from all other values to correct nonspecific antibody binding. Antibody uptake. Likewise, in HeLa cells, expression of auxilin binding assays were routinely performed in duplicate, but markedly inhibited transferrin uptake (Fig. 2C,D). occasionally were done in quadruplicate. Quantification of this effect (Table 1) showed that 10% of the control cells had little or no transferrin uptake compared to 65% of the transfected cells. Interestingly, while in both RESULTS transfected HeLa and Cos cells the expressed auxilin was cytosolic, its distribution did not appear to be uniform. Effect of auxilin on transferrin endocytosis Although the expressed auxilin varied from a grainy To determine the effect of expression of auxilin in cultured appearance to obvious speckles which ranged in size from tiny cells, we first expressed auxilin with a N-terminal Flag epitope particles to large granules, its cellular appearance did not seem in Cos and HeLa cells. Fig. 2A shows the fluorescence to be related to the inhibition of transferrin uptake. In contrast, obtained when Cos cells were stained with an anti-Flag the uptake of FITC-dextran, a marker for bulk fluid-phase antibody to detect the expressed auxilin. Transferrin uptake in endocytosis, was comparable in auxilin transfected and control the same cells was also imaged by immunofluorescence cells (Fig. 2E,F). These results establish that expression of microscopy as shown in Fig. 2B. Comparison of Fig. 2A and auxilin specifically affects clathrin-dependent receptor B shows that, in the non-transfected cells, the transferrin was mediated endocytosis in transfected cells, but not clathrin- mainly localized to the recycling endosome, whereas the cells independent fluid phase uptake. expressing auxilin showed marked inhibition of transferrin To further verify that auxilin inhibits transferrin uptake, we

Table 1. Percentage of cells with reduced transferrin uptake Expressed protein J-domain Cells None Auxilin Auxilin-C Auxilin-N auxilin mutant AP180 AP180-C AP180-N 10 69 50 19 65 94 91 22 HeLa (n=199) (n=86) (n=195) (n=203) (n=57) (n=112) (n=160) (n=117) 10 67 55 7 66 98 98 13 Cos (n=151) (n=39) (n=87) (n=41) (n=70) (n=56) (n=48) (n=77) Transfection, transferrin uptake and immunoﬂuorescence microscopy were performed on cells as described in Materials and Methods. The total number of transfected cells on the slides were counted as well as the number of transfected cells that showed signiﬁcantly reduced transferrin uptake. The results are expressed as the percentage of transfected cells that displayed reduced transferrin internalization. About 10% of control cells show very little uptake of transferrin in a typical experiment under our experimental conditions. 356 JOURNAL OF CELL SCIENCE 114 (2)

Fig. 2. Effect of auxilin expression on transferrin and fluid phase uptake. Cells grown on coverslips were transiently transfected with Flag-tagged auxilin (A-F), auxilin C-terminal fragment (G,H), or full length auxilin with a nonfunctional J- domain mutant (I,J). Transferrin uptake assay and fluid phase uptake using FITC- labeled dextran were performed as described in Materials and Methods. (A,C,E,G,I) indicate transfected cells which was detected using mAb M5 anti-Flag antibody. (B,D,H,J) Transferrin internalization; (F) FITC-labeled dextran. (A,B), Cos cells; (C-J), HeLa cells. biochemically compared the uptake of biotinylated transferrin in mock and auxilin transfected Cos cells. Using Cos cells in which about 30% of the population was transfected with auxilin, we found that these cells took up about 30% less transferrin than the mock-transfected cells in good agreement with the 30% transfection efficiency (data not shown). Therefore, both biochemical and fluorescence microscopy studies showed that transient expression of auxilin markedly inhibits clathrin-mediated endocytosis. Since auxilin contains three domains, we next investigated which of these domains is required for inhibition of endocytosis. We expressed either the N- terminal tensin domain, the C-terminal portion of auxilin lacking the tensin domain but containing the clathrin- binding domain and the J-domain, or full-length auxilin with the critical residues, HPDK, of the J-domain (Sell et al., 1990; Tsai and Douglas, 1996) mutated to AAAK, so that, in vitro, we found that the mutated auxilin no longer supported uncoating (data not shown). As shown in Table 1, expression of the N-terminal tensin domain of auxilin in both HeLa and Cos cells had no significant effect on transferrin uptake, whereas, the C-terminal portion of auxilin, which acts like intact auxilin in vitro in uncoating clathrin coated vesicles, also acted like intact auxilin in vivo, inhibiting endocytosis and forming auxilin granules in the cytosol (Fig. 2G,H). We next tested whether the expressed auxilin had to interact with Hsc70 in order to inhibit endocytosis by expressing auxilin with its J-domain mutated so that it could no longer interact with Hsc70 in vitro. Unexpectedly, we found that just like expression of intact auxilin (Fig. 2I,J; Table 1), raising expression of this mutated auxilin inhibited transferrin uptake the possibility that inhibition of endocytosis by expression of Expression of auxilin or AP180 inhibits endocytosis 357

Fig. 3. Effect of AP180 expression on transferrin and ﬂuid phase uptake. Transferrein internalization was measured in Cos cells (A,B) and HeLa cells (C-D) transfected with AP180. (A,C) Transfected cells; (B,D) transferrin internalization. (E,F) Fluid phase uptake of HeLa cells transfected with AP180 stained for AP180(E) or FITC-labeled dextran (F). (G,H) Transferrin receptor distribution in HeLa cells expressing AP180. (G) Transfected cells using a rabbit polyclonal anti Flag antibody; (H) transferrin receptor localization. (I,J) Transferrin internalization in AP180 transfected N2A cells. (I) Transfected cells; (J) transferrin internalization. auxilin is not related to its involvement in uncoating of clathrin but rather to its activity as an assembly protein. Interestingly, however, in contrast to what is observed with expression of intact auxilin or the C-terminal portion of auxilin with an intact J-domain, there was a morphological difference in that none of the cells expressing auxilin with a mutated J- domain showed formation of auxilin granules. Effect of AP180 on transferrin endocytosis The observation that expression of auxilin inhibits endocytosis is intriguing because expression of numerous other intact proteins involved in endocytosis such as Eps15, dynamin, amphiphysin, rho, rac, and β-arrestin do not inhibit endocytosis (Benmerah et al., 1998; Damke et al., 1994; Lamaze et al., 1996; Goodman et al., 1996; Wigge et al., 1997); endocytosis is inhibited only by expression of domains or mutants of these proteins that interfere with the function of the parent proteins (Damke et al., 1994; Wigge et al, 1997; Benmerah et al., 1998; Benmerah et al., 1999; Owen et al., 1999; Nesterov et al., 1999). It therefore seems possible that expression of auxilin inhibits endocytosis by overwhelming the regulatory mechanisms in place to prevent inappropriate polymerization of clathrin in the cytosol. If so, inhibition of endocytosis by well. To investigate this question we determined whether expression of auxilin may be a general phenomenon that not only endocytosis is inhibited by expression of the nerve-speciﬁc occurs with auxilin but with other clathrin-binding proteins as AP180, which, like auxilin, is monomeric. 358 JOURNAL OF CELL SCIENCE 114 (2)

120

100

Fig. 4. GLUT4 endocytosis in transfected cells. Primary 60 culture of rat adipocytes were co-transfected with HA-tagged 40 GLUT4 and various DNA constructs of Flag-tagged AP180, auxilin, their fragments and mutant as indicated. Noted as G4, 20 control cells were transfected with HA-GLUT4 only. Cell 0 Cell Surface HA-Glut4 (% max. control) surface GLUT4 in absence (basal) and presence of insulin G4 Auxilin-N Auxilin Auxilin-C AP180-N AP180 AP180-C were measured using mAb anti-HA antibody followed by the incubation with 125I-sheep anti-mouse antibody. Basal Insulin

As we observed with expression of auxilin, expression of about 20-fold more AP180 than normal neuro-2A cells (data AP180 markedly inhibited transferrin uptake in both Cos (Fig. not shown). Therefore, even in cells that normally express 3A,B) and HeLa cells (Fig. 3C,D), although like auxilin with AP180, over-expression of this protein markedly inhibits a mutated J-domain, the expressed AP180 did not form transferrin uptake. granules. Quantification of the inhibition of transferrin uptake We next examined whether it is, in fact, the clathrin binding (Table 1) showed that expression of AP180 reduced transferrin domain of AP180 that is causing inhibition of transferrin uptake in about 95% of the transfected population of Cos and uptake or whether this inhibition is due to the ability of AP180 HeLa cells, which shows that regardless of the level of to inhibit phospholipase D activity (Lee et al., 1997). The expression of AP180, it causes a marked reduction in clathrin latter activity is localized to the N-terminal fragment of AP180 mediated endocytosis. This is a greater inhibition of transferrin (Lee et al., 1997), while the C-terminal fragment has the uptake than we observed with auxilin and, in agreement with clathrin assembly activity (Ye and Lafer, 1995). Like this observation, we found that in the transfected Cos cells expression of intact AP180, expression of the C-terminal much of the transferrin was localized on the plasma membrane fragment of AP180 inhibited transferrin uptake in HeLa cells, (Fig. 3A,B), an effect that we did not observe with expression while expression of the N-terminal fragment of AP180 had no of auxilin. Similarly, expression of AP180 in HeLa cells may effect. Table 1 quantifies the effect of the C- and N-terminal also have caused much of the transferrin to accumulate on the fragments of AP180 on transferrin uptake in a large population plasma membrane as shown by the diffuse staining of the of transfected HeLa and Cos cells. These results clearly show transferrin in the transfected cells (Fig. 3C,D). By acid washing that it is the clathrin-assembly activity of AP180 that is the cells briefly in 0.5% acetic acid/0.5 M NaCl, pH 2.4, to responsible for inhibiting transferrin uptake, not its ability to remove cell surface-bound transferrin, the transferrin inhibit phospholipase D activity. associated with the AP180 transfected HeLa cells was completely removed (data not shown). This establishes that the Effect of AP180 and auxilin on GLUT4 glucose transferrin is associated with the plasma membrane. Similar to transporter endocytosis the results obtained in cells expressing auxilin, the expression If expression of AP180 and auxilin or their clathrin binding of AP180 was specific to clathrin-mediated endocytosis since domains indeed inhibits transferrin uptake by inhibiting fluid phase uptake, as measured by uptake of FITC-dextran, endocytosis non-specifically, uptake of proteins other than was unaffected by expression of AP180 (Fig. 3E,F). transferrin receptor should also be affected by this expression. The association of transferrin with the plasma membrane of To test this prediction we investigated the effect of expression the AP180 transfected cells predicts that there should be an of AP180, auxilin and their clathrin-binding domains on the increase in transferrin receptor on the plasma membrane in level of GLUT4 glucose transporter present on the plasma transfected cells expressing AP180. Fig. 3G,H show that membrane of adipocytes. In its cycle between the plasma the transferrin receptors in non-transfected HeLa cells are membrane and an intracellular compartment, the GLUT4 localized to coated pits and endosomal compartments, while in glucose transporter is thought to be internalized by clathrin- the AP180 transfected cells much of the receptor appears to be mediated endocytosis (Robinson et al., 1992; Chakrabarti et al., localized diffusely on the plasma membrane. Therefore, our 1994; Volchuk et al., 1998). Therefore if expression of AP180 data strongly suggest that expression of AP180 is inhibiting and auxilin inhibits internalization of GLUT4 in a primary internalization of the transferrin receptor rather than a later step culture of rat adipocytes, transfected cells should display a in endocytosis. higher level of GLUT4 on the cell surface in the absence of Since neither Cos nor HeLa cells normally express AP180, insulin. Fig. 4 shows that this is indeed the case. As we we carried out a similar experiment using mouse neuro-2A observed for internalization of transferrin, expression of cells after first demonstrating by western blot analysis, using AP180 had a somewhat greater effect than expression of an antibody specific for AP180, that these cells indeed auxilin. In fact, expression of the clathrin binding portion of express AP180 (data not shown). As we observed for Cos and AP180 brought the basal level of GLUT4 glucose transporters HeLa cells, the transfected neuro-2A cells expressing AP180 on the plasma membrane up to the level observed in the showed markedly reduced transferrin uptake (Fig. 3I,J). presence of insulin while expression of the N-terminal Western blot experiments showed that the transfected neuro- fragments of AP180 and auxilin had almost no effect. These 2A cells produced, after correcting for transfection efficiency, data confirm that, for uptake of GLUT4 transporter as well as Expression of auxilin or AP180 inhibits endocytosis 359 transferrin receptor, expression of the APs auxilin or AP180 with the trans-Golgi network in the cells expressing AP180, interferes with clathrin-mediated endocytosis. there was no change in the distribution of the γ chain of AP1; it remained bound to the trans-Golgi network even in the Effect of AP180 on the distributions of clathrin and absence of clathrin (Fig. 5G,H). As expected, the Golgi coat APs We next investigated whether expression of AP180 affects the distribution of clathrin in HeLa cells since our data strongly suggested that it is the clathrin-binding ability of AP180 that is required for inhibition of clathrin-mediated endocytosis. Fig. 5 shows the localization of clathrin in HeLa cells expressing either intact AP180 (Fig. 5A,B) or the C-terminal fragment of AP180 (Fig. 5C,D). Normally clathrin is associated with both the plasma membrane and the trans- Golgi network but in the transfected cells, there seemed to be a loss of clathrin from the trans-Golgi network and an appearance of aggregated clathrin in the cytosol. In agreement with the observed loss of clathrin from the trans-Golgi network, using a chimeric protein, the IL-2 receptor α chain (Tac) containing a signal localization sequence to the lysosome (Marks et al., 1995), we found that over- expression of AP180 or its C- terminal fragment increased plasma membrane association of the chimeric Tac, indicating inhibition of transport of this fusion protein from the trans-Golgi network to the lysosome (data not shown). On the other hand, expression of the N- terminal fragment of AP180 had no effect on the distribution of clathrin (Fig. 5E,F). The observation that expression of AP180 or its clathrin binding domain removed clathrin from the trans-Golgi network allowed us to investigate whether this affected the distribution of AP1 on the trans- Golgi network. Strikingly, despite the decrease in clathrin associated

Fig. 5. Effect of AP180 expression on localization of clathrin, AP1 and βCOP in HeLa cells. HeLa cells were transfected with Flag-tagged AP180 (A,B and G-J), Flag-tagged AP180 C- terminal fragment (C,D), or Flag- tagged AP180 N-terminal fragment (E,F), ﬁxed, and stained for Flag using a rabbit polyclonal antibody (A,C,E,G) or mAb M5 (I), clathrin (B,D,F), AP1 (H), and βCOP (J). 360 JOURNAL OF CELL SCIENCE 114 (2)

Fig. 6. (A-C) Confocal microscopic photograph of clathrin localization in cells transfected with Flag-tagged AP180. (A) The ﬁeld where z cut was performed. (B,C) z cut photographs show clathrin distribution. Green, AP180; Red, clathrin. Only clathrin is shown in B and C. Arrows indicate transfected cells. (D-G) AP2 localization in cells expressing AP180. (D,F) Transfected cell; (E,G) AP2 localization. Expression of auxilin or AP180 inhibits endocytosis 361

Fig. 7. Effect of auxilin expression on localization of clathrin, Hsc70, AP2 and AP1 in HeLa cells. The transfected cells, labeled by either M5 antibody (C) or a rabbit antiserum against Flag (A,E,G,I), were stained for clathrin (B), Hsc70 (D), AP2 (F,H) and AP1 (J). protein β-coatomer also appeared normal in AP180 transfected cells (Fig. 5I,J). We also investigated whether AP180 affects the localization of clathrin and AP2 on the plasma membrane. The presence of aggregated clathrin in the cytosol partially obscured the amount of clathrin associated with the plasma membrane, but confocal microscopy suggested that there was indeed less clathrin associated with the plasma membrane of HeLa cells expressing AP180 than with the plasma membrane of untransfected cells (Fig. 6A- C). Furthermore, in agreement with our observation that AP1 localization on the trans-Golgi network is unaffected by expression of AP180, we observed no significant difference in the amount of AP2 associated with the plasma membranes of the transfected and untransfected cells (Fig. 6D-G). This lack of effect of AP180 on AP2 localization was further confirmed by comparing the fluorescence intensity per unit area in control and AP180 expressing cells. Using the Metamorph imaging computer program, we found that the fluorescence intensity was 18.01±4.90 and 18.68±4.20 in control and AP180 expressing cells, respectively. These results suggest that the AP2 pit density was not significantly affected due to expression of AP180. Therefore, in a result that has important implications for the mechanism of clathrin- coated pit formation, the sequestration of clathrin does 362 JOURNAL OF CELL SCIENCE 114 (2)

Fig. 8. Effect of GFP-GAK expression in HeLa cells on transferrin internalization and localization of clathrin, Hsc70, AP2, and AP1. (A,C,E,G,I) Transfected cells and the corresponding panels show transferrin internalization (B), clathrin (D), Hsc70 (F), AP2 (H) and AP1 (J). not significantly affect the localization of the key APs involved in receptor recruitment and clathrin polymerization at the plasma membrane and trans-Golgi network suggesting that these APs bind to nascent pits independently of clathrin. Effect of auxilin and GAK on distributions of clathrin and APs To investigate further whether expression of clathrin APs affect clathrin distribution without affecting the localization of AP2 and AP1, we investigated the effect of auxilin on the localization of clathrin and the APs. Strikingly, we found that, in both Cos cells (data not shown) and HeLa cells, auxilin granules always contained clathrin (Fig. 7A,B) and Hsc70 (Fig. 7C,D). Using colocalization with a Lamp-1 antibody, we determined that these proteins are not localized in the lysosomes (data not shown). The association of clathrin with the auxilin granules was accompanied by a marked decrease of clathrin in the cytosol, which, in turn, made it easier to discern than in the cells expressing AP180, that there was a marked decrease in clathrin associated with the clathrin-coated pits on the plasma membrane as well as on the trans-Golgi network. On the other hand, there was no apparent association of either AP2 or AP1 with the granules. And consistent with this observation, we did not observe significant redistribution of either AP2 (Fig. 7E-H) or AP1 (Fig. 7I,J) in these cells confirming that, as in cells expressing AP180, nascent pits containing APs form on the plasma membrane and the trans- Golgi network of these cells in the absence of clathrin. independent of clathrin binding comes from studies with the Further support that nascent pits containing APs can form auxilin homolog, GAK, which, in contrast to auxilin, is an on the plasma membrane and the trans-Golgi network of cells endogenous protein in HeLa cells. Cells transfected with GFP- Expression of auxilin or AP180 inhibits endocytosis 363 GAK showed decreased transferrin uptake, an effect that was AP1 and AP2. Strikingly, we did not observe a decrease in the particularly dramatic in the cells showing formation of GAK level of AP1 associated with the trans-Golgi network, nor did granules (Fig. 8A,B). Furthermore, as with the auxilin we observe a change in the distribution of AP2 on the plasma granules, clathrin was associated with the GAK granules (Fig. membrane. Interestingly, when Tebar et al. (Tebar et al., 1999) 8C,D), and in cells with GAK granules, there was a marked expressed CALM, they observed a similar depletion of clathrin decrease in clathrin associated with the trans-Golgi network from the trans-Golgi network with no effect on AP1 and clathrin-coated pits on the plasma membrane. In fact, in distribution, but did not observe a decrease in clathrin at the some cases, almost all of the clathrin in the cell was associated plasma membrane. Therefore, our results show for the first with the GAK granules making it particularly clear that, time that, even in the absence of clathrin binding, AP2 compared to the dramatic changes in clathrin distribution, there apparently forms nascent pits on the plasma membrane. was no significant change in the distribution of AP1 and AP2. Both APs and clathrin are present in the cytosol as well as Specifically, the AP1 was still associated with the trans-Golgi on cellular membranes and therefore, when clathrin-coated pits network (Fig. 8I,J), while the AP2 retained its punctate form, both APs and clathrin must be recruited to the appearance on the plasma membrane although some cytosolic membrane. There has been speculation that formation of AP2 appeared to be associated with the GAK-clathrin granules clathrin-coated pits involves co-assembly of clathrin, AP2 and in the cytosol (Fig. 8G,H). Interestingly, when the cells receptors on the plasma membrane (Pearse and Crowther, transfected with either auxilin or GAK were stained for Hsc70, 1987) but our data suggest that AP recruitment is independent we found that the granules that contained clathrin and auxilin of clathrin recruitment. In this regard, there is strong evidence or GAK also contained Hsc70 (Fig. 7C,D and Fig. 8E,F), that AP1 is recruited to the trans-Golgi network by the binding which explains why we did not observe these granules in cells of ARF1, which then dissociates once the AP1 and clathrin are expressing auxilin with a mutated J-domain that could not bound (Zhu et al., 1998), but it is not yet understood what interact with Hsc70. Therefore, in the cells expressing GAK as causes recruitment of AP2 to the plasma membrane. In any well as auxilin, nascent pits containing APs form on the plasma case our results strongly suggest that nascent pits containing membrane and the trans-Golgi network even though clathrin is APs can form in the absence of clathrin binding. These data not associated with these pits. are consistent with the observation that when clathrin is removed from existing coated pits by potassium depletion or treatment with hypertonic solution, AP2 remains behind DISCUSSION (Hansen et al., 1993; Brown et al., 1999). They are also consistent with the finding of Hannan et al. (Hanna et al., 1998) There is strong evidence that Hsc70, acting with the J-domain who found that clathrin and AP2 are independently uncoated proteins auxilin or GAK, plays a major role in uncoating from clathrin-coated vesicles. Finally, they are consistent with clathrin-coated vesicles both in vitro and in vivo, but does not the results of Liu et al. (Liu et al., 1998) who found that, when uncoat clathrin-coated pits (Heuser and Steer, 1989). We were, they over-expressed clathrin hubs, not only was endocytosis therefore, interested in whether expression of auxilin or GAK inhibited but, in addition, there was increased clathrin heavy in cultured cells increased or decreased clathrin-mediated chain associated with the plasma membrane. Yet despite this endocytosis. In the present study we found that expression of increase, there was no change in the distribution of AP2. either auxilin or over-expression of GAK inhibited clathrin- Since many of the proteins involved in endocytosis shuttle mediated endocytosis in Cos and HeLa cells. However, even between cytosolic and membrane-bound pools, a key question expression of auxilin with a mutated J-domain inhibited in the regulation of endocytosis is what keeps these proteins clathrin-mediated endocytosis. Furthermore, expression of the from polymerizing in the cytosol. There is evidence that clathrin assembly protein AP180 also inhibited endocytosis phosphorylation may regulate clathrin polymerization in the and here too it was the clathrin-binding domain of AP180 that cell (Wilde and Brodsky, 1996) and there is also evidence that was responsible for this inhibition. Since this work was Hsc70 acting as a chaperone may form a complex with clathrin completed, Tebar et al. (Tebar et al., 1999) showed that CALM, triskelions and APs that prevent them from polymerizing in the a homolog of AP180 expressed in non-neuronal cells, also cytosol (Eisenberg and Greene, 1998; Black et al., 1991). In inhibited clathrin-mediated endocytosis when it was expressed this regard, our observation that GAK- or auxilin-clathrin- in Cos cells where it is normally present, again supporting the Hsc70 granules form in cells expressing auxilin or over- view that expression of proteins or domains of proteins that act expressing GAK provides the first direct evidence that clathrin, as clathrin assembly proteins inhibit clathrin-mediated Hsc70, and auxilin indeed form a complex in vivo as well as endocytosis. in vitro (Jiang et al., 2000), although we could only Studies on localization of clathrin provided an explanation demonstrate complex formation in cells over-expressing for the inhibition of clathrin-mediated endocytosis by over- auxilin or GAK. The data presented in this paper also show expression of clathrin-binding proteins. In cells expressing that the mechanisms that prevent clathrin from polymerizing auxilin, GAK, AP180, or their clathrin-binding domains, in the cytosol can be overwhelmed by increasing the levels of clathrin was either aggregated in the cytosol or, in the case of APs in the cell suggesting that polymerization of clathrin in GAK or auxilin with an intact J-domain, in GAK- or auxilin- clathrin-coated pits rather than in the cytosol depends on clathrin-Hsc70 granules. At the same time there was a decrease multiple regulatory factors including the concentration of APs in the level of clathrin associated with the trans-Golgi network present in the cell. and the plasma membrane. This led to an opportunity to The observation that expression of AP180 or auxilin inhibits determine whether the absence of clathrin from clathrin-coated clathrin-mediated endocytosis provides a simple method of pits affected the distribution of the clathrin assembly proteins testing whether a given process in the cell involves clathrin- 364 JOURNAL OF CELL SCIENCE 114 (2) mediated endocytosis. Using this method we confirmed that the Damke, H., Baba, T., Warnock, D. E. and Schmid, S. L. (1994). Induction GLUT4 glucose transporters are internalized from the plasma of mutant dynamin specifically blocks endocytic coated vesicle formation. J. membrane by clathrin-mediated endocytosis and, at the same Cell Biol. 127, 915-934. David, C., McPherson, P. S., Mundigl, O. and De Camilli P. (1996) A role time, demonstrated that expression of AP180 and auxilin not of amphiphysin in synaptic vesicle endocytosis suggested by its binding to only inhibited endocytosis in immortalized cells but also in dynamin in nerve terminals. Proc. Nat. Acad. Sci. USA 93, 331-335. primary tissue culture cells. Our studies on the GLUT4 Dell’Angelica, E. C., Mullins, C. and Bonifacino, J. S. (1999). AP4, a novel transporter show that the strongest inhibition of clathrin- protein complex related to clathrin adaptors. J. Biol. Chem. 274, 7278-7285. Dreyling, M. H., Martinez-Climent, J. A., Zheng, M., Mao, J., Rowley, J. mediated endocytosis occurred with the clathrin-binding D. and Bohlander, S. K. (1996). The t(10;11)(p13;14) in the U937 cell line domain of AP180. In fact, expression of this domain inhibited results in the fusion of the AF10 gene and CALM, encoding a new member clathrin-mediated endocytosis so strongly that the basal level of the AP3 clathrin assembly protein family. Proc. Nat. Acad. Sci. USA 93, of the GLUT4 transporter on the plasma membrane of 4804-4809. transfected adipocytes almost reached the same level as in cells Eisenberg, E. and Greene, L. E. (1998). Disassembly of protein complexes I: clathrin uncoating. In Molecular Biology of Chaperones and Folding treated with insulin. On the other hand, although it has been Catalysts. (ed. B. Bukau), pp. 329-3346. Harwood Academic Publishers. reported that the GLUT4 glucose transporter interacts with Gaidarov, I., Krupnick, J. G., Falck, J. R., Benovic, J. L. and Keen, J. H. AP1 and AP3 (Gillingham et al., 1999), over-expression of (1999). Arrestin function in G protein-coupled receptor endocytosis requires AP180 or its clathrin binding domain had no effect on the phosphoinositide binding. EMBO J. 18, 871-881. transport of GLUT4 glucose transporters to the plasma Gillingham, A. K., Koumanov, F., Pryor, P. R., Reaves, B. J., and Holman, G. D. (1999). Association of AP1 adaptor complexes with GLUT4 vesicles. membrane in the presence of insulin suggesting that clathrin is J. Cell Sci. 112, 4793-4800. not involved in this transport. Therefore, in future studies, Goodman, Jr. O. B., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. expression of the clathrin-binding domain of AP180 should B., Gagnon, A. W., Keen, J. H. and Benovic J. L. (1996). β-Arrestin acts provide a simple method of determining whether clathrin- as a clathrin adaptor in endocytosis of the β2-adrenergic receptor. Nature 383, 447-450. mediated endocytosis is involved in various processes in the Goodman, Jr. O. B., Krupnick, J. G., Gurevich, V. V., Benovic, J. L. and cell, a method that will compliment the use of clathrin hubs to Keen, J. H. (1997). Arrestin/clathrin interaction. Localization of the arrestin inhibit endocytosis (Liu et al., 1998). Since one method binding locus to the clathrin terminal domain. J. Biol. Chem. 6, 1501-1522. decreases the level of clathrin heavy chain associated with the Greener, T., Zhao, X., Nojima, H., Eisenberg, E. and Greene, L. E. (2000). plasma membrane while the other increases it, agreement Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells. J. Biol. Chem. 275, 1365-1370. between the effects of these two methods will strengthen the Hannan, L. A., Newmyer, S. L. and Schmid, S. L. (1998). ATP- and cytosol- conclusion that clathrin-mediated endocytosis is required for a dependent release of adaptor proteins from clathrin-coated vesicles: A dual particular process. role for Hsc70. Mol. Biol. Cell 9, 2217-2229. Hansen, S. H., Sandvig, K. and van Deurs, B. (1993). Clathrin and HA2 We thank Drs Julie Donaldson and Harish Radhakrishna for their adaptors: effects of depletion, hypertonic medium, and cytosol acidification. many helpful discussions, Dr Kenneth Yamada for the GFP-vector, Dr J. Cell Biol. 121, 61-72. Heuser, J. and Steer, C. J. (1989). Turmeric binding of the 70-kD uncoating Ivan Bonifacino for the Tac construct, and Dr Xufeng Wu for her ATPase to the vertices of clathrin triskelia: a candidate intermediate in the valuable help with the confocal microscopy work. vesicle uncoating reaction. J. Cell Biol. 108, 1457-1466. Hirst, J., Bright, N. A., Rous, B. and Robinson, M. S. (1999). Characterization of a fourth adaptor-related protein complex. Mol. Biol. Cell REFERENCES 10, 2787-2802. Jiang, R. F., Greener, T., Barouch, W., Greene, L. and Eisenberg, E. (1997). Ahle, S. and Ungewickell, E. (1990). Auxilin, a newly identified clathrin- Interaction of auxilin with the molecular chaperone, Hsc70. J. Biol. Chem. associated protein in coated vesicles from bovine brain. J. Cell Biol. 111, 19- 272, 6141-6145. 29. Jiang, R., Gao, B., Prasad, K., Greene, L. E. and Eisenberg, E. (2000). Al-Hasani H., Hinck C. S. and Cushman S. W. (1998). Endocytosis of the Hsc70 chaperones clathrin and primes it to interact with vesicle membranes. glucose transporter GLUT4 is mediated by the GTPase dynamin. J. Biol. J. Biol. Chem. 275, 8439-8447. Chem. 273, 17504-17510. Kanaoka, Y., Kimura, S. H., Okazaki, I., Ikeda, M. and Nojima, H. (1997). Benmerah, A., Lamaze, C., Begue, B., Schmid, S. L., Dautry-Versat, A., GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain. and Cerf-Bensussan, N. (1998). AP2/Eps15 interaction is required for FEBS Lett. 402, 73-80. receptor-mediated endocytosis. J. Cell Biol. 140, 1055-1062. Keen, J. H. (1990). Clathrin and associate assembly and disassembly proteins. Benmerah, A., Bayrou, M., Cerf-Bensussan, N. and Dautry-Versat, A. Annu. Rev. Biochem. 59, 415-438. (1999). Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Kioka, N., Sakata, S., Kawauchi, T., Amachi, T., Akiyama, S. K., Okazaki, Sci. 112, 1303-1311. K., Yaen, C., Yamada, K. M. and Aota, S.-I. (1999). Vinexin: A novel Black, M. M., Chestnut, M. H., Pleasure, I. T. and Keen, J. H. (1991). Stable vinculin-binding protein with multiple SH3 domains enhances actin clathrin-uncoating protein (Hsc70) complexes in intact neurons and their cytoskeletal organization. J. Cell Biol. 144, 59-69. axonal-transport. J. Neurosci. 11, 1163-1172. Lamaze, C., Chuang, T. H., Terlecky, L. J., Bokoch, G. M. and Schmid, S. Brown, C. M., Roth, M. G., Henis, Y. I. and Petersen, N. O. (1999). An L. (1996). Regulation of receptor-mediated endocytosis by Rho and Rac. internalization-competent influenza hemagglutinin mutant causes the Nature 382, 117-179. redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in Lamaze C., Fujimoto, L. M., Yin, H. L. and Schmid, S. L. (1997). The actin clathrin free clusters. Biochemistry 38, 15166-15173. cytoskeleton is required for receptor-mediated endocytosis in mammalian Chakrabarti, R., Buxton, J., Joly, M. and Corvera, S. (1994). Insulin- cells. J. Biol. Chem. 272, 20332-20335. sensitive association of GLUT-4 with endocytic clathrin-coated vesicles Lee, C. H., Kang, S. H., Chung, J. K., Sekiya, F., Kim, J. R., Han, J. S., revealed with the use of brefeldin A. J. Biol. Chem. 269, 7926-7933. Kim, S. R., Bae, Y. S., Morris, A. J. and Rhee, S. G. (1997). Inhibition of Chen, H., Fre, S., Slepnev, V. I., Capua M. R., Takei, K., Butler, M. H., Di phospholipase D by clathrin assembly protein 3 (AP3). J. Biol. Chem. 272, Fiore, P. P. and De Camilli, P. (1998). Epsin is an EH-domain-binding 15986-15992. protein implicated in clathrin-mediated endocytosis. Nature 394, 793-797. Liu, S. H., Marks, M. S. and Brodsky, F. M. (1998). A dominant-negative Cremona, O., Di Paolo, G., Wenk, M. R., Luthi, A., Kim, W. T., Takei, K., clathrin mutant differentially affects trafficking of molecules with distinct Daniell, L., Nemoto, Y., Shears, S. B., Flavell, R. A., McCormick, D. A. sorting motifs in the class II major histocompatibility complex (MHC) and De Camilli, P. (1999). Essential role of phosphoinositide metabolism in pathway. J. Cell Biol. 140, 1023-1037. synaptic vesicle recycling. Cell 99, 179-188. Marks, M. S., Roche, P. A., van Donselaar, E., Woodruff, L., Peters, P. J. Expression of auxilin or AP180 inhibits endocytosis 365

and Bonifacino, J. S. (1995). A lysosomal targeting signal in the cytoplasmic Simpson, F., Peden, A. A., Christopolou, L. and Robinson, M. S. (1997). tail of the β chain directs HLA-DM to MHC class II compartments. J. Cell Characterization of the adaptor-related protein complex, AP3. J. Cell Biol. Biol. 131, 351-369. 137, 835-845. McLauchlan, H., Newell, J., Morrice, N., Osborne, A., West, M. and Slepnev, V. I., Ochoa, G. C., Butler, M. H., Grabs, D. and De Camilli, P. Smythe, E. (1998). A novel role for Rab5-GDI in ligand sequestration into (1998). Role of phosphorylation in regulation of the assembly of endocytic clathrin-coated pits. Curr. Biol. 8, 34-45. coat complexes. Science 281, 821-824. Munn, A. L., Stevenson, B. J., Geli, M. I. and Riezman, H. (1995). End5, Smythe, E., Redelmeier, T. E. and Schmid, S. L. (1992). Receptor-mediated end6, and end7-mutations that cause actin delocalization and block the endocytosis in semiintact cells. Meth. Enzymol. 219, 223-234. internalization step of endocytosis in Saccharomyces-cerevisiae. Mol. Biol. Takei, K., McPherson, P. S., Schmid, S. L. and De Camilli, P. (1995). Tubular Cell. 6, 1721-1742. membrane invaginations coated by dynamin rings are induced by GTP- Nesterov, A., Carter, R. E., Sorkina, T., Gill, G. N. and Sorkin, A. (1999). gamma S in nerve terminals. Nature 9, 186-190. Inhibition of the receptor-binding function of clathrin adaptor protein AP2 Takei, K., Slepnev, V. I., Haucke, V. and De Camilli, P. (1998). Amphiphysin by dominant-negative mutant mu 2 and its effects on endocytosis. EMBO J. tubulates protein-free liposomes and regulates the formation of clathrin- and 18, 2489-2499. dynamin-coated structures in vitro. Mol. Biol. Cell (suppl.) 9, 750. Owen, D. J., Vallis, Y., Noble, M. E., Hunter, J. B., Dafforn, T. R., Evans, Tebar, F., Sorkina, T., Sorkin A., Ericsson, M. and Kirchhausen, T. (1996). P. R. and McMahon, H. T. (1999). A structural explanation for the binding Eps15 is a component of clathrin-coated pits and vesicles and is located at of multiple ligands by the alpha-adaptin appendage domain. Cell 97, 805- the rim of coated pits. J. Biol. Chem. 271, 28727-28730. 815. Tebar, F., Bohlander, S. K. and Sorkin, A. (1999). Clathrin assembly Pearse, B. M. and Crowther, R. A. (1987). Structure and assembly of coated lymphoid myeloid leukemia (CALM) protein: localization in endocytic- vesicles. Annu. Rev. Biophys. Biophys. Chem. 16, 49-68. coated pits, interactions with clathrin, and the impact of overexpression on Pearse, B. M. and Robinson, M. S. (1990). Clathrin, adapters, and sorting. clathrin-mediated traffic. Mol. Biol. Cell. 10, 2687-2702. Annu. Rev. Cell Biol. 6, 151-171. Tsai, J. and Douglas, M. G. (1996). A conserved HPD sequence of the J- Prasad, K., Barouch, W., Greene, L. and Eisenberg, E. (1993). A protein domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site cofactor is required for uncoating of clathrin baskets by uncoating ATPase. distinct from substrate binding. J. Biol. Chem. 271, 9347-9354. J. Biol. Chem. 268, 23758-23761. Ungewickell, E. and Oestergaard, L. (1989). Identiﬁcation of the clathrin Qualmann, B., Roos, J., DiGregorio, P. J. and Kelly, R. B. (1999). Syndapin assembly protein AP180 in crude calf brain extracts by two-dimensional I, a synaptic dynamin binding protein that associates with the neural Wiskott- sodium dodecyl-sulfate polyacrylamide-gel electrophoresis. Analyt. Aldrich syndrome protein. Mol. Biol. Cell 10, 501-513. Biochem. 179, 352-356. Rapoport, I., Miyazaki, M., Boll, W., Duckworth, B., Cantley, L. C., Ungewickell, E., Ungewickell, H., Holstein, S., Lindner, R., Prasad, K., Shoelson, S. and Kirchhausen T. (1997). Regulatory interactions in the Barouch, W., Martin, B., Greene, L. E. and Eisenberg, E. (1995). Role recognition of endocytic sorting signals by AP2 complexes. EMBO J. 16, of auxilin in uncoating clathrin-coated vesicles. Nature 378, 632-635. 2240-2250. Van der Bliek, A. M. Redelmeier, T. E., Damke, H., Tisdale, E. J., Ringstad, N., Gad, H., Low, P., Di Paolo, G., Brodin, L., Shupliakov, O. and Meyerowitz, E. M. and Schmid, S. L. (1993). Mutations in human dynamin De Camilli, P. (1999). Endophilin/SH3p4 is required for the transition from block an intermediate stage in coated vesicle formation. J. Cell Biol. 122, early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron 553-563. 24, 143-154. Volchuk, A., Narine, S., Foster, L. J., Grabs, D., De Camilli, P. and Klip, Robinson, M. S. and Kreis, T. E. (1992). Recruitment of coat proteins onto A. (1998). Perturbation of dynamin II with an amphiphysin SH3 domain Golgi membranes in intact and permeabilized cells – effects of brefeldin-A increases GLUT4 glucose transporters at the plasma membrane in 3T3-L1 and G-protein activators. Cell 69, 129-138. adipocytes. Dynamin II participates in GLUT4 endocytosis. J. Biol. Chem. Robinson, L. J., Pang, S., Harris, D. S., Heuser, J. and James, D. E. 273, 8169-8176. (1992). Translocation of the glucose transporter (GLUT4) to the cell Wigge P., Kohler, K., Vallis, K. Doyle, C. A., Owen, D., Hunt, S. P. And surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and McMahon, H. T. (1997) Amphiphysin heteodimers: Potential role in clathrin GTP gamma S and localization of GLUT4 to clathrin lattices. J. Cell Biol. mediated endocytosis. Mol. Biol. Cell 8, 2003-2015. 117, 1181-1196. Wilde, A. and Brodsky, F. M. (1996). In vivo phosphorylation of adaptors Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A. regulates their interaction with clathrin. J. Cell Biol. 135, 635-645. V., Witke, W., Huttner, W. B. and Soling, H. D. (1999). Endophilin I Ye, W. L. and Lafer, E. M. (1995). Clathrin binding and assembly activities mediates synaptic vesicle formation by transfer of arachidonate to of expressed domains of the synapse-speciﬁc clathrin assembly protein AP3. lysophosphatidic acid. Nature 40, 133-141. J. Biol. Chem. 270, 10933-10939. Sell, S. M., Eisen, C., Ang, D., Zylicz, M. and Georgopoulos, C. (1990). Zhu, Y., Traub, L. M. and Kornfeld, S. (1998). ADP-ribosylation factor 1 Isolation and characterization of dnaJ null mutants of Escherichia coli. J. transiently activates high-affinity adaptor protein complex AP-1 binding sites Bacteriol. 172, 4827-4835. on Golgi membranes. Mol. Biol. Cell 9, 1323-1337.