INTRODUCTION

During receptor-mediated endocytosis, clathrin triskelionspolymerize and form clathrin-coated pits on the plasmamembrane 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-Golginetwork. In addition to clathrin and receptors, these pitscontain assembly proteins (APs) that catalyze thepolymerization of clathrin triskelions and in some cases bindthe receptors localized in the clathrin-coated pits. A number ofdifferent APs have been described. AP1, AP2, AP3 and AP4are 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 onthe trans-Golgi network, AP2 on the plasma membrane, AP3on both the trans-Golgi membrane and endosomes, and AP4 onperinuclear structures. AP180 (Ungewickell and Oestergaard,1989) and auxilin (Ahle and Ungewickell, 1990) are neuronalspecific APs that consist of single subunits of 92 kDa and 100kDa, respectively; CALM and GAK, which have recently beendescribed, are the non-neuronal homologs of AP180 (Dreylinget 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 butunlike 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 beendiscovered that are involved in the formation, invagination, andpinching off of clathrin coated vesicles including dynamin,amphiphysin, epsin, eps15, endophilin, syndapin I and thesmall GTPase protein, Rab5-GDI (Van der Bliek et al., 1993;Takei et al., 1995; David et al., 1996; Chen et al., 1998; Tebaret 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 coatassembly 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 isdephosphorylation of many of the proteins involved information 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 byHsc70 and its partner proteins, auxilin or cyclin G-associatedkinase (GAK). These partner proteins not only assembleclathrin but also have J-domains that enable them to interactwith Hsc70 (Prasad et al., 1993; Ungewickell et al., 1995; Jianget al., 1997; Greener et al., 2000). Recently, Cremona et al.

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Although uncoating of clathrin-coated vesicles is a keyevent in clathrin-mediated endocytosis it is unclear whatprevents uncoating of clathrin-coated pits before they pinchoff to become clathrin-coated vesicles. We have shown thatthe J-domain proteins auxilin and GAK are required foruncoating by Hsc70 in vitro. In the present study, weexpressed auxilin in cultured cells to determine if thiswould block endocytosis by causing premature uncoatingof clathrin-coated pits. We found that expression of auxilinindeed inhibited endocytosis. However, expression ofauxilin with its J-domain mutated so that it no longerinteracted with Hsc70 also inhibited endocytosis as didexpression of the clathrin-assembly protein, AP180, or itsclathrin-binding domain. Accompanying this inhibition, weobserved a marked decrease in clathrin associated with theplasma membrane and the trans-Golgi network, which

provided us with an opportunity to determine whether theabsence of clathrin from clathrin-coated pits affected thedistribution of the clathrin assembly proteins AP1 andAP2. Surprisingly we found almost no change in theassociation of AP2 and AP1 with the plasma membraneand the trans-Golgi network, respectively. This wasparticularly obvious when auxilin or GAK was expressedwith functional J-domains since, in these cases, almost allof the clathrin was sequestered in granules that alsocontained Hsc70 and auxilin or GAK. We conclude thatexpression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it isno longer available to bind to nascent pits but that assemblyproteins bind to these pits independently of clathrin.

Key words: Auxilin, AP180, Endocytosis

SUMMARY

Expression of auxilin or AP180 inhibits endocytosisby mislocalizing clathrin: evidence for formation ofnascent pits containing AP1 or AP2 but not clathrinXiaohong Zhao 1, Tsvika Greener 1, Hadi Al-Hasani 2, Samuel W Cushman 2, Evan Eisenberg 1 and Lois E. Greene 1,*1Laboratory of Cell Biology, NHLBI and 2Experimental Diabetes, Metabolism and Nutrition Section, NIDDK, NIH, Bethesda, MD, USA*Author for correspondence (e-mail: greenel@helix.nih.gov)

Accepted 27 October 2000Journal of Cell Science 114, 353-365 © The Company of Biologists Ltd

RESEARCH ARTICLE

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(Cremona et al., 1999) have shown that hydrolysis of PIP2 bysynaptojanin is also important for uncoating in vivo.

An important question regarding regulation of uncoating byHsc70 is why premature uncoating of clathrin-coated pits doesnot occur before they pinch-off to form clathrin-coatedvesicles. Since the J-domain proteins auxilin and GAK arecritical for uncoating it seemed possible that the level of auxilinor GAK present in the cell would not only affect the rate andextent of uncoating of clathrin-coated vesicles but also whetheror not clathrin-coated pits were uncoated by Hsc70; if excessauxilin or GAK caused premature uncoating of clathrin-coatedpits before they pinched off to form clathrin-coated vesicles, itmight markedly inhibit clathrin-mediated endocytosis. On theother hand, quantitative western blot analysis showed that thelevel of auxilin present in neuronal cells is almost 10 times thelevel of GAK present in non-neuronal cells (Greener et al.,2000), probably because recycling of synaptic vesicles requiresclathrin-mediated endocytosis to occur much more rapidly inneuronal cells than in non-neuronal cells. Therefore, markedlyincreasing the level of auxilin or GAK present in cultured cellsmight actually increase the rate of uncoating of clathrin-coatedvesicles and thereby increase the rate of clathrin-mediatedendocytosis rather than inhibit it.

In the present study, we found that expression of auxilin orGAK markedly decreased clathrin-mediated endocytosis inHeLa and Cos cells and, at the same time, in many of the cellsled to the formation of clathrin-Hsc70-auxilin granules in thecytosol and a decrease in clathrin associated with clathrin-coated pits on the plasma membrane and the trans-Golginetwork. However, clathrin-mediated endocytosis was alsoinhibited by auxilin with its J-domain mutated so that it nolonger supported uncoating by Hsc70 in vitro although in thiscase the clathrin in the cytosol did not form granules butappeared to become aggregated in the cytosol. A similar effectoccurred when AP180 or its clathrin-binding domain wasexpressed. Surprisingly, however, in none of these cases waslocalization of AP1 or AP2 affected despite the mislocalizationof clathrin suggesting first, that expression of clathrin-bindingproteins inhibits endocytosis by causing mislocalization ofclathrin away from nascent pits, and second, that the bindingof APs to these pits occurs independently of clathrin.

MATERIALS AND METHODS

Cell culture and transfectionHeLa and Cos cells were purchased form ATCC. Mouseneuroblastoma N2A cells were a gift from Dr Y. Peng Loh (NICHD,NIH). Cells were maintained in DMEM supplemented with 10%fetal bovine serum, 2 mM glutamine, penicillin (100 unit/ml), andstreptomycin (100 unit/ml) in a humidified incubator with 5% CO2 at37°C. All media and supplements were obtained from Biofluids, Inc.(Rockville, MD, USA). For the purpose of immunofluorescencestudies calcium phosphate precipitation was used to transfect cell. Inorder to get higher transfection efficiency in biochemical assays,SuperFect (QIAGEN, Valencia, CA, USA) was used as transfectionreagent.

AntibodiesMonoclonal antibody M5 against Flag was obtained from KodakScientific Imaging System (Rochester, NY USA). A rabbit antiserumto Flag was from ZYMED Laboratories, Inc. (San Francisco, CA,

USA). Monoclonal anti-HA antibody (HA. 11) was purchased fromBerkeley Antibody Co. (Richmond, CA, USA). Rabbit antibodyagainst Golgi β coatomer (β-COP), monoclonal anti-clathrin heavychain (X22), monoclonal anti-α-adaptin (AP.6) are from AffinityBioReagents, Inc. (Golden, CO, USA). Monoclonal antibody againstγ-adaptin of AP1 (100/3) was obtained from Sigma (St Louis, MO,USA). Monoclonal mouse anti-human transferrin receptor antibodywas purchased from Biomeda Corp. (Foster City, CA, USA). A rabbitantiserum to human transferrin was obtained from BoehringerMannhein (Indianapolis, IN, USA). Monoclonal and polyclonalantibodies against Hsc70 were purchased from StressgenBiotechnologies Corp. (Victoria, BC, Canada). Fluorescence-conjugated secondary antibodies were from JacksonImmunoResearch laboratories, Inc. (West Grove, PA, USA). 125I-sheep anti-mouse antiserum was from Amersham Pharmacia Biotech(Piscataway, NJ, USA).

Plasmid constructionAuxilin and AP180 and their truncated mutants were prepared as Flagfusion proteins (Fig. 1A-B) using a pFlag-CMV-2 expression vectorfrom Kodak Scientific Imaging System (Rochester, NY, USA).Auxilin and AP180 cDNA were subcloned to give pTG176 andpTG135 expressing wild-type AP180 and wild-type auxilin,respectively. This wild-type construct of auxilin was later used tomake pTG177 expressing mutated auxilin contains a non-active J-domain, where the HPDK conserved motif was changed to AAAK.N- and C-terminal fragments of auxilin were made as follows. Thefirst 1,224 base pairs of auxilin cDNA were subcloned to give pTG168expressing the 45 kDa N-terminal fragment of auxilin which containsthe tensin domain. The last 1,521 base pairs of auxilin cDNA weresubcloned to give pTG197 expressing the 56 kDa C-terminal fragmentof auxilin which contains the clathrin binding and J-domains. N- andC-terminal fragments of AP180 were made as follows. The first 1,674base pairs of AP180 cDNA were subcloned to give pTG190expressing the 61 kDa N-terminal domain of AP180 containing thedomain which interacts with phospholipase D. The last 1,791 basepairs of AP180 cDNA were subcloned to give pTG192 expressing the65 kDa C-terminal domain of AP180 containing its clathrin bindingdomain (Lee et al., 1997). GAK was subcloned into GFP vector(Kioka et al., 1999) with the epitope tag at the N-terminal of GAK.

Endocytosis assaysFor immunofluorescence microscopy studies, cells were grown onglass coverslips and transfected 24-48 hours before the assay. Cellswere washed three times with PBS and incubated with DMEMcontaining 0. 5% BSA for 30 minutes at 37°C. Human transferrin wasthen added to the media at the final concentration of 30 µg/ml.Incubation was continued at 37°C for 5 to 10 minutes. To measurebulk fluid-phase endocytosis, 1 mg/ml lysine-fixable FITC-dextran(70,000) from Molecular Probes was added to the cells in DMEMcontaining 0.5% BSA for 1 hour at 37°C. Cells were washed quicklywith PBS, fixed in 2% formaldehyde and processed forimmunofluorescence microscopy.

To measured transferrin internalization biochemically, a modifiedbiotinylated transferrin uptake assay (Smythe et al., 1992) was used.Briefly, Cos cells grown in 6-well plates were first depleted ofendogenous transferrin. Biotinylated transferrin was then added andincubated with cells for minutes. After removing free biotinylatedtransferrin by washing, avidin was added to the plates to mask surface-bound biotinylated transferrin. Internalized biotin activity in celllysates were then assayed quantitatively using streptavidin-horseradish peroxidase in an ELISA plate coated with a rabbitantibody against transferrin. Experiments were performed intriplicate.

Immunofluorescence microscopy Cells were treated as indicated in each experiment, fixed in 2%

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355Expression of auxilin or AP180 inhibits endocytosis

formaldehyde at room temperature for 15minutes. After washing the cells three timewith PBS containing 10% FBS, cellswere incubated with primary antibodies for1 hour at room temperature. Cells werewashed again three times and incubatedwith fluorescence-conjugated secondaryantibodies.

GLUT4 glucose transporterendocytosis Rat adipose cells from male rats wereprepared and GLUT4 endocytosis assayswere conducted as previously described (Al-Hasani et al., 1998). Briefly, cells weretransfected by electroporation with HA-GLUT4 alone or cotransfected with HA-GLUT4 and Flag-AP180, auxilin or theirmutants as indicated. Cell surface GLUT4 inabsence or presence of insulin (1×104

microunits/ml) were measured by thebinding of monoclonal anti-HA antibody tocell surface HA-GLUT4 followed by theaddition of 125I-sheep anti-mouse antibody.Cell surface associated radioactivity wascounted in a γ-counter. Unless statedotherwise, the values obtained fromtransfected cells were subtracted from allother values to correct nonspecific antibody binding. Antibodybinding assays were routinely performed in duplicate, butoccasionally were done in quadruplicate.

RESULTS

Effect of auxilin on transferrin endocytosisTo determine the effect of expression of auxilin in culturedcells, we first expressed auxilin with a N-terminal Flag epitopein Cos and HeLa cells. Fig. 2A shows the fluorescenceobtained when Cos cells were stained with an anti-Flagantibody to detect the expressed auxilin. Transferrin uptake inthe same cells was also imaged by immunofluorescencemicroscopy as shown in Fig. 2B. Comparison of Fig. 2A andB shows that, in the non-transfected cells, the transferrin wasmainly localized to the recycling endosome, whereas the cellsexpressing auxilin showed marked inhibition of transferrin

uptake. Likewise, in HeLa cells, expression of auxilinmarkedly inhibited transferrin uptake (Fig. 2C,D).Quantification of this effect (Table 1) showed that 10% of thecontrol cells had little or no transferrin uptake compared to65% of the transfected cells. Interestingly, while in bothtransfected HeLa and Cos cells the expressed auxilin wascytosolic, its distribution did not appear to be uniform.Although the expressed auxilin varied from a grainyappearance to obvious speckles which ranged in size from tinyparticles to large granules, its cellular appearance did not seemto be related to the inhibition of transferrin uptake. In contrast,the uptake of FITC-dextran, a marker for bulk fluid-phaseendocytosis, was comparable in auxilin transfected and controlcells (Fig. 2E,F). These results establish that expression ofauxilin specifically affects clathrin-dependent receptormediated endocytosis in transfected cells, but not clathrin-independent fluid phase uptake.

To further verify that auxilin inhibits transferrin uptake, we

Fig. 1.Auxilin and AP180 DNA constructsused in the study. (A) Auxilin; (B) AP180.All constructs are N-terminal Flag-tagged.

Table 1. Percentage of cells with reduced transferrin uptakeExpressed protein

J-domainCells None Auxilin Auxilin-C Auxilin-N auxilin mutant AP180 AP180-C AP180-N

10 69 50 19 65 94 91 22HeLa (n=199) (n=86) (n=195) (n=203) (n=57) (n=112) (n=160) (n=117)

10 67 55 7 66 98 98 13Cos (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 oftransfected cells on the slides were counted as well as the number of transfected cells that showed significantly reduced transferrin uptake. The results areexpressed as the percentage of transfected cells that displayed reduced transferrin internalization. About 10% of control cells show very little uptake of transferrinin a typical experiment under our experimental conditions.

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biochemically compared the uptake ofbiotinylated transferrin in mock andauxilin transfected Cos cells. Using Coscells in which about 30% of thepopulation was transfected with auxilin,we found that these cells took upabout 30% less transferrin than themock-transfected cells in goodagreement with the 30% transfectionefficiency (data not shown). Therefore,both biochemical and fluorescencemicroscopy studies showed thattransient expression of auxilin markedlyinhibits clathrin-mediated endocytosis.

Since auxilin contains three domains,we next investigated which of thesedomains is required for inhibition ofendocytosis. We expressed either the N-terminal tensin domain, the C-terminalportion of auxilin lacking the tensindomain but containing the clathrin-binding domain and the J-domain, orfull-length auxilin with the criticalresidues, HPDK, of the J-domain (Sellet al., 1990; Tsai and Douglas, 1996)mutated to AAAK, so that, in vitro, wefound that the mutated auxilin no longersupported uncoating (data not shown).As shown in Table 1, expression of theN-terminal tensin domain of auxilin inboth HeLa and Cos cells had nosignificant effect on transferrin uptake,whereas, the C-terminal portion ofauxilin, which acts like intact auxilin invitro in uncoating clathrin coatedvesicles, also acted like intact auxilin invivo, inhibiting endocytosis andforming auxilin granules in the cytosol(Fig. 2G,H). We next tested whether theexpressed auxilin had to interact withHsc70 in order to inhibit endocytosis byexpressing auxilin with its J-domainmutated so that it could no longerinteract with Hsc70 in vitro. Unexpectedly, we found thatexpression of this mutated auxilin inhibited transferrin uptake

just like expression of intact auxilin (Fig. 2I,J; Table 1), raisingthe possibility that inhibition of endocytosis by expression of

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Fig. 2.Effect of auxilin expression ontransferrin and fluid phase uptake. Cellsgrown on coverslips were transientlytransfected with Flag-tagged auxilin (A-F),auxilin C-terminal fragment (G,H), or fulllength auxilin with a nonfunctional J-domain mutant (I,J). Transferrin uptakeassay and fluid phase uptake using FITC-labeled dextran were performed asdescribed in Materials and Methods.(A,C,E,G,I) indicate transfected cells whichwas detected using mAb M5 anti-Flagantibody. (B,D,H,J) Transferrininternalization; (F) FITC-labeled dextran.(A,B), Cos cells; (C-J), HeLa cells.

357Expression of auxilin or AP180 inhibits endocytosis

auxilin is not related to itsinvolvement in uncoating of clathrinbut rather to its activity as anassembly protein. Interestingly,however, in contrast to what isobserved with expression of intactauxilin or the C-terminal portion ofauxilin with an intact J-domain,there was a morphologicaldifference in that none of the cellsexpressing auxilin with a mutated J-domain showed formation of auxilingranules.

Effect of AP180 on transferrinendocytosisThe observation that expressionof auxilin inhibits endocytosis isintriguing because expression ofnumerous other intact proteinsinvolved in endocytosis such asEps15, dynamin, amphiphysin, rho,rac, and β-arrestin do not inhibitendocytosis (Benmerah et al., 1998;Damke et al., 1994; Lamaze et al.,1996; Goodman et al., 1996; Wiggeet al., 1997); endocytosis is inhibitedonly by expression of domains ormutants of these proteins thatinterfere with the function of theparent proteins (Damke et al., 1994;Wigge et al, 1997; Benmerah etal., 1998; Benmerah et al., 1999;Owen et al., 1999; Nesterov etal., 1999). It therefore seems possiblethat expression of auxilin inhibitsendocytosis by overwhelming theregulatory mechanisms in place toprevent inappropriate polymerizationof clathrin in the cytosol. If so, inhibition of endocytosis byexpression of auxilin may be a general phenomenon that not onlyoccurs with auxilin but with other clathrin-binding proteins as

well. To investigate this question we determined whetherendocytosis is inhibited by expression of the nerve-specificAP180, which, like auxilin, is monomeric.

Fig. 3. Effect of AP180 expression ontransferrin and fluid phase uptake.Transferrein internalization wasmeasured in Cos cells (A,B) and HeLacells (C-D) transfected with AP180.(A,C) Transfected cells; (B,D)transferrin internalization. (E,F) Fluidphase uptake of HeLa cells transfectedwith AP180 stained for AP180(E) orFITC-labeled dextran (F).(G,H) Transferrin receptor distributionin HeLa cells expressing AP180.(G) Transfected cells using a rabbitpolyclonal anti Flag antibody;(H) transferrin receptor localization.(I,J) Transferrin internalization inAP180 transfected N2A cells.(I) Transfected cells; (J) transferrininternalization.

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As we observed with expression of auxilin, expression ofAP180 markedly inhibited transferrin uptake in both Cos (Fig.3A,B) and HeLa cells (Fig. 3C,D), although like auxilin witha mutated J-domain, the expressed AP180 did not formgranules. Quantification of the inhibition of transferrin uptake(Table 1) showed that expression of AP180 reduced transferrinuptake in about 95% of the transfected population of Cos andHeLa cells, which shows that regardless of the level ofexpression of AP180, it causes a marked reduction in clathrinmediated endocytosis. This is a greater inhibition of transferrinuptake than we observed with auxilin and, in agreement withthis observation, we found that in the transfected Cos cellsmuch of the transferrin was localized on the plasma membrane(Fig. 3A,B), an effect that we did not observe with expressionof auxilin. Similarly, expression of AP180 in HeLa cells mayalso have caused much of the transferrin to accumulate on theplasma membrane as shown by the diffuse staining of thetransferrin in the transfected cells (Fig. 3C,D). By acid washingthe cells briefly in 0.5% acetic acid/0.5 M NaCl, pH 2.4, toremove cell surface-bound transferrin, the transferrinassociated with the AP180 transfected HeLa cells wascompletely removed (data not shown). This establishes that thetransferrin is associated with the plasma membrane. Similar tothe results obtained in cells expressing auxilin, the expressionof AP180 was specific to clathrin-mediated endocytosis sincefluid phase uptake, as measured by uptake of FITC-dextran,was unaffected by expression of AP180 (Fig. 3E,F).

The association of transferrin with the plasma membrane ofthe AP180 transfected cells predicts that there should be anincrease in transferrin receptor on the plasma membrane intransfected cells expressing AP180. Fig. 3G,H show thatthe transferrin receptors in non-transfected HeLa cells arelocalized to coated pits and endosomal compartments, while inthe AP180 transfected cells much of the receptor appears to belocalized diffusely on the plasma membrane. Therefore, ourdata strongly suggest that expression of AP180 is inhibitinginternalization of the transferrin receptor rather than a later stepin endocytosis.

Since neither Cos nor HeLa cells normally express AP180,we carried out a similar experiment using mouse neuro-2Acells after first demonstrating by western blot analysis, usingan antibody specific for AP180, that these cells indeedexpress AP180 (data not shown). As we observed for Cos andHeLa cells, the transfected neuro-2A cells expressing AP180showed markedly reduced transferrin uptake (Fig. 3I,J).Western blot experiments showed that the transfected neuro-2A cells produced, after correcting for transfection efficiency,

about 20-fold more AP180 than normal neuro-2A cells (datanot shown). Therefore, even in cells that normally expressAP180, over-expression of this protein markedly inhibitstransferrin uptake.

We next examined whether it is, in fact, the clathrin bindingdomain of AP180 that is causing inhibition of transferrinuptake or whether this inhibition is due to the ability of AP180to inhibit phospholipase D activity (Lee et al., 1997). Thelatter activity is localized to the N-terminal fragment of AP180(Lee et al., 1997), while the C-terminal fragment has theclathrin assembly activity (Ye and Lafer, 1995). Likeexpression of intact AP180, expression of the C-terminalfragment of AP180 inhibited transferrin uptake in HeLa cells,while expression of the N-terminal fragment of AP180 had noeffect. Table 1 quantifies the effect of the C- and N-terminalfragments of AP180 on transferrin uptake in a large populationof transfected HeLa and Cos cells. These results clearly showthat it is the clathrin-assembly activity of AP180 that isresponsible for inhibiting transferrin uptake, not its ability toinhibit phospholipase D activity.

Effect of AP180 and auxilin on GLUT4 glucosetransporter endocytosisIf expression of AP180 and auxilin or their clathrin bindingdomains indeed inhibits transferrin uptake by inhibitingendocytosis non-specifically, uptake of proteins other thantransferrin receptor should also be affected by this expression.To test this prediction we investigated the effect of expressionof AP180, auxilin and their clathrin-binding domains on thelevel of GLUT4 glucose transporter present on the plasmamembrane of adipocytes. In its cycle between the plasmamembrane and an intracellular compartment, the GLUT4glucose transporter is thought to be internalized by clathrin-mediated endocytosis (Robinson et al., 1992; Chakrabarti et al.,1994; Volchuk et al., 1998). Therefore if expression of AP180and auxilin inhibits internalization of GLUT4 in a primaryculture of rat adipocytes, transfected cells should display ahigher level of GLUT4 on the cell surface in the absence ofinsulin. Fig. 4 shows that this is indeed the case. As weobserved for internalization of transferrin, expression ofAP180 had a somewhat greater effect than expression ofauxilin. In fact, expression of the clathrin binding portion ofAP180 brought the basal level of GLUT4 glucose transporterson the plasma membrane up to the level observed in thepresence of insulin while expression of the N-terminalfragments of AP180 and auxilin had almost no effect. Thesedata confirm that, for uptake of GLUT4 transporter as well as

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G4 Auxilin-N Auxilin Auxilin-C AP180-N AP180 AP180-C

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Fig. 4.GLUT4 endocytosis in transfected cells. Primaryculture of rat adipocytes were co-transfected with HA-taggedGLUT4 and various DNA constructs of Flag-tagged AP180,auxilin, their fragments and mutant as indicated. Noted as G4,control cells were transfected with HA-GLUT4 only. Cellsurface GLUT4 in absence (basal) and presence of insulinwere measured using mAb anti-HA antibody followed by theincubation with 125I-sheep anti-mouse antibody.

359Expression of auxilin or AP180 inhibits endocytosis

transferrin receptor, expression of the APs auxilin or AP180interferes with clathrin-mediated endocytosis.

Effect of AP180 on the distributions of clathrin andAPsWe next investigated whetherexpression of AP180 affects thedistribution of clathrin in HeLa cellssince our data strongly suggestedthat it is the clathrin-binding abilityof AP180 that is required forinhibition of clathrin-mediatedendocytosis. Fig. 5 shows thelocalization of clathrin in HeLa cellsexpressing either intact AP180 (Fig.5A,B) or the C-terminal fragment ofAP180 (Fig. 5C,D). Normallyclathrin is associated with both theplasma membrane and the trans-Golgi network but in the transfectedcells, there seemed to be a loss ofclathrin from the trans-Golginetwork and an appearance ofaggregated clathrin in the cytosol. Inagreement with the observed loss ofclathrin from the trans-Golginetwork, using a chimeric protein,the IL-2 receptor α chain (Tac)containing a signal localizationsequence to the lysosome (Marks etal., 1995), we found that over-expression of AP180 or its C-terminal fragment increased plasmamembrane association of thechimeric Tac, indicating inhibitionof transport of this fusion proteinfrom the trans-Golgi network to thelysosome (data not shown). On theother hand, expression of the N-terminal fragment of AP180 had noeffect on the distribution of clathrin(Fig. 5E,F).

The observation that expression ofAP180 or its clathrin bindingdomain removed clathrin from thetrans-Golgi network allowed us toinvestigate whether this affected thedistribution of AP1 on the trans-Golgi network. Strikingly, despitethe decrease in clathrin associated

with the trans-Golgi network in the cells expressing AP180,there was no change in the distribution of the γ chain of AP1;it remained bound to the trans-Golgi network even in theabsence of clathrin (Fig. 5G,H). As expected, the Golgi coat

Fig. 5. Effect of AP180 expression onlocalization of clathrin, AP1 and βCOPin HeLa cells. HeLa cells weretransfected 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 usinga rabbit polyclonal antibody (A,C,E,G)or mAb M5 (I), clathrin (B,D,F), AP1(H), and βCOP (J).

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

361Expression of auxilin or AP180 inhibits endocytosis

protein β-coatomer alsoappeared normal in AP180transfected cells (Fig. 5I,J).We also investigated whetherAP180 affects the localizationof clathrin and AP2 onthe plasma membrane. Thepresence of aggregated clathrinin the cytosol partiallyobscured the amount ofclathrin associated with theplasma membrane, but confocalmicroscopy suggested thatthere was indeed less clathrinassociated with the plasmamembrane of HeLa cellsexpressing AP180 than withthe plasma membrane ofuntransfected cells (Fig. 6A-C). Furthermore, in agreementwith our observation that AP1localization on the trans-Golginetwork is unaffected byexpression of AP180, weobserved no significantdifference in the amount ofAP2 associated with theplasma membranes of thetransfected and untransfectedcells (Fig. 6D-G).

This lack of effect of AP180on AP2 localization wasfurther confirmed bycomparing the fluorescenceintensity per unit area incontrol and AP180 expressingcells. Using the Metamorphimaging computer program,we found that the fluorescenceintensity was 18.01±4.90 and18.68±4.20 in control andAP180 expressing cells,respectively. These resultssuggest that the AP2 pitdensity was not significantlyaffected due to expression ofAP180. Therefore, in a resultthat has important implicationsfor the mechanism of clathrin-coated pit formation, thesequestration of clathrin does

Fig. 7.Effect of auxilinexpression on localization ofclathrin, Hsc70, AP2 and AP1 inHeLa cells. The transfected cells,labeled by either M5 antibody (C)or a rabbit antiserum against Flag(A,E,G,I), were stained forclathrin (B), Hsc70 (D), AP2(F,H) and AP1 (J).

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not significantly affect thelocalization of the key APsinvolved in receptor recruitmentand clathrin polymerization at theplasma membrane and trans-Golginetwork suggesting that these APsbind to nascent pits independentlyof clathrin.

Effect of auxilin and GAK ondistributions of clathrin andAPsTo investigate further whetherexpression of clathrin APs affectclathrin distribution withoutaffecting the localization of AP2and AP1, we investigated theeffect of auxilin on the localizationof clathrin and the APs. Strikingly,we found that, in both Cos cells(data not shown) and HeLa cells,auxilin granules always containedclathrin (Fig. 7A,B) and Hsc70(Fig. 7C,D). Using colocalizationwith a Lamp-1 antibody, wedetermined that these proteins arenot localized in the lysosomes(data not shown). The associationof clathrin with the auxilingranules was accompanied by amarked decrease of clathrin in thecytosol, which, in turn, made iteasier to discern than in the cellsexpressing AP180, that there was amarked decrease in clathrinassociated with the clathrin-coatedpits on the plasma membrane aswell as on the trans-Golgi network.On the other hand, there was noapparent association of either AP2or AP1 with the granules. Andconsistent with this observation,we did not observe significantredistribution of either AP2 (Fig.7E-H) or AP1 (Fig. 7I,J) in thesecells confirming that, as in cellsexpressing AP180, nascent pitscontaining APs form on theplasma membrane and the trans-Golgi network of these cells in the absence of clathrin.

Further support that nascent pits containing APs can formon the plasma membrane and the trans-Golgi network of cells

independent of clathrin binding comes from studies with theauxilin homolog, GAK, which, in contrast to auxilin, is anendogenous protein in HeLa cells. Cells transfected with GFP-

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Fig. 8.Effect of GFP-GAK expressionin HeLa cells on transferrininternalization and localization ofclathrin, Hsc70, AP2, and AP1.(A,C,E,G,I) Transfected cells and thecorresponding panels show transferrininternalization (B), clathrin (D), Hsc70(F), AP2 (H) and AP1 (J).

363Expression of auxilin or AP180 inhibits endocytosis

GAK showed decreased transferrin uptake, an effect that wasparticularly dramatic in the cells showing formation of GAKgranules (Fig. 8A,B). Furthermore, as with the auxilingranules, clathrin was associated with the GAK granules (Fig.8C,D), and in cells with GAK granules, there was a markeddecrease in clathrin associated with the trans-Golgi networkand clathrin-coated pits on the plasma membrane. In fact, insome cases, almost all of the clathrin in the cell was associatedwith the GAK granules making it particularly clear that,compared to the dramatic changes in clathrin distribution, therewas no significant change in the distribution of AP1 and AP2.Specifically, the AP1 was still associated with the trans-Golginetwork (Fig. 8I,J), while the AP2 retained its punctateappearance on the plasma membrane although some cytosolicAP2 appeared to be associated with the GAK-clathrin granulesin the cytosol (Fig. 8G,H). Interestingly, when the cellstransfected with either auxilin or GAK were stained for Hsc70,we found that the granules that contained clathrin and auxilinor GAK also contained Hsc70 (Fig. 7C,D and Fig. 8E,F),which explains why we did not observe these granules in cellsexpressing auxilin with a mutated J-domain that could notinteract with Hsc70. Therefore, in the cells expressing GAK aswell as auxilin, nascent pits containing APs form on the plasmamembrane and the trans-Golgi network even though clathrin isnot associated with these pits.

DISCUSSION

There is strong evidence that Hsc70, acting with the J-domainproteins auxilin or GAK, plays a major role in uncoatingclathrin-coated vesicles both in vitro and in vivo, but does notuncoat clathrin-coated pits (Heuser and Steer, 1989). We were,therefore, interested in whether expression of auxilin or GAKin cultured cells increased or decreased clathrin-mediatedendocytosis. In the present study we found that expression ofeither auxilin or over-expression of GAK inhibited clathrin-mediated endocytosis in Cos and HeLa cells. However, evenexpression of auxilin with a mutated J-domain inhibitedclathrin-mediated endocytosis. Furthermore, expression of theclathrin assembly protein AP180 also inhibited endocytosisand here too it was the clathrin-binding domain of AP180 thatwas responsible for this inhibition. Since this work wascompleted, Tebar et al. (Tebar et al., 1999) showed that CALM,a homolog of AP180 expressed in non-neuronal cells, alsoinhibited clathrin-mediated endocytosis when it was expressedin Cos cells where it is normally present, again supporting theview that expression of proteins or domains of proteins that actas clathrin assembly proteins inhibit clathrin-mediatedendocytosis.

Studies on localization of clathrin provided an explanationfor the inhibition of clathrin-mediated endocytosis by over-expression of clathrin-binding proteins. In cells expressingauxilin, GAK, AP180, or their clathrin-binding domains,clathrin was either aggregated in the cytosol or, in the case ofGAK or auxilin with an intact J-domain, in GAK- or auxilin-clathrin-Hsc70 granules. At the same time there was a decreasein the level of clathrin associated with the trans-Golgi networkand the plasma membrane. This led to an opportunity todetermine whether the absence of clathrin from clathrin-coatedpits affected the distribution of the clathrin assembly proteins

AP1 and AP2. Strikingly, we did not observe a decrease in thelevel of AP1 associated with the trans-Golgi network, nor didwe observe a change in the distribution of AP2 on the plasmamembrane. Interestingly, when Tebar et al. (Tebar et al., 1999)expressed CALM, they observed a similar depletion of clathrinfrom the trans-Golgi network with no effect on AP1distribution, but did not observe a decrease in clathrin at theplasma membrane. Therefore, our results show for the firsttime that, even in the absence of clathrin binding, AP2apparently forms nascent pits on the plasma membrane.

Both APs and clathrin are present in the cytosol as well ason cellular membranes and therefore, when clathrin-coated pitsform, both APs and clathrin must be recruited to themembrane. There has been speculation that formation ofclathrin-coated pits involves co-assembly of clathrin, AP2 andreceptors on the plasma membrane (Pearse and Crowther,1987) but our data suggest that AP recruitment is independentof clathrin recruitment. In this regard, there is strong evidencethat AP1 is recruited to the trans-Golgi network by the bindingof ARF1, which then dissociates once the AP1 and clathrin arebound (Zhu et al., 1998), but it is not yet understood whatcauses recruitment of AP2 to the plasma membrane. In anycase our results strongly suggest that nascent pits containingAPs can form in the absence of clathrin binding. These dataare consistent with the observation that when clathrin isremoved from existing coated pits by potassium depletion ortreatment with hypertonic solution, AP2 remains behind(Hansen et al., 1993; Brown et al., 1999). They are alsoconsistent with the finding of Hannan et al. (Hanna et al., 1998)who found that clathrin and AP2 are independently uncoatedfrom clathrin-coated vesicles. Finally, they are consistent withthe results of Liu et al. (Liu et al., 1998) who found that, whenthey over-expressed clathrin hubs, not only was endocytosisinhibited but, in addition, there was increased clathrin heavychain associated with the plasma membrane. Yet despite thisincrease, there was no change in the distribution of AP2.

Since many of the proteins involved in endocytosis shuttlebetween cytosolic and membrane-bound pools, a key questionin the regulation of endocytosis is what keeps these proteinsfrom polymerizing in the cytosol. There is evidence thatphosphorylation may regulate clathrin polymerization in thecell (Wilde and Brodsky, 1996) and there is also evidence thatHsc70 acting as a chaperone may form a complex with clathrintriskelions and APs that prevent them from polymerizing in thecytosol (Eisenberg and Greene, 1998; Black et al., 1991). Inthis regard, our observation that GAK- or auxilin-clathrin-Hsc70 granules form in cells expressing auxilin or over-expressing GAK provides the first direct evidence that clathrin,Hsc70, and auxilin indeed form a complex in vivo as well asin vitro (Jiang et al., 2000), although we could onlydemonstrate complex formation in cells over-expressingauxilin or GAK. The data presented in this paper also showthat the mechanisms that prevent clathrin from polymerizingin the cytosol can be overwhelmed by increasing the levels ofAPs in the cell suggesting that polymerization of clathrin inclathrin-coated pits rather than in the cytosol depends onmultiple regulatory factors including the concentration of APspresent in the cell.

The observation that expression of AP180 or auxilin inhibitsclathrin-mediated endocytosis provides a simple method oftesting whether a given process in the cell involves clathrin-

364

mediated endocytosis. Using this method we confirmed that theGLUT4 glucose transporters are internalized from the plasmamembrane by clathrin-mediated endocytosis and, at the sametime, demonstrated that expression of AP180 and auxilin notonly inhibited endocytosis in immortalized cells but also inprimary tissue culture cells. Our studies on the GLUT4transporter show that the strongest inhibition of clathrin-mediated endocytosis occurred with the clathrin-bindingdomain of AP180. In fact, expression of this domain inhibitedclathrin-mediated endocytosis so strongly that the basal levelof the GLUT4 transporter on the plasma membrane oftransfected adipocytes almost reached the same level as in cellstreated with insulin. On the other hand, although it has beenreported that the GLUT4 glucose transporter interacts withAP1 and AP3 (Gillingham et al., 1999), over-expression ofAP180 or its clathrin binding domain had no effect on thetransport of GLUT4 glucose transporters to the plasmamembrane in the presence of insulin suggesting that clathrin isnot involved in this transport. Therefore, in future studies,expression of the clathrin-binding domain of AP180 shouldprovide a simple method of determining whether clathrin-mediated endocytosis is involved in various processes in thecell, a method that will compliment the use of clathrin hubs toinhibit endocytosis (Liu et al., 1998). Since one methoddecreases the level of clathrin heavy chain associated with theplasma membrane while the other increases it, agreementbetween the effects of these two methods will strengthen theconclusion that clathrin-mediated endocytosis is required for aparticular process.

We thank Drs Julie Donaldson and Harish Radhakrishna for theirmany helpful discussions, Dr Kenneth Yamada for the GFP-vector, DrIvan Bonifacino for the Tac construct, and Dr Xufeng Wu for hervaluable help with the confocal microscopy work.

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