THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 27, Issue of September 25, pp. 16514-16520,199O Printed in Ll. S. A.

Clathrin-coated Pits Contain an Integral Membrane Protein That Binds the AP-2 Subunit with High Affinity*

(Received for publication, May 14, 1990)

David T. Mahaffey@, John S. Peeler*, Frances M. BrodskyYl, and Richard G. W. Anderson$)( From the *Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 and the YDepartment of Pharmacy, University of California at San Francisco, San Francisco, California 74143

Coated pits will assemble onto purified plasma mem- branes that are attached to a poly-L-lysine coated sub- stratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558- 563: Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108,1615- 1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 sub- units from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached mem- branes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (& = 3 % lo-’ M) to salt stripped membranes. Binding is saturable and abolished by treating mem- branes with ~20 Mg/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer- washed membranes that bear endogenous AP-2 bound triskelions with high affinity (& = 3 x lOme M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.

The clathrin-coated pit is the site of internalization for a wide variety of macromolecules. Within tissue culture cells, ~3000 coated pits are converted to endocytic vesicles every minute (l), which accounts for the rapid uptake of receptors carrying ligands into the cell (2, 3). Since there is not any decline in the rate of receptor internalization when cells are incubated for several hours in the presence of protein synthe- sis inhibitors (4), all of the coated pit machinery must be disassembled and reassembled multiple times.

Like coated vesicles (5), the coated pit is assembled from two heteromolecular subunits: the triskelion and the AP sub-

* This work was supported by National Institutes of Health Grants HL 20948 (to R. G. W. A.) and GM 38093 (to F. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be-hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Present address: Dept. of Biochemistry, University of Utah School of Medicine, 50 N. Medical Dr., Salt Lake City, UT 84132.

11 To whom correspondence should be addressed.

unit. Each triskelion is a trimeric structure composed of three clathrin heavy chain molecules (180 kDa) and three clathrin light chain molecules (33-35 kDa). This subunit is the build- ing block for the clathrin polygonal lattice (6). Intercalated between the lattice and the plasma membrane (7) is the AP complex. First designated assembly proteins (9) and then adaptor proteins (16), this subunit is a brick shaped unit that is composed of two copies each of three different molecules with molecular weights of lOO,OOO-116,000, 46,000-48,000, and 14,000-17,000 (10). There are at least two different spe- cies of AP subunits, designated AP-1 (or HA-I (11)) and AP- 2 (or HA-II (11)). The former is found preferentially associ- ated with the Golgi region of the cell (12), whereas the latter is found in coated pits at the cell surface (13).

Within a cell, a coated vesicle forms from a coated pit. For this reason, it is important to understand the molecular interactions that are required for pit assembly. Several years ago we developed an in vitro membrane preparation that allows us to study this process (17). Membranes that are attached to a poly-L-lysine coated surface have abundant coated pits (17). When the triskelions are removed from these membranes by treatment with pH 9.0 buffer, new lattices easily form when the membranes are incubated either with cytosol(l7) or coat proteins (18) as a source of clathrin. These studies showed that there are a limited number of coated pit assembly sites and that these sites not only initiate lattice assembly but also control the size of the lattice.

The AP subunit in the coated vesicle coat structure (7) is situated so that it could hold the lattice on the membrane by linking the triskelion to an integral membrane protein of the vesicle. In support of this idea, Virshup and Bennett (15) reported that purified APs will bind to either coated vesicle membranes or membranes that were partially purified from brain. Moreover, Keen and co-workers (9, 22) have shown that APs will bind to triskelions. There is no information available, however, about whether these same interactions occur during coated pit formation, which is vital knowledge for understanding the assembly of this organelle. For this reason, we have employed the in vitro coated pit assembly assay we developed (17,18) to measure the interaction of AP- 2 with the inner membrane surface, as well as the interaction of triskelions with AP-2 that is bound to these membranes. These studies have revealed the presence of an integral mem- brane protein in the coated pit that binds AP-2 with high affinity.

EXPERIMENTAL PROCEDURES

Materials-Immulon I Removawell 96-well plates (011-010-6301) were purchased from Dynatech Laboratories, Inc. (Alexandria, VA). Human plasma fibronectin was obtained from the New York Blood Center. Minimal essential medium (330-1435), Dulbecco’s phosphate- buffered saline (310-4190), Dulbecco’s modified Eagle’s medium (320- 1885), fetal calf serum (200-6140), and trypsin-EDTA (610-5300)

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An Integral Membrane Protein That Binds AP-2 Subunit 16515

were from Gibco Laboratories (Grand Island, NY). Poly-I.-lysine (P- 1524), Hepes’ (H-3375), NaCl (S-9625), Mes (M-8250), EGTA (E- 4378), orthophenanthroline (P-9375), benzamidine (B-6506), soybean trypsin inhibitor (T-9128), Taps (T-5130), BSA (A-7909), crystalline bovine serum albumin (C-BSA; A-7638). Tris-HCI (T-3253), NH.,Cl (A-4514), and NaF (S-1504) were from Sigma. Leupeptin (62070), MgCI,.6H,O (63065), KCI (FOlBO), DTT (43819), and paraformal- dehyde (76240) were purchased from Fluka (Ronkonkoma, NY). Dowex l-X8 (1904-l) and NaH,PO.,.HzO (5-3818) were from J. T. Baker Chemical Co. (Phillipsburg, NJ). NazHP0.,.7Hr0 (S-373), glass coverslips (12548A), and NaN,, (S-227) were from Fisher. Glycine (161-0718) was from Bio-Rad. Normal horse serum (S-2000), biotinylated horse anti-mouse IgG (BA-2000), and wheat agglutinin conjugated to rhodamine tetramethylisothiocyanate (RL-1022) were from Vector Laboratories, Inc. (Burlingame, CA). Glutaraldehyde (16320) was from Electron Microscopy Sciences (Fort Washington, PA). Elastase (LS06363) was from Worthington. Phenylmethanesul- fonyl fuoride (PMSF, 837091) was from Boehringer Mannheim. Rab- bit anti-mouse IgG conjugated to fluorescein isothiocyanate (61-6011) was from Zymed Laboratories, Inc. (South San Francisco, CA). ““I- Labeled streptavidin (IM-236) at a specific activity of 20-40 pCi/ag, was purchased from Amersham Corp. A mixture of insulin, transfer- rin, and selenium (ITS Premix) was purchased from Collaborative Research Inc. (Lexington, MA). Human lipoprotein-deficient serum (d > 1.215 g/ml) was prepared by ultracentrifugation of human plasma (19). Monoclonal anticlathrin heavy chain IgG (designated X-22) was prepared as previously described (20). Monoclonal anti- AP-2 IgG (designated AP.6) is directed against the (Y lOO-kDa subunit of the AP-2 complex (21). Superose 6, preparation grade (17-0489- 01), was from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Hydroxylapaptite (391948) was from Behring Diagnostics. Centricon 90 microconcentrators (4209) were from Amicon (Beverly, MA). Micro BCA protein assay was from Pierce Chemical Co.

Buffers-The following buffers were used where indicated. The pH for each buffer was adjusted with either NaOH or KOH, as shown in parentheses. Buffer A: 50 mM Hepes, 100 mM NaC1, pH 7.4 (NaOH). Buffer B: 20 mM Mes, 2.5 mM EGTA, 2.5 mM MgCl?, 100 mM KCl, 1 mM DTT, 10 I.IM leupeptin, 1 mM orthophenanthroline, 0.5 mM henzamidine, 2 PI/ml soybean trypsin inhibitor, pH 6.2 (NaOH). Buffer C: 20 mM Hepes, 3 mM EGTA, 5 mM MgCl,, 100 mM KCl, pH 6.8 (NaOH). Buffer D: 20 mM Taps, 1 mM DTT, 10 FM leupeptin, 1 mM orthophenanthroline, 0.5 mM benzaminidine, 2 fig/ml soybean trypsin inhibitor, pH 9.0 (NaOH). Buffer E: 36.4 mM Hepes, 68.2 mM KCI, 4.1 mM Mg acetate, 1 mM DTT, 10 pM leupeptin, 1 mM orthophenanthroline, 0.5 mM benzamidine, 2 pg/ml soybean trypsin inhibitor, pH 7.2 (KOH). Buffer F: 0.5 M Tris-HCl, 1 mM DTT, 10 PM leupeptin, 1 mM orthophenanthroline, 0.5 mM benzaminidine, 2 r/ml soybean trypsin inhibitor, pH 7.0 (NaOH). Buffer G: mixture (l:l, v/v) 1.0 M Tris-HCl, pH 7.0 (NaOH), and 0.1 M Mes, 1.0 mM EGTA, 0.5 mM MgCI,, 3.0 mM NaNzI, 10 IJM leupeptin. Buffer H: 10 mM K,HPO.,, pH 8.4, 0.1 M NaCl, 10% glycerol, 0.2 mM DTT, 10 PM leupeptin. Buffer I: minimal essential medium, 20 mM Hepes, pH 7.4 (NaOH). Bufffer J: 100 mM Mes (pH 6.5), 1.0 mM EGTA, 0.5 mM MgCl,, 0.02% (w/v) NaN:%.

Cell Culture-Cultured fibroblasts were derived from a skin biopsy obtained from a normal subject. Cells were grown in monolayer and set up for experiments according to a standard format. On day 0, 7 X 10” cells were seeded onto each Petri dish (100 x 15 mm) containing 10 ml of Dulbecco’s modified Eagle medium supplemented with 100 units/ml penicillin, 100 pg/ml streptomycin, and 10% (v/v) fetal calf serum. Fresh medium of the same composition was added on day 3. On day 5 of cell growth, each monolayer received 8 ml of Dulbecco’s modified Eagle’s medium supplemented with penicillin, streptomycin, 6 pg/ml transferrin, 5 fig/ml selenium, 5 pg/ml insulin (ITS Premix), and 10% (v/v) human lipoprotein-deficient serum. On day 7 of cell growth, the media was removed and 3 ml of trypsin-EDTA was added to each dish and the cells were incubated for 5 min at room temper- ature. The cell suspension was removed and added to an equal volume of buffer I containing 10% (v/v) fetal calf serum. The suspended cells were centrifuged at 600 x g for 5 min and washed three times by repeated, alternate resuspension and centrifugation in buffer I. This

’ The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; Taps, 3-I [2- hydroxy-l,l-i~is(hydroxymethyl)ethyl]amino~-l-propanesulfonic acid; DTT, dithiothreitol; BSA, bovine serum albumin; C-BSA, crys- talline bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride.

cell suspension was used as described below. Purification of AI’- I, AP2, and Clathrin Trishelions-Coated ves-

icles were purified as previously described (18) and stored overnight at 4 “C. Coat proteins were stripped from coated vesicles with 0.5 M Tris, pH 7.0, as described previously (18) with the modification that 10 PM leupeptin was included in the buffers. The resulting coat protein mixture was subjected to gel filtration chromatography by a modification of the procedure of Keen (22). The column (2.6 X 53.5 cm) containing Superose 6 (preparation grade) was equilibrated with buffer G. Elution downward at 4 “C was at 1.0 ml/min; 4.0-ml frac- tions were collected. Gel filtration yielded a characteristic ALXll profile (22); clathrin containing fractions were held in buffer G at 4 “C, whereas the AP fractions were dialyzed against buffer H at 4 “C overnight. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to document the protein makeup of each fraction (Fig. 1) and clathrin containing fractions were concentrated in Centricon 30 microconcentrators and stored at 4 “C until use.

The AP fractions were pooled and further fractionated by hydrox- ylapatite chromatography as described (8, 11). A 1. X 6.5~cm hydrox- ylapaptite column was equilibrated with buffer H, 4 “C, at a flow rate of 0.3 ml/min. The column was washed overnight after the AP fractions were added. The column was eluted with 50 ml of a lo-500 mM K,HPO1 gradient in buffer H. One milliliter fractions were collected and AP-1 and AP-2 containing fractions (Fig. 1) were dialyzed into buffer E containing 10% glycerol and stored at 4 “C until use.

Indirect I”‘I-Streptauidin Binding Assay-Membranes were pre- pared in 96-well plates as previously described (18) with the exception that immediately prior to sonication, cells were not allowed to sit for an extended time in buffer A. Instead, each triplicate set of wells was washed 3 times with buffer A (250 rl/well/wash) and then immedi- ately washed with buffer B before sonication.

To remove endogenous triskelions, membranes were subjected to the following protocol: 4 washes (350 rl/well/wash) with buffer D; 5 min incubation in buffer D; 1 wash with buffer D; 7 washes with buffer E containing 0.1% C-BSA. To remove AP complexes, these membranes were further processed: 4 washes with buffer F; 5 min incubation in buffer F; 1 wash with buffer F; 7 washes with buffer E containing 0.1% C-BSA. Membranes in some experiments were washed by the same protocol except that buffer F was replaced with buffer E containing 0.6 M NaCl. For AP-2 binding, lower nonspecific binding was observed when buffer F was used.

After the wash procedure described above was completed, all wells were incubated with buffer E containing 1.0% C-BSA (100 Fcl/well) for 15 min at 4 “C. AP complex or triskelions were diluted into buffer E containing 1.0% C-BSA about 10 min prior to incubation with membranes (50 pi/well at the indicated concentration).

Either clathrin or AP-2 was measured on the attached membranes using a iY”I-streptavidin binding assay as previously described (18). In each assay either 1 fig/ml of X-22 or 1 rg/ml of AP.6 monoclonal antibody was used.

Other Procedures-Protein determinations were made either by the method of Lowry et al. (23) or a modification of the bicinchoninic

Chlhlill

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FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis analysis of triskelions (clathrin) and AP complexes (AP-1, AP-2). Triskelions and AP complexes were purified by gel filtration and hydroxylapatite chromatography as described. Each lane contains 15 pg of protein.

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An Integral Membrane Protein That Binds AP-2 Subunit

acid method (32). Preparation of membranes for rapid-freeze deep etch electron microscopy was carried out as previously described (18) using 4% glutaraldehyde in buffer C as the fixative. Polyacrylamide gel electrophoresis was carried out according to the method of Laem- mli (24) using a 2.5% stacking gel and a 5.0-12.5% gradient running gel. Conversion of protein concentration to molarity was based on a triskelion molecular weight of 640,000 (25) and an AP-2 average molecular weight of 300,000 (15, 22). Clathrin cage reassembly was carried out according to the method of Keen (22).

RESULTS

The Membrane System-Cells were allowed to attach at 37 “C to a poly-L-lysine/fibronectin-coated substratum as pre- viously described (18) and three types of membrane prepara- tions were prepared: (a) untreated membranes, (b) membranes washed with a pH 9.0 buffer, and (c) membranes washed either with 0.5 M Tris or 0.6 M NaCl (Tris salt). When untreated membranes were assayed for the presence of either clathrin or AP-2, an equal amount of both subunits was present (Fig. 2). Membranes that had been treated with high pH buffer had lost clathrin but not AP-2, whereas the Tris salt-treated membranes had diminished amounts of both clathrin and AP-2 (Fig. 2). The ability to control the molecular composition of the coated pits allowed us to determine how purified clathrin and AP-2 interacted with membranes that contained either clathrin and AP-2, only AP-2, or neither of these subunits.

AP-2 Binds with High Affinity to Tris Salt-stripped Mem- branes-If APs link the triskelion to the plasma membrane, then they in turn must bind to a specific component(s) of the membrane. Moreover, this binding site should be within a coated pit. To see if the inner surface of membranes has an AP-binding site, purified AP-2 was prepared (Fig. 1) and Tris salt-stripped membranes were incubated with various concen- trations of the complex at 4 “C. As shown in Fig. 3, with increasing concentrations of protein membranes bound more AP-2 until binding plateaued at ~30 pg/ml of AP-2. At saturation there was ~50% more AP-2 bound to the mem- brane than was initially present (compare with 0 on the ordinate, Fig. 3). We consistently found that more AP-2 bound to the membranes than was initially present (rebound AP-2/ initial AP-2 = 1.81 f 0.6, n = 13) even though the magnitude of the difference was quite variable. From these experiments we determined that the concentration of AP-2 required for half-maximal binding was =3 X lo-’ M.

The membranes were subjected to various treatments to determine the chemical identity of the AP-2-binding site. Treatment of Tris salt-stripped membranes with mild non- ionic detergents had little effect on binding; however, binding was completely destroyed by ionic detergents. The binding

/iP 2 Cwnrln *p-z Clam”” AP-2 cam” NO Treatment pli 9.0 0.5 M Trls

FIG. 2. Effect of high pH and Tris on the amount of triske- lion (clathrin) and AP complex (AP-2) bound to membranes. Attached membranes were prepared and either not treated (no treat- ment), washed with buffer D (pH 9.0), or washed with buffer D and buffer F (0.5 M Tris) before fixation with formaldehyde. Fixed mem- branes were assayed for the presence of either triskelions or AP-2 using an indirect ‘251-streptavidin binding assay as described. All values are the average of triplicate wells.

1

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01 5 10 20 30 40 50 AP-2 (wg/ml)

FIG. 3. Dependence of AP-2 binding on protein concentra- tion. Membranes (Cl) were prepared in individual wells of a 96-well plate and treated sequentially with buffer D, pH 9.0, and then buffer E containing 0.6 M NaCl to remove endogenous AP-2. Wells were incubated with the indicated concentration of purified AP-2 for 30 min at 4 “C and assayed for bound AP-2 as described. The amount of AP-2 on untreated membranes (0) and the amount of AP-2 remaining on the membranes after the salt wash (v) are indicated on the ordinate. Values represent the amount bound to membranes minus the amount bound to wells that did not contain membranes; no cell well values ranged from 6,000 cpm (at 0.7 rg/ml AP-2) to 30,000 cpm (at 52.3 @g/ml AP-2). All values are the average of triplicate wells.

Elastase + PMSF

NO Membranes ---’ 0

0 5 10 15 20 Elastase Concentration

h.WfW FIG. 4. Effect of elastase (0) or elastase plus PMSF (A) on

AP-2 binding to Tris salt-stripped membranes. Membranes were prepared in individual wells of a 96-well plate, washed with buffer F (0.5 M Tris), and incubated in the presence of the indicated concentration of elastase (0) or elastase plus 1 mM PMSF (A) in buffer E for 30 min at 4 ‘C. Proteolysis was terminated by washing the membranes with buffer E containing 1 mM PMSF. Following the elastase, the membranes were washed again with buffer F and incu- bated for 30 min at 4 “C with 10 pg/ml of coat proteins (prepared by Tris extraction (18)) in buffer E containing 1% C-BSA and 1 mM PMSF. The membranes were then assayed for AP-2 binding as described. All values are the average of duplicate wells. The amount of endogenous AP-2 on the membranes before (x) and after (0) stripping are shown on the ordinate.

site was also sensitive to the serine proteases elastase, trypsin, and chymotrypsin. Fig. 4 shows that with increasing amounts of elastase, there was a decrease in the binding of AP-2 to Tris salt-treated membranes. The elastase effect was due to proteolysis because loss of binding was prevented by PMSF. By contrast, neither carboxypeptidase A and Y, thermolysin, papain, thrombin, nor endopeptidase Arg-C, Lys-C, and Gly- C had any effect on binding. Since the binding site was insensitive to either high salt or carbonate washing (100 mM sodium carbonate, pH 11.5), these results infer that the bind-

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ing site is an integral membrane protein. If these binding sites are involved in coated pit assembly,

then they should be occupied by endogenous AP-2 within coated pits and be oriented exclusively towards the inside surface of the membrane. To test the first assumption, we compared the amount of AP-2 that would bind to salt-stripped membranes with the amount that would bind to unstripped membranes that contain coated pits (Fig. 5). After incubating each set of membranes with 50 pg/ml of AP-2, there was not any difference in the amount of AP-2 that was bound. Al- though, more AP-2 was bound to both membranes than was initially present, the magnitude of the difference was the same for both stripped and unstripped membranes. These results indicate that a substantial portion of the binding sites on stripped membranes are occupied by endogenous AP-2 on unstripped membranes. The second assumption was tested by using immunofluorescence to assay for the presence of binding sites on the outside surface of the cell. Either sonicated membranes or whole cells were incubated at 4 “C with satu- rating amounts of AP-2, fixed, and processed for immunoflu- orescence. Whereas the membranes gave a bright anti-AP-2 IgG signal, there was not any evidence of binding to whole cells (data not shown).

We further explored the coated pit localization of the AP- 2-binding site using elastase as a probe. Tris salt-stripped membranes were incubated with increasing amounts of elas- tase, Tris salt-washed again, and then assayed for AP-2 binding. As seen in Fig. 6A there was a concentration-de- pendent loss of binding sites. By contrast, when the same protocol was applied to unstripped membranes, there was not any loss of AP-2-binding sites. The protection was not due to some other factor that was removed by the stripping procedure because Fig. 6B shows that when present, the excess AP-2- binding sites (compare with initial values, x on ordinate, Fig. 6B) detected in most experiments were destroyed by elastase even though these membranes were not initially washed with Tris salt. In other words, only the AP-e-binding sites that were occupied by coated pits were resistant to elastase.

The selective localization of AP-2 in coated pits (13) and

FIG. 5. Binding of AP-2 to either salt stripped or unstripped membranes. Membranes were prepared in individual wells of a 96- well plate and either not treated or washed sequentially with buffer D, pH 9.0, and then buffer E containing 0.6 M NaCl to remove endogenous AP-2. Membranes were incubated with 10 rg/ml of purified AP-2 for 30 min at 4 “C and assayed for bound AP-2 as described. Values represent the amount bound to membranes minus the amount that bound to wells that did not contain membranes. All values are the average of triplicate wells.

16517

0 2.5 5 7.5 lo Elastase @g/ml)

FIG. 6. Effect of elastase on AP-2 binding to membranes that either have (Cl) or do not have (B) coated pits. Membranes were prepared in individual wells of a 96-well plate and treated with elastase either before (0) or after (m) treatment with buffer F (0.5 M Tris) to remove endogenous triskelions and AP-2. Membranes were washed with buffer E prepared without protease inhibitors and then incubated with the indicated concentrations of elastase for 30 min at 4 “C. Proteolysis was terminated by washing the membranes with buffer E containing 1 mM PMSF. Following the elastase, the mem- branes were washed again with buffer F (0.5 M Tris) and incubated for 30 min at 4 “C with 10 rg/ml of coat proteins (prepared by Tris extraction (18)) in buffer E containing 1% C-BSA and 1 mM PMSF. The membranes were then assayed for AP-2 binding as described. The values represent the amount bound to membranes minus the amount bound to wells that did not contain membranes. All values are the average of duplicate wells. The amount of endogenous AP-2 on the membranes before (x) and after (0) stripping are shown on the ordinate. A and B are from separate experiments that differed in the number of excess AP-2-binding sites on the membrane.

AP-1 in the Golgi (12) could be due to differences in the affinity of binding to their respective membranes. When we tested the ability of AP-1 to compete for AP-2 binding to plasma membranes (Fig. 7), we found that there was weak competition. About a lo-fold higher level of AP-1 was required to reduce AP-2 binding by 50%. The unavailability of enough AP-1 made it impossible to extend the concentration curve. This was specific competition because neither 10 mg/ml of albumin nor 10 mg/ml of crude bacterial extract diminished AP-2 binding (data not shown).

Triskelions Will Not Bind to Rebound AP-2-If the high affinity binding of AP-2 to the cytoplasmic surface of plasma membranes is the first step in the initiation of coated pit assembly, then the next step should be triskelion binding to AP-2. To see if this second binding event could be assayed in vitro, we prepared Tris salt-stripped membranes and incu- bated them sequentially with purified AP-2 and triskelions using various concentrations and protocols. Regardless of the conditions, we failed to see any significant binding of triske- lions, even though normal amounts of AP-2 bound (data not shown).

One explanation for these results is that the triskelions were rendered incompetent to bind to the AP-2 subunits during the purification; therefore, additional experiments were performed. We measured the ability of triskelions to bind to pH-stripped membranes since these membranes have

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To rule out the possibility that the AP-2 subunits were damaged during the purification procedure, we prepared coat proteins and analyzed their binding activity without further purification. The AP-2 subunit from these preparations bound to Tris salt-stripped membranes as well as purified AP-2 (Fig. 6). Moreover, these same subunits promoted clathrin cage formation at physiologic pH and ionic strength (Fig. lo), whereas purified triskelions did not assemble under these conditions. Since the triskelions present in the coat protein preparations would not bind to AP-2 that had rebound to the plasma membrane (data not shown), we conclude that our AP-2 preparations retained the abilty to carry out all the interactions previously described by other investigators but are unable to bind to the triskelions when they are rebound to Tris salt-stripped membranes.

DISCUSSION

High Affinity Binding of AP-2-Isolated plasma membranes stripped of endogenous coat proteins display high affinity binding sites for the AP-2 complex. Binding is saturable, but at saturation a typical membrane preparation has substan- tially more (up to 2-fold) AP-2 bound than was initially present on the membrane. The differences between initial levels and rebound levels of AP-2 were the same regardless of whether or not the membranes were stripped (Fig. 7). This implies that the additional bound AP-2 is on high affinity sites that either are not occupied within the cell or become unoccupied during the initial preparation of the membranes.

There are several reasons to think that AP-2 binding is specific. First, a binding constant of 3 x lo-’ M is comparable to the binding affinity of ankyrn (26) and band 4.1 (27), two soluble proteins that bind to the cytoplasmic portion of band 3 in erythrocyte membranes. These two proteins tether the spectrin-actin complex to the plasma membrane (28). Second, a major portion of the binding sites are occupied by coated pits because when coated pits are left intact, they mask AP-2 binding and protect the binding site from protease treatment. Third, the binding site is very sensitive to elastase, which indicates that it is a protein. Finally, the binding site behaves as if it is an integral membrane protein, since it is not removed by high salt or carbonate treatment.

We were unable to measure AP-1 binding directly. Never-

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WC. 10. Formation of clathrin cages by coat proteins (0) but not pure triskelions (0). Clathrin cage reassembly was per- formed using the method of Keen (22). Coat proteins were removed from bovine brain-coated vesicles with buffer F (0.5 M Tris). Column- purified clathrin were prepared as described and centrifuged at 100,000 X g for 60 min to remove aggregated proteins. Either 800 pg of pure clathrin (in buffer G) or 800 rg coat proteins (in buffer F) were diluted to 1 ml with buffer J and dialyzed against buffer J overnight at 4 “C. Samples were layered onto 15.ml linear gradients of 5-20% (v/v) glycerol in buffer J and were centrifuged at 26,000 rpm for 90 min. The gradients were fractionated into 0.5-ml fractions and the protein concentration of each fraction was measured using the bicinchoninic acid method (32).

theless, in a competition assay there was evidence that AP-1 did bind weakly to the AP-2-binding site. We cannot rule out the possibility that our AP-1 preparation was contaminated with AP-2, which accounted for the apparent lower affinity of AP-1; nevertheless, these results imply that whatever me- diates the binding of AP-1 to membranes in the Golgi region of a cell may be related to the AP-2-binding site on the plasma membrane.

The identity of the AP-P-binding site is not known. Based on their data, Pearse and Crowther (6) have suggested that the cytoplasmic tail of receptors that utilize the coated pit pathway interact with APs and that AP-1 and AP-2 recognize different sets of receptor tails. Whereas their model implies that the tail domain binds the AP subunit and initiates coated pit assembly, another interpretation is that AP subunits first bind to an integral membrane protein that is a structural component of coated pits and that receptor tails subsequently interact with APs during the clustering event. Further char- acterization of the high affinity AP-2-binding site we have identified should allow us to distinguish between these and other models for coated pit assembly.

AP-2 Is Necessary But Not Sufficient for Coated Pit Assem- bly-Despite the testing of a number of different conditions, we were unable to assemble purified triskelions onto Tris salt- washed membranes that had rebound AP-2 subunits. At first we attributed this failure to difficulties associated with puri- fying the various subunits. This seems not to be the case: (a) membranes that contained endogenous AP-2 avidly bound triskelions (Fig. 9), (b) AP-2 subunits promoted cage assembly under conditions where triskelions would not form cages (Fig. lo), (c) the AP-2 subunits of a crude coat protein preparation would bind to membranes, but once bound would not bind triskelions (Fig. 6; data not shown). Taken together, these results indicate that the various subunits are competent to interact if the conditions are suitable. Apparently we have been unable to recreate in vitro the proper environment for triskelions to bind to AP-2 that has rebound to membranes. There are several potential explanations for these results including the possibility that the stripping treatment has either rearranged the binding sites or removed an essential ingredient from the membrane.

The high affinity binding of triskelions to pH-stripped membranes that contained endogenous AP-2 is worth noting. The calculated half-maximal binding (3.4 x lo-’ M) was nearly identical to that reported for triskelion binding to pH- stripped coated vesicles (14). Associated with the binding was the formation of a planar clathrin lattice on the membrane. Therefore, the binding affinity has two components: (a) the contribution of AP-2/triskelion interactions and (b) the con- tribution of triskelion/triskelion interactions. In this assay, it is not possible to determine which of these interactions gov- erns the assembly sequence.

Goud et al. (29), found that clathrin made up about 0.2% of the total cellular protein in tibroblasts and that 50% of this was assembled on coated membranes. If an average cell is 20 pm in diameter, then the concentration of soluble triskelion in the cell would be -90 nM. This concentration is 30 times the amount required for half-maximal binding, which indi- cates that at anytime all of the binding sites in a cell should be occupied.

Multiple Steps in Coated Pit Assembly-We can identify at least three steps in the coated pit assembly process. 1) The plasma membrane contains a class of membrane proteins that initiates the assembly of a coated pit by binding AP-2. 2) Binding of AP-2 is followed by an activation step that makes the complex competent to bind clathrin triskelions, which in

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16520 An Integral Membrane Protein That Binds AP-2 Subunit

turn leads to assembly. The activation step could involve a modification (e.g. phosphorylation) of the AP-2 and/or a spatial rearrangement (e.g. clustering) of the complex to make it assembly competent. 3) The final step is the assembly of the clathrin lattice. Once the AP-2 complexes are in the proper state, this step proceeds without additional nonstructural molecules.

The AP-2 activation event may be what controls coated pit assembly. During each endocytic cycle, all of the coated pit proteins must recycle. Presumably the AP-2 binding site is like other membrane proteins and recycles through the en- docytic pathway (4). There is some evidence that AP-2 is associated with endosomes (30) even though these compart- ments are devoid of clathrin. If this is the case, then both AP- 2 and its binding site may recycle together. Upon returning to the plasma membrane, triskelion binding and assembly is only permissive after the AP-2 and its receptor cluster to- gether and assume an activated state. Recent studies by Beck and Keen (31) suggest that the AP-2 complex aggregates under physiological conditions. This aggregation behavior, when taking place in the plane of the membrane, may lead to efficient clustering, which in turn renders AP-2 competent for lattice assembly.

One of the peculiar aspects of the in vitro coated pit assem- bly reaction is the vastly different affinity triskelions exhibit towards endogenous as compared to exogenously added mem- brane bound AP-2. Keen and co-workers (22), have shown that the AP complex will bind to triskelions and that AP-2 binds much more avidly to immobilized triskelions than does AP-1. Yet in our membrane assay we cannot detect AP-2/ triskelion interactions when AP-2 is bound to membranes in an inactive form. These results suggest that the high affinity binding of triskelions (3.4 x lo-’ M) to membranes bearing activated AP-2 is due largely to interactions between the legs of the triskelions. Exactly what makes AP-2 competent to promote this interaction is an important topic for future investigation.

Acknowledgments-We would like to thank Bill Donzell for pre- paring the rapid-freeze deep etch images. We are also indebted to Renee Bianchi for preparing the purified triskelions and AP com- plexes. This work would not have been possible without the support of the members of the laboratory and the secretarial assistance of Mary Surovik.

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D T Mahaffey, J S Peeler, F M Brodsky and R G Andersonsubunit with high affinity.

Clathrin-coated pits contain an integral membrane protein that binds the AP-2

1990, 265:16514-16520.J. Biol. Chem. 

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