©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Chain of the AP-2 Adaptor Is a Clathrin Binding Subunit (*)

(Received for publication, April 28, 1995; and in revised form, July 13, 1995)

Oscar B. Goodman , Jr. (§) James H. Keen (¶)

From the Department of Pharmacology and the Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated beta2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated alpha(a) exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of alpha(a) bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a approx29-kDa alpha(a) clathrin-binding fragment that remained tightly cage-associated. Translated alpha(a) also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated beta2 subunit. Clathrin binding by both the alpha and beta subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H.(1987) Cell Biol. 105, 1989). It also [Abstract] raises the possibility that the alpha-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other alpha functions.


INTRODUCTION

Receptor-mediated endocytosis is a multi-step process involving membrane invagination, coated pit formation, and budding of these pits to form coated vesicles(2) . A major protein implicated in endocytosis is clathrin, a triskelion-shaped protein that forms the structural basis for the regular polygonal lattice of coated pits and vesicles(1, 3) . These coated membranes also contain additional protein components that have been referred to as assembly, adaptor, or associated proteins (APs). (^1)One probable function of APs is to promote polymerization of the clathrin lattice at defined sites and times. APs are also likely to interact with receptor cytoplasmic tails resulting in the selective inclusion of various receptors into coated pits (reviewed in Refs. 2, 4, 5).

APs vary in structure and intracellular localization. The best characterized examples include AP-1, a Golgi-associated heterotetramer consisting of , beta1, AP47, and AP19 polypeptides; AP-2, a plasma membrane-associated heterotetramer of alpha, beta2, AP50, and AP17 polypeptides; and AP-3/AP180, a neuronspecific monomer(2, 4) . This study concerns the AP-2 complex and focuses on the interactions of its alpha subunit with clathrin. Two genetically distinct isoforms of alpha subunit exist: alpha(c), an isoform which is expressed ubiquitously, and alpha(a), an isoform believed to be expressed primarily in neurons. The isoforms are 84% identical and differ predominantly in their C-terminal portions. The alpha(a) isoform contains a unique 42 amino acid insert beginning at position 704(6) .

Although AP-2-clathrin interactions have been studied in detail(1, 7, 8, 9, 10, 11, 12, 13) , it has been difficult to ascertain the contributions of individual AP-2 subunits. Fractionation of AP-2 polypeptides with urea and guanidinium chloride was used to study these interactions, indicating that the alpha and beta2 subunits alone were necessary and sufficient for coat assembly activity(13) . Ahle and Ungewickell(7) , using mild denaturation to purify beta2 subunit from AP-2, demonstrated that the former was capable of competitively inhibiting AP-2 binding to preassembled clathrin cages. This work was extended by Gallusser and Kirchhausen (14) who demonstrated that recombinant beta2 subunit purified by denaturation-renaturation from Escherichia coli inclusion bodies was capable of promoting clathrin assembly. Collectively, these results support the hypothesis that the beta2 subunit plays an important role in AP-2-driven clathrin assembly in vivo, but the role of the alpha subunit remains undefined.

We have previously reported that an alpha/AP50/AP17 complex prepared by mild denaturation-renaturation was capable of binding to preformed clathrin cages, suggesting that one or more of the other subunits, most likely the alpha subunit, also recognizes and binds clathrin(13) . We adopt the approach of in vitro translation of the individual large AP-2 subunits to further explore this issue. The findings reported here indeed demonstrate that the alpha subunit can bind tightly to clathrin, consistent with a role in coat assembly or other coat-associated functions.


MATERIALS AND METHODS

The T(N)T rabbit reticulocyte lysate transcription-translation kit and pSP65 cloning vector were purchased from Promega. Translabel was obtained from ICN Biomedicals, Inc. Sepharose CL-4B and Superose 6B resins were from Sigma, and CN-Br activated Sepharose CL-4B was purchased from Pharmacia Biotech Inc. Clathrin and assembly proteins were prepared from calf brains as described previously(1, 15) . L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-trypsin was from Worthington Biochemical, Inc. HEPES was purchased from Boehringer Mannheim. All other chemicals were reagent grade or better.

Buffers used are as follows: Buffer A: 0.1 M sodium MES, 1.0 mM EGTA, 0.5 mM MgCl(2), 0.02% NaN(3), pH 6.50; Buffer B, Buffer A: 1.0 M Tris-HCl, pH 7.0, glycerol (4.5:4.5:1 (v/v/v)); Buffer C, 5 mM sodium MES, 2 mM CaCl(2), pH 6.15; Buffer T, 100 mM dipotassium tartrate, 10 mM HEPES, 1 mM EGTA, 0.5 mM MgCl(2), pH 7.0

In Vitro Transcription-Translation

Polypeptides were expressed in vitro using a T(N)T rabbit reticulocyte lysate transcription-translation kit. The alpha(a) cDNA, kindly provided by M. S. Robinson (Oxford University) (6) was subcloned into the EcoRI site of the pSP65 vector (Promega), under the sp6 promotor. Constructs containing inserts in both the sense and antisense orientations were confirmed with restriction enzyme analysis and purified on cesium chloride gradients.

The cDNA template for the C-terminal deletion mutant alpha was produced by restricting the pSP65alpha(a) sense construct with AvaI (alpha(a) nucleotide 2021). The mutant was then generated by runoff transcription-translation. Full-length beta2 subunit were transcribed from the pBluescript SK+ T3 promotor, using a construct kindly provided by T. Kirchhausen (Harvard University)(16) . Luciferase was translated with the sp6 T(N)T control construct (Promega). beta-Galactosidase was produced using a pSP65-based construct provided by V. Gurevich (Thomas Jefferson University). Transcription-translation reactions were assembled according to the manufacturer's instructions and incubated 90-120 min. Proteins were translated in the presence of [S]Translabel: although the reagent contained both [S]L-methionine and -cysteine, a large excess of unlabeled cysteine in the translation kit blocked the latter's incorporation. Prior to use, translation reactions were centrifuged at 100,000 revolutions/min for 20 min at 2 °C in a Beckman TLA 100 rotor to clear ribosomes and aggregates. All experiments utilized freshly translated subunits.

Proteolysis of Translated alpha(a) and beta2 Subunits

A fresh 1.0 mg/ml trypsin stock solution was prepared in 1 mM HCl. An aliquot of the stock solution was diluted at least 10-fold into 50 mM Tris-HCl, pH 7.4, immediately before use. A 5-µl translation reaction aliquot and an appropriate amount of diluted trypsin stock were combined with 50 mM Tris-HCl, pH 7.4, in a final volume of 100 µl, according to the protocol of Matsui and Kirchhausen(12) . Reactions were incubated 15 min at room temperature and quenched with either SDS sample buffer or a 3-fold molar excess of soybean trypsin inhibitor.

Digests of alpha(a) bound to cages were carried out in Buffer A. Cage high speed pellets (see below) with bound alpha(a) were resuspended in Buffer A and an aliquot of this suspension combined with trypsin stock freshly diluted in buffer A. Reactions were incubated 15 min at room temperature and quenched with a 3-fold molar excess of soybean trypsin inhibitor.

Cage Binding Assays

Preparation of clathrin cages and binding of APs to preformed clathrin cages has been described elsewhere (10, 11) . All steps were performed at 4 °C except where noted. In this work, cage aliquots were supplemented with 100 mM Tris-HCl and spun 2 min at 13,000 times g just prior to use. Clathrin concentrations were determined spectrophotometrically in 0.5 M Tris-HCl using the calculated extinction coefficient of 1.0 mg ml cm. Typically, 20-60 µg of cages and 5 µl of translation reaction were incubated for 30 min in Buffer A supplemented with 100 mM Tris-HCl, pH 6.5, in a final volume of 300 µl. Samples were centrifuged at 75,000 revolutions/min for 7 min in a TLA 100.2 rotor. Pellets were resuspended in 60 µl of sample buffer, and supernatants were precipitated with 10% (w/v) trichloroacetic acid and similarly resuspended. Equal aliquots of pellets and supernatant were analyzed on 7.5% SDS-polyacrylamide minigels.

Clathrin-Sepharose Binding Assays

Preparation of clathrin-Sepharose and its binding by APs have been described previously(1) . We modified the protocol to accommodate a small scale binding assay. Into Bio-Rad BioSpin Chromatography columns, 300 µl of resin was aliquoted. Columns were equilibrated with several column volumes of Buffer A containing 0.05 mg/ml bovine serum albumin, leupeptin, antipain, and pepstatin (10 µg/µl). A 3-µl aliquot of translation reaction was mixed with 97 µl of the equilibration buffer, loaded, and incubated on the column for 30 min, rinsed four times with one column volume each of equilibration solution, and eluted with four column volumes of Buffer B supplemented with 0.05 mg/ml bovine serum albumin. Unbound and eluted fractions were pooled separately, precipitated in 10% trichloracetic acid, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).

SDS-PAGE Gel Band Analysis

SDS-PAGE gels were Coomassie stained, destained, and treated with EN^3HANCE (DuPont NEN), dried, and subjected to fluorography for 0.5-16 h on Kodak X-0MAT film. Radioactive bands were excised from the dried gels, dissolved in H(2)O(2), and counted in a Wallack LKB scintillation counter.


RESULTS

In Vitro Translated Polypeptides

Two different alpha(a) polypeptides were synthesized in vitro (Fig. 1): the full-length polypeptide consisting of 977 amino acids (lane 1) and a C-terminal truncation mutant (alpha) possessing only the N-terminal 605 amino acids (lane 2). The full-length alpha(a) polypeptide has a calculated molecular mass of 107,655 Da but migrated on SDS-PAGE as a band of approx114,000 M(r), coincident with the Coomassie-stained alpha (a) band of bovine brain AP-2. The yield of the deletion mutant (M(r) = 67,500) was substantially lower than that of the full-length product, as approximately a 10-fold longer autoradiographic exposure was required for comparable detectability on film. As a negative control, we translated alpha(a) in the presence of the antisense alpha(a) construct, obtaining a greatly attenuated yield as expected (lane 3). The expressed beta2 subunit (lane 4, M(r) = 105,133) was readily resolved from translated alpha(a) by gel electrophoresis. Like translated alpha(a), the beta2 polypeptide comigrated with a corresponding Coomassie-stained band of AP-2. Finally, in some experiments, translated luciferase (lane 5, M(r) = 62,000) and beta-galactosidase (lane 6, M(r) = 116,000) were utilized as controls.


Figure 1: In vitro translated proteins. Translated reaction mixtures were analyzed by SDS, 7.5% PAGE and autoradiography as described under ``Materials and Methods.'' Lane 1, full-length AP-2 alpha(a) expressed from the pSP65alpha(a) plasmid; the 107.7-kDa polypeptide migrates as M(r) = 114,000 band. Lane 2, alpha (C-terminal deletion mutant) expressed from the AvaI-restricted pSP65alpha(a) plasmid; 67.5-kDa polypeptide migrates as M(r) = 65,000 band. Lane 3, cotranscription-translation of pSP65alpha(a) sense and pSP65alpha(a) antisense plasmids. Lane 4, full-length AP-2 beta2 polypeptide translated using the pBluescriptSKbeta2 plasmid; 105.1-kDa polypeptide migrates as M(r) = 105,000 band. Lane 5, luciferase (M(r) = 62,000). Lane 6, beta-galactosidase (M(r) = 116,000).



Proteolysis of in Vitro Translated alpha(a) and beta2 Polypeptides

Limited proteolysis of AP-2 reveals two major stable protein domains: one containing 60-66-kDa N-terminal core domains of the alpha and beta2 subunits associated with intact AP50 and AP17 intact subunits, and the other consisting of 30-40-kDa domains corresponding to smaller C-terminal appendages of the alpha and beta subunits (10, 12, 17, 18, and data not shown).

Similarly, proteolysis of the in vitro translated alpha(a) or beta2 subunits alone yielded fragments of approx58-66 kDa and approx40 kDa (Fig. 2). Identical results were obtained when translated proteins were cleaved in the presence of carrier AP-2, as assessed by comigration of Coomassie Blue-stained and radiolabeled bands on SDS-PAGE (data not shown). Quantitative analysis of the changes in the full-length alpha(a) product and the appendage domain confirm a precursor-product relationship (Fig. 3). The coincidence of the two curves in Fig. 3demonstrates that at all trypsin concentrations the fraction of full-length alpha(a) cleaved is virtually identical to that of appendage generated. Furthermore, from the published sequence of the alpha(a) cDNA(6) , the C-terminal 40 kDa portion of alpha(a) (i.e. amino acids 610-977) is predicted to contain five of the 17 methionine residues of alpha(a), or 27% of the total alpha(a) radiolabel. In close agreement with this prediction, 24% of the undigested full-length counts were found in the 40-kDa proteolytic product upon complete digestion of full-length alpha(a). Hence, we conclude that the initial cleavage of translated alpha(a) by trypsin occurs in a region corresponding to the relatively exposed linker of an alpha subunit in the intact AP-2 complex.


Figure 2: Limited proteolysis of translated alpha and beta polypeptides. The formation of labile core (C) and stable appendage (A) domains from the full-length subunit (F) are indicated (see text for details). Two-µl aliquots of alpha(a) (lanes 1-6) and beta2 (lanes 7-12) translation reactions were digested for 15 min at 22 °C with the following trypsin concentrations and analyzed by SDS-PAGE: lanes 1 and 7, no trypsin; lanes 2 and 8, 75 ng/ml; lanes 3 and 9, 150 ng/ml; lanes 4 and 10, 300 ng/ml; lanes 5 and 11, 600 ng/ml; lanes 6 and 12, 9600 ng/ml.




Figure 3: Quantitative analysis of in vitro translated alpha polypeptide cleavage. Comparison of progress of cleavage of the full-length polypeptide (plotted as percent of maximal cleavage: closed circles, solid line) with appearance of the appendage domain (plotted as percent of maximal change: open diamonds, dashed line) on limited trypsin digestion of in vitro translated alpha(a) polypeptide. Coincidence of the two curves supports a precursor-product relationship (see text for details).



The translated beta2 subunit was slightly more resistant to proteolysis initially than the alpha(a) subunit, as has been observed by others(17) . The initial proteolytic susceptibility of translated alpha(a), resulting in the generation of cores and appendages (Fig. 2, lanes 2 and 3) was comparable to that of bovine brain alpha(a) in an AP-2 complex as assessed by digestion of translated alpha(a) in the presence of AP-2 (data not shown). However, further digestion of both translated subunits revealed significant differences from those in brain AP-2. The 58-66-kDa core domains of translated alpha(a), and especially of beta2, were much more labile than those of AP-2. While virtually no 60-66-kDa fragments of the translated subunits remained at trypsin concentrations greater than 170 ng/ml (Fig. 2, lanes 4-6 and 10-12), AP-2 derived N-terminal products were stable at much higher trypsin concentrations, in excess of 840 ng/ml (21 and data not shown). In contrast, the 40-kDa C-terminal appendages of both the translated alpha and beta polypeptides were similar to AP-2 in their relative stability, resistant even at trypsin concentrations of 9,600 ng/ml (Fig. 2, lanes 6 and 12).

Elsewhere, we have reported that a high affinity inositol polyphosphate-binding site exists near the N terminus of the alpha subunit. (^2)Inclusion of 1 mM phytic acid (1,2,3,4 5,6-IP(6)) did not alter the proteolytic pattern of alpha cleavage described above (data not shown).

Binding of Full-length Translated Proteins to Preformed Clathrin Cages

To examine the interaction of translated polypeptides with assembled clathrin, we utilized a cage binding assay. In preliminary experiments, we ascertained that the addition of the translation reaction had no effect on the sedimentability of clathrin cages (data not shown). Furthermore, we determined that both translated luciferase, a standard for the in vitro expression system, and beta-galactosidase, a polypeptide of similar size to the AP-2 large subunits, showed essentially no affinity for the assembled clathrin cages in the binding assay (Fig. 4, lanes 1-8). As expected from previous work(7) , translated beta2 subunit did bind to clathrin cages (data not shown). As shown in Fig. 4, the majority of translated alpha polypeptide cosedimented with the clathrin cages (lane 13), while a small amount of translated and apparently aggregated alpha polypeptide was sedimented by low speed centrifugation (lane 12). Typically, sedimentation of translated alpha(a) in the absence of cages, taken as a measure of nonspecific binding, was 10-15% of the total binding (lane 10).


Figure 4: In vitro translated alpha(a), but not luciferase or beta-galactosidase, binds to assembled clathrin cages. Aliquots (5 µl) of translation mixes containing controls, luciferase (lanes 1-4), or beta-galactosidase (lanes 5-8), or alpha(a) (lanes 9-14) were incubated in the absence (lanes 1, 3, 5, 7, and 9-11) or presence (lanes 2, 4, 6, 8, and 12-14) of 60 µg of clathrin cages for 30 min at 4 °C. The samples were then centrifuged and equal proportions of high speed pellets (lanes 1 and 2, 5 and 6, 10, and 13) and supernatants (lanes 3, and 4, 7 and 8, 11, and 14) were electrophoresed; for alpha(a), low speed pellets (lanes 9 and 12) were also analyzed.



By incubating the in vitro translation mixture with increasing concentrations of clathrin cages we obtained a dissociation constant of 1.1 times 10M (Fig. 5). From the asymptote of the binding curve, the maximal alpha fraction bound was 0.71, implying that not all of the translated protein was capable of binding clathrin. The binding was not inhibited by 1 mM phytic acid, a potent inhibitor of AP-2 self-association(9) , further evidence that binding was specific and not due to aggregation or self-association (data not shown).


Figure 5: Binding of translated full-length alpha(a) and alpha as a function of clathrin cage concentration. Aliquots of translation reaction containing in vitro translated full-length alpha(a) (filled squares, solid line) or alpha (open squares, dashed line) were incubated with clathrin cages at the indicated concentrations for 30 min and fractionated by centrifugation. The fraction bound was determined by excision and counting of the high speed pellet and supernatant bands and was corrected for nonspecific sedimentation. The data were fit to rectangular hyperbolas (Kaleidagraph) yielding values of K approx 1.1 times 10M and 71% maximal binding (r = 0.999) for the full-length alpha(a), and K approx 3.1 times 10M and 36% maximal binding (r = 0.955) for alpha.



Clathrin cage binding by translated alpha(a) subunit was inhibited by saturating quantities of AP-2, confirming that the interaction was specific (Fig. 6). From the apparent IC and the published K(d) for the AP-2-clathrin interaction of 10M(17, 20) , we calculate a K(d) for the alpha(a)-clathrin interaction of 0.7 times 10M, in reasonable agreement with the value estimated by direct binding.


Figure 6: Binding of translated alpha(a) to clathrin cages is blocked by brain AP. Cages (20 µg) were incubated with a bovine brain AP preparation containing the indicated concentration of AP-2 in Buffer T for 90 min, followed by incubation with 4 µl of translated alpha(a) for an additional 30 min. Cage-associated alpha(a) was determined after centrifugation. The data were corrected for nonspecific sedimentation and are expressed relative to binding in the absence of exogenous brain AP.



Both clathrin-clathrin and AP-clathrin interactions are readily reversed by high concentrations of protonated amines such as Tris-HCl (1, 6, 15) . The alpha-clathrin interaction was also reversible. Brief (5 min) treatment of the sedimented cages, to which translated alpha(a) was bound, with 500 mM Tris-HCl, pH 7, followed by a high speed spin released most (>80%) of the alpha(a) into the supernatant. Furthermore, the solubilized alpha(a) again cosedimented with the clathrin cages reformed by stepwise dialysis of the dissociated preparation into Buffer C and then into Buffer A (data not shown).

Binding of Translated Proteins to Clathrin Trimers Immobilized on Sepharose CL-4B Beads

We used clathrin-Sepharose to assess the ability of alpha and beta subunits to bind clathrin triskelia (Fig. 7). Underivatized Sepharose CL-4B bound little if any of either of the translation products. The translated beta2 polypeptide bound tightly to clathrin-Sepharose, providing the first direct demonstration of its ability to bind disassembled clathrin as well as cage structures. Binding of the alpha(a) polypeptide was also observed, although it was not as complete as that of the beta2 polypeptide under these conditions. Interestingly, while beta2 bound with relatively little sensitivity to pH, alpha(a) binding to clathrin trimers was considerably more sensitive to increasing pH throughout the range 6.5-7.5.


Figure 7: pH dependence of the binding of in vitro translated alpha (a) and beta2 to clathrin-Sepharose. A, aliquots (3 µl) of translation reactions of alpha(a) (lanes 1-6) or beta2 (lanes 7-12) were incubated for 30 min with 300 µl of clathrin-Sepharose (lanes 2-5 and 8-11) or underivatized Sepharose (lanes 1, 6, 7, and 12) which had been pre-equilibrated with buffer A adjusted to the indicated pH (see below). Columns were washed with 1200 µl of the equilibration buffer (panel W) and the remaining bound protein eluted with 1200 µl of Buffer B supplemented with 0.05 mg/ml bovine serum albumin (panel E). All fractions were precipitated with 10% trichloroacetic acid, and equal proportions of washes and eluants were analyzed by electrophoresis and autoradiography. Lanes 1, 2, 7, and 8, pH 6.5; lanes 3 and 9, pH 6.8; lanes 4 and 10, pH 7.2; lanes 5, 6, 11, and 12, pH 7.6. B, data from panel A for each polypeptide are quantified and plotted as the percentage of maximal binding. Maximal binding for each polypeptide was observed at pH 6.5 and was 80% for alpha(a) and essentially 100% for beta2.



Functional Domains of Translated Proteins

Previous studies have shown that following controlled proteolysis, only the large core fragments of AP-2 retain an ability to bind to clathrin cages(10) . The smaller C-terminal appendages fail to interact appreciably with clathrin cages. We carried out related experiments to evaluate the functional properties of domains of isolated alpha subunit and its individual domains. We observed that only full-length alpha(a) bound clathrin cages de novo; in contrast the 58-66- and 40-kDa proteolytic products remained predominantly in the supernatant (data not shown).

Alternatively, to assess the ability of the isolated N-terminal region of translated alpha(a) to bind to preformed clathrin cages, we also produced a truncated polypeptide, designated alpha, by runoff transcription-translation (Fig. 1). This protein did bind in a saturable manner to clathrin cages, although the apparent binding affinity (K(d) = 3 times 10M) was somewhat lower that of the full-length protein (Fig. 5). Only 36% of the total protein was capable of binding, suggesting either that a greater proportion of the translated protein was misfolded or that a binding equilibrium had not been established.

In contrast to the failure of either core or appendage domain of translated and digested alpha(a) to bind, when cages with bound alpha(a) were incubated with trypsin the core fragments were preferentially retained by the cages while the appendage domain was released into the supernatant (Fig. 8). A similar result has been obtained with native AP-2(12) . Interestingly, on more vigorous proteolysis of the cage-bound translated alpha(a) a discrete 29-kDa cage-associated fragment became evident and was prominent only in the presence of clathrin cages (compare Fig. 8, lane 2, with Fig. 2). This fragment likely corresponds to the clathrin-binding domain of the alpha subunit.


Figure 8: Proteolysis of in vitro translated alpha(a) subunit bound to clathrin cages reveals a unique approx29-kDa fragment. Clathrin cages containing bound in vitro translated alpha(a) in Buffer A were incubated for 15 min at 22 °C in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of trypsin (100 ng/ml). Following sedimentation, cage-associated and released alpha(a) fragments were analyzed by electrophoresis and autoradiography. Lanes 1 and 2, high speed pellets; lanes 3 and 4, supernatants.




DISCUSSION

AP-2 is capable of binding clathrin trimers with high affinity, an interaction representing an initial step in the coat assembly process. Following treatment with urea or guanidinium chloride, the AP-2 complex has been fractionated by gel filtration or hydroxylapatite chromatography, yielding partially purified alpha, beta, and 50 kDa/17 kDa subunits. Of these, only the large alpha and beta2 subunits of AP-2 were required for clathrin coat assembly in vitro(13) . Dissociated beta2 subunits from such preparations were shown to bind to clathrin cages but could not alone sponsor clathrin assembly(7) . Recent studies using recombinant protein have reported that beta2 alone is capable of inducing clathrin assembly(14) , and beta1 has been implicated in clathrin recruitment in the trans-Golgi network(22) . Previous work from this laboratory suggested that alpha subunits could bind to clathrin trimers and cages(23) . However, these experiments suffered from potential limitations in that the alpha fractions contained small quantities of beta and 50-kDa/17-kDa polypeptides, preventing an unambiguous assignment of clathrin binding activity to the alpha subunit.

To further examine the issue of clathrin-binding subunits, we have translated the alpha(a) and beta2 subunits of AP-2 in vitro in a rabbit reticulocyte lysate system to assess their respective clathrin binding capabilities de novo. The approach of in vitro translation has several important advantages. The individual subunits are generated without resort to the strong denaturants that make it extremely difficult to be certain that the native state has been reattained. In contrast, the translation system produces polypeptides in a physiological environment with the appropriate folding factors, more closely resembling the intracellular milieu. Further, readily detectable radioactive polypeptides are generated that can be used at tracer levels (leq10M) in functional assays. This is a major advantage in the study of AP-2 structure and function because the protein and its subunits are prone to aggregation and self-association (8) even at relatively low protein concentrations (geqµg/ml or 10M). Finally, the study of individual polypeptides of multisubunit proteins by in vitro transcription-translation (24) may be particularly appropriate for the APs. Although these proteins have been assumed to function only as intact tetrameric complexes, there is recent evidence that the AP50 functions independently of the AP-2 complex as an activator of the vacuolar proton pump(25, 26) . The structural and functional attributes of the isolated alpha and beta polypeptides reported here and previously (7, 14) suggest that they, too, could have independent roles.

Our results show that readily detectable amounts of AP-2 alpha(a) and beta2 polypeptides can be expressed in a functional form in vitro. Both appear to assume the proper secondary and tertiary conformation by folding into the core and appendage domains that are characteristic of the intact AP-2 protein. The fragments obtained on limited proteolysis correspond well to those expected from the large subunits of bovine brain AP-2, though there are differences. While the C-terminal appendage fragment is resistant to further proteolysis, the core fragments appear to be more heterogenous and extremely labile. This is consistent with the proposed quaternary structure of isolated AP-2(10, 21) . The alpha and beta2 C-terminal appendages do not display stable intermolecular contacts with other subunits of AP-2 and likely function as independently folded and stable domains. In contrast, in native AP-2 protein the N-terminal core domains of alpha and beta are in proximity to each other and to the AP50 and AP17 polypeptides. These interactions do not occur with the translated polypeptides. Consequently, the in vitro translated subunits may be relatively unprotected and more prone to proteolysis, yielding the results seen in Fig. 2.

Upon proteolysis of cage-bound alpha(a), the appendage fragments are preferentially released, while the core is almost entirely retained. Of particular interest is the appearance of a novel 29-kDa fragment when cage-bound alpha(a) is proteolyzed (Fig. 8). The appearance of this band correlates well with the disappearance of the core 58-66-kDa fragments, suggesting that further digestion is blocked by tight association and stabilization by clathrin. Conversely, if dissociated this fragment may be more rapidly degraded: in the absence of clathrin, heterogeneous bands of this size are barely detectable (Fig. 2). It seems likely that this fragment comprises part of a discrete clathrin-binding domain within the alpha subunit.

In contrast to the tight retention of the core and 29-kDa fragment when the full-length protein is proteolyzed, alpha and proteolytic core fragments generated prior to cage binding interact with much lower affinity. This may indicate that the appendage and/or C-terminal linker regions of alpha(a) are required to maintain the free core domain in a conformation in which it is able to interact with clathrin. Alternatively, the appendage or linker regions may interact with clathrin directly.

The observation that both alpha(a) and beta2 subunits of AP-2 have clathrin-binding sites supports the concept that coat assembly proceeds by bivalent binding and stabilization of overlapping clathrin triskelia in a conformation that leads to polygon formation, essentially the cross-linking model proposed earlier(1, 2) . Whether this hypothetical mechanism extends to other proteins such as AP-3/AP180, auxillin (27, 28) and a novel AP-20 (29) that have been reported to promote clathrin assembly in vitro remains to be determined. In any case, the expanding group of proteins capable of promoting polymerization suggests that coat assembly may be invoked by different effectors under a variety of different circumstances in vivo.

Though both alpha(a) and beta2 subunits bind strongly to assembled clathrin lattices, alpha(a) subunit binding to clathrin trimers is much more sensitive to pH in the physiological range than is the beta subunit. This seems unlikely to be a consequence of lability of the isolated alpha conformation in solution, as we detect no change in either the proteolysis pattern or susceptibility of translated alpha subunit over this pH range (data not shown). The increased affinity of the alpha subunit for clathrin with decreasing pH correlates with the increased ability of AP-2 to drive coat formation with decreasing pH(21) , arguing for a role of the alpha subunit in lattice assembly. In addition, cytoplasmic acidification to pH 6.3-6.5 also results in ``freezing'' of clathrin lattices with increased curvature (32) thereby arresting receptor-mediated endocytosis(19, 21, 30, 31) . These observations suggest that the alpha-clathrin interaction may also be involved in lattice shape changes during vesiculation and endocytosis, or conceivably, that through this binding clathrin may affect other alpha functions.


FOOTNOTES

*
This research was funded by National Institutes of Health GM-28526 (to J. H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by NIH National Research Service Award #CA09662 and the Foerderer Foundation.

To whom correspondence should be addressed. Tel.: 215-955-4624; Fax: 215-923-1098; keen{at}lac.jci.tju.edu.

(^1)
The abbreviations used are: APs, associated proteins; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

(^2)
I. Gaidarov, Q. Chen and J. H. Keen, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. V. V. Gurevich for his helpful discussions and assistance with in vitro translation reactions, as well as for the beta-galactosidase control plasmid. We also thank Dr. M. S. Robinson for the alpha(a) adaptin clone and Dr. T. Kirchhausen for the full-length beta2-adaptin clone. We are grateful to Drs. F. Santini, I. Gaidarov, and J. Yuan for their thoughtful feedback concerning the manuscript.

Note Added in Proof-We have recently found that the translated mouse subunit of the AP-1 complex binds to clathrin cages with K approx 0.15 µM, comparable to the alpha(a) results shown in Fig. 5. In view of published results(22) , this suggests that AP-1, like AP-2, can bind bivalently to clathrin.


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