From the Department of Cell Biology, Harvard Medical School and The Center for Blood Research, Boston, Massachusetts 02115
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ABSTRACT |
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Eps15 is a phosphorylation substrate of the epidermal growth factor receptor kinase. In vivo, it is largely found in complex with AP-2, the plasma membrane clathrin adaptor protein complex. Although AP-2 is uniformly distributed across the surface of clathrin-coated pits and vesicles, Eps15 is preferentially found in the rims of endocytic clathrin-coated pits (1). This observation suggests that Eps15 may disengage from AP-2 during coat formation. Here we use two new anti-Eps15 antibodies to show that, contrary to our own earlier suggestion, coated vesicles isolated from brain do not contain detectable amounts of Eps15. Furthermore, when AP-2 complexes that are saturated with Eps15 are used for in vitro assembly of clathrin-AP-2 coats, normal structures are formed that contain the expected amounts of clathrin and AP-2, but the amount of Eps15 present is dramatically lower than that of AP-2. We propose that during coated pit formation, addition of clathrin to the growing edge at the rim of the pit releases Eps15 from AP-2.
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INTRODUCTION |
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During the past decade, several groups have extensively studied
the protein components and the mechanism responsible for the formation
of clathrin-coated pits. Clathrin and its tetrameric adaptors AP-1 and
AP-2 are among the best characterized structural elements of the coat
(for recent reviews, see Refs. 2-4). The APs recruit clathrin, and
they facilitate coat formation through its association with clathrin.
The interaction involves a short segment located in the hinge region of
the chain of AP-2 (5) and the terminal domain of
clathrin.1 Recently, it was
suggested that Eps15 (6), a ~100-kDa molecule that forms complexes
with AP-2 (7-9), is involved in the clathrin-mediated internalization
pathway. Eps15-AP-2 complexes are found both in the cytosol and at the
plasma membrane (1, 9, 10). In solution, the interaction between Eps15
and AP-2 involves a short segment toward the C terminus of Eps15 and
the C-terminal ear domain of the
chain of AP-2 (7, 8). Most of the
Eps15 in the cell is bound to AP-2, whereas ~80% of AP-2 is free
(1). The membrane-bound Eps15/AP-2 complexes are mainly found within clathrin-coated pits (1, 10), usually at the rims and growing edges of
coated pits (1).
The formation of coated pits at the plasma membrane requires AP-2 complexes and clathrin to be recruited to the membrane and a poorly understood nucleation event that initiates clathrin coat assembly. After nucleation, new clathrin and AP-2 complexes presumably add to the free edges of the pit, so that an AP-2 complex that is added to the pit early on will move progressively deeper into the pit. How is it, then, that Eps15 is concentrated at the edges of pits, regardless of the state of completion of the pit? We previously examined whether Eps15 is present in coated vesicles (1) and found a band of the approximately correct molecular weight in gels of purified bovine brain vesicle preparations. However, this band was not conclusively identified as Eps15, and in the absence of purified Eps15 quantitation was impossible. We recently used recombinant Eps15 to generate polyclonal anti-Eps15 antibodies (11). Here we readdress the question of the quantity of Eps15 present in complete vesicles. We estimate that less than 1 in 1000 AP-2 complexes in vesicles carry Eps15.
To further examine how Eps15 behaves during the process of coat formation, we made 1:1 complexes between recombinant Eps15 and purified AP-2 and determined their ability to incorporate into clathrin coats assembled in vitro. We found that the coats formed by Eps15-AP-2 complexes are indistinguishable from coats formed in the absence of Eps15 but that the AP-2 complexes present in the coats are significantly depleted in Eps15. We suggest that assembly of clathrin into the coat results in the disengagement of Eps15 from AP-2.
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EXPERIMENTAL PROCEDURES |
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Coat Proteins and Antibodies--
Clathrin-coated vesicles were
isolated from calf brains, and clathrin and AP-2 were purified using
previously described procedures (12-14). Recombinant Eps15 was made in
Escherichia coli and purified by nickel-nitriloacetic acid
chromatography (11). Stock solutions of clathrin (3 mg/ml), Eps15 (1 mg/ml), and AP-2 (0.7 mg/ml) were used as starting materials for the
experiments described here. Protein amounts were estimated by BCA or by
SDS-PAGE2 and Coomassie Blue
staining using purified clathrin and bovine serum albumin as standards.
Polyclonal antibodies specific for the N-terminal 538 amino acids or
for the C-terminal 368 residues of Eps15 were generated using the
corresponding glutathione S-transferase fusion proteins as
antigens. The monoclonal antibody specific for the 1/
2 subunits
of AP-2 was 6A (15).
Gel Filtration Chromatography-- Prior to gel filtration, the samples were dialyzed overnight at 4 °C against coat preassembly buffer (33 mM MES, 8 mM Hepes, 167 mM Tris, 100 mM NaCl, 0.67 mM EGTA, 0.02% NaN3, 0.08% Triton X-100, pH 7.4) or against coat assembly buffer (100 mM MES, 2 mM EDTA, 2 mM DTT, 0.05% Triton X-100, pH 6.6). After centrifugation at 15,000 rpm at 4 °C for 10 min, the supernatants (250 µl) were applied to a preparative grade Superose 6 gel filtration column (H10/30, Pharmacia Biotech Inc.) equilibrated with coat preassembly or with coat assembly buffer. The samples were eluted at a flow of 0.5 ml/min. 0.5-ml fractions were collected and processed for SDS-6% PAGE and Coomassie Blue staining or Western blot analysis.
Coat Assembly-- The in vitro assembly of coats was performed as described earlier (12) using 1 mg/ml clathrin and 0.25 mg/ml AP-2, in the absence or the presence of 0.3-0.6 mg/ml Eps15. After overnight dialysis at 4 °C against coat assembly buffer, the samples were subjected to a low speed centrifugation (15,000 rpm for 10 min; Eppendorf centrifuge) followed by a high speed centrifugation step (60,000 rpm for 12 min; TLA 100.4 rotor Beckman). The pellets were resuspended into the same starting volume with coat assembly buffer and processed for SDS-PAGE and Coomassie Blue staining or for negative staining. Assembly of coats was confirmed by direct electron microscopic observation of samples negatively stained with 1.5% uranyl acetate (5). Images were obtained at a primary magnification of 50,000.
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RESULTS AND DISCUSSION |
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The Amount of Eps15 in Coated Vesicles Is Surprisingly Low-- Electron microscopic observations of cell sections indicate that, regardless of coat size, the Eps15 present in a coated pit is concentrated at the rim (1). The question of how much Eps15 is present in a coated vesicle was left open, however. A band with a molecular weight appropriate for Eps15 was observed on a Western blot, but no purified Eps15 standards were available to allow firm identification or quantitation of the signal. Because recombinant Eps15 is now available (6, 7, 11), we combined Coomassie Blue staining and Western blot analysis to estimate the content of Eps15 in coated vesicles by comparison with known amounts of Eps15. The results, depicted in Fig. 1, indicate a content of less than 3 ng of Eps15 for ~20 µg of clathrin in the coated vesicle sample (lane 9). The amount of Eps15 was estimated by comparison with the signal elicited by Western blot in the Eps15 standard (lane 8). The amount of AP-2 in the same coated vesicle sample (lane 9) is ~3-5 µg. Because the proportion of AP-2 complexes in the cytoplasm that carry Eps15 is ~10%, it appears that the AP-2 complexes recruited into the vesicle have either been selected to lack Eps15 or have lost Eps15 as part of the assembly process. The ratio of AP-2 complexes to Eps15 is ~1,000:1 in the coated vesicle sample, and because each coated vesicle contains an average of ~30 AP-2 complexes, only one vesicle in 30 contains an Eps15 molecule.
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Eps15 Associates Stably with AP-2-- To determine whether the association between Eps15 and AP-2 s is perturbed by the assembly process, we first made Eps15-saturated AP-2. Recombinant Eps15 at 0.3 mg/ml was mixed with purified bovine brain AP-2 complexes at 0.25 mg/ml and assayed by gel filtration. After mixing, the AP-2 complexes quantitatively co-elute with Eps15 (Eps15 + AP-2, Fig. 2A); Eps15 is in excess over AP-2 by Coomassie Blue stain. Thus, all of the AP-2 complexes in this preparation are bound to Eps15 under the initial coat preassembly buffer conditions. The AP-2-Eps15 complexes elute at the same fractions as free Eps15 (Eps15; Fig. 2A), indicating that the Stokes radius of the complex is not significantly different from that of Eps15 itself (16). We have recently shown that Eps15 forms parallel dimers and anti-parallel tetramers of ~31 nm in length (11), and so this result is not surprising. Presumably this result indicates that the globular AP-2 complexes (~60-nm diameter) (17) do not bind at the extreme ends of the molecule.
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Eps15 Is Lost during Coat Assembly-- We next asked whether the Eps15-saturated AP-2 complexes can participate in the formation of clathrin coats. Under the saturating conditions used above, the presence of Eps15 does not interfere with the efficiency of coat formation. Negative stain electron microscopy (Fig. 3) shows that the normal barrel-shaped geometry of the clathrin-AP-2 coats was maintained in the presence of Eps15. Eps15 also had no effect on the efficiency of coat formation as estimated by counting the number of coats present in four random fields in the absence of Eps15 (91 ± 11 coats/field) or in the presence of Eps15 (86 ± 1 coats/field). We isolated the coats by high speed centrifugation and determined the relative amounts of Eps15, clathrin, and AP-2 by SDS-PAGE and Coomassie staining. The amount of AP-2 in the pellet is unaffected by the presence of excess Eps15 (compare lane 8 with lanes 10 and 12 in Fig. 4), but the ratio of Eps15 to AP-2 is very low, far from the stoichiometric ratio expected if Eps15-saturated AP-2 complexes are incorporated into coats without perturbation. Doubling the amount of Eps15 present in the coat-forming reaction may slightly increase the ratio of Eps15 to AP-2 in the pellet, but the ratio is clearly still far from 1:1. We propose, therefore, that the assembly of a clathrin coat interferes with the interaction between Eps15 and AP-2, such that the majority of AP-2 complexes loose their associated Eps15s. Glycerol gradient rate zonal centrifugation analysis confirms that the clathrin coats formed under these conditions have very little Eps15 compared with the amount of AP-2 present in the fractions containing the coats (not shown).
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Conclusions--
We have shown that the interaction between AP-2
complexes and clathrin is severely disrupted by the coat assembly
process. Several interpretations of this observation are possible. For example, it is possible that the coat forming process selects only AP-2
complexes that are free of Eps15 and that this selection drives the
dissociation of AP-2 and Eps15. However, such an interpretation would
be inconsistent with the electron microscopic observations that show
Eps15 located at the rims of growing coated pits (1). A second
possibility is that AP-2-Eps15 complexes can bind at the rim of a
coated pit, captured (for example) by AP-AP or AP-transmembrane protein
cytosolic tail interactions and that clathrin binding then displaces
Eps15 from the AP-2 complex. This displacement could be the result of
an allosteric change in AP-2 due to clathrin binding (14).
Alternatively, it could be due to steric or competitive interactions
between clathrin and Eps15 on AP-2 upon binding to each other that
results in the disruption of the contact between AP-2 and Eps15. Eps15
and clathrin bind to sites in AP-2 that are very close to each other
(17): the ear, the binding site for Eps15 (7, 8), is only 2-10 nm
from the
hinge, the binding site for clathrin (5). Eps15, at 31 nm
in length (11), could easily span this distance, and the terminal
domain of clathrin is ~7 nm in diameter (19). It is known, however,
that clathrin induces a conformational change in the AP-2 complex upon
binding and co-assembly to form the coat (14); this conformational
change may also be involved in the release of Eps15 from the AP
complexes at the growing face of the vesicle.
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ACKNOWLEDGEMENTS |
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We thank the members of our laboratory for stimulating discussions, especially E. Ter Haar. We also thank I. Rapoport and W. Boll for help with the purification of coat proteins.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM36548, by Special Funds from The Center for Blood Research and the Department of Cell Biology (to H. M. S.), and by a NATO fellowship (to P. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence addressed: Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3140; Fax: 617-278-3131; E-mail: kirchhausen{at}crystal.harvard.edu.
1 A. Contreras and T. Kirchhausen, manuscript in preparation.
2 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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