The α Chain of the AP-2 Adaptor Is a Clathrin Binding Subunit

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 β2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated αa exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of αa bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a ≈29-kDa αa clathrin-binding fragment that remained tightly cage-associated. Translated αa also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated β2 subunit. Clathrin binding by both the α and β 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 α-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other α functions. 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 β2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated αa exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of αa bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a ≈29-kDa αa clathrin-binding fragment that remained tightly cage-associated. Translated αa also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated β2 subunit. Clathrin binding by both the α and β 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 α-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other α functions. INTRODUCTIONReceptor-mediated endocytosis is a multi-step process involving membrane invagination, coated pit formation, and budding of these pits to form coated vesicles(2Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar). 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(1Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, 3Brodsky F.M. Science. 1988; 242: 1396-1402Crossref PubMed Scopus (205) Google Scholar). These coated membranes also contain additional protein components that have been referred to as assembly, adaptor, or associated proteins (APs). 1The abbreviations used are: APsassociated proteinsMES4-morpholineethanesulfonic acidPAGEpolyacrylamide gel electrophoresis. 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 γ, β1, AP47, and AP19 polypeptides; AP-2, a plasma membrane-associated heterotetramer of α, β2, AP50, and AP17 polypeptides; and AP-3/AP180, a neuronspecific monomer(2Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar, 4Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (535) Google Scholar). This study concerns the AP-2 complex and focuses on the interactions of its α subunit with clathrin. Two genetically distinct isoforms of α subunit exist: αa, an isoform which is expressed ubiquitously, and αa, an isoform believed to be expressed primarily in neurons. The isoforms are 84% identical and differ predominantly in their C-terminal portions. The αa isoform contains a unique 42 amino acid insert beginning at position 704(6Robinson M.S. J. Cell Biol. 1989; 108: 833-842Crossref PubMed Scopus (108) Google Scholar).Although AP-2-clathrin interactions have been studied in detail(1Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, 7Ahle S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar, 8Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4437-4441Abstract Full Text PDF PubMed Google Scholar, 9Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar, 10Keen J.H. Beck K.A. Biochem. Biophys. Res. Commun. 1989; 158: 17-23Crossref PubMed Scopus (18) Google Scholar, 11Keen J.H. Beck K.A. Kirchhausen T. Jarrett T. J. Biol Chem. 1991; 266: 7950-7956Abstract Full Text PDF PubMed Google Scholar, 12Matsui W. Kirchhausen T. Biochemistry. 1990; 29: 10791-10798Crossref PubMed Scopus (80) Google Scholar, 13Prasad K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar), 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 α and β2 subunits alone were necessary and sufficient for coat assembly activity(13Prasad K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar). Ahle and Ungewickell(7Ahle S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar), using mild denaturation to purify β2 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 (14Gallusser A. Kirchhausen T. EMBO J. 1993; 12: 5237-5244Crossref PubMed Scopus (124) Google Scholar) who demonstrated that recombinant β2 subunit purified by denaturation-renaturation from Escherichia coli inclusion bodies was capable of promoting clathrin assembly. Collectively, these results support the hypothesis that the β2 subunit plays an important role in AP-2-driven clathrin assembly in vivo, but the role of the α subunit remains undefined.We have previously reported that an α/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 α subunit, also recognizes and binds clathrin(13Prasad K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar). 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 α subunit can bind tightly to clathrin, consistent with a role in coat assembly or other coat-associated functions.MATERIALS AND METHODSThe TNT 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(1Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, 15Keen J.H. Willingham M.C. Pastan I.H. Cell. 1979; 16: 303-312Abstract Full Text PDF PubMed Scopus (297) Google Scholar). 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 MgCl2, 0.02% NaN3, pH 6.50; Buffer B, Buffer A: 1.0 M pH Buffer mM sodium MES, mM pH Buffer mM mM mM EGTA, 0.5 mM MgCl2, pH were expressed in vitro using a TNT rabbit reticulocyte lysate transcription-translation The αa by S. Robinson M.S. J. Cell Biol. 1989; 108: 833-842Crossref PubMed Scopus (108) Google Scholar) was into the of the pSP65 vector the in both the and were confirmed with and purified on chloride for the C-terminal was by the with The was by β2 subunit were from the using a by T. Kirchhausen T. W. A. Keen J.H. S. A. 1989; PubMed Scopus Google Scholar). was translated with the TNT was using a by were assembled to the and were translated in the of although the reagent both and a large of in the translation kit the to translation were at for at in a to and All utilized translated of Translated αa and β2 1.0 was prepared in mM of the was at into mM pH A translation and an of were with mM pH in a of to the of and W. Kirchhausen T. Biochemistry. 1990; 29: 10791-10798Crossref PubMed Scopus (80) Google Scholar). were at and with or a of of αa bound to cages were in Buffer A. with bound αa were in Buffer A and an of this with in A. were at and with a of of clathrin cages and binding of APs to preformed clathrin cages has been described J.H. Beck K.A. Biochem. Biophys. Res. Commun. 1989; 158: 17-23Crossref PubMed Scopus (18) Google Scholar, 11Keen J.H. Beck K.A. Kirchhausen T. Jarrett T. J. Biol Chem. 1991; 266: 7950-7956Abstract Full Text PDF PubMed Google Scholar). All were at this were with mM and at to Clathrin were in 0.5 M using the of 1.0 of cages and of translation were for in Buffer A with mM pH in a of were at for in a were in of and were with acid and of and were on of and its binding by APs have been described previously(1Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar). We the to a binding of was were with of Buffer A and A of translation was with of the and on the for with one each of and with of Buffer with and were in and by gel were and with and to for on were from the in and in a Translated αa polypeptides were in vitro the consisting of amino and a C-terminal the N-terminal amino The αa has a of but on as a of with the αa of The of the was than that of the as a was for on As a translated αa in the of the αa a as The expressed β2 subunit 4, was from translated αa by gel electrophoresis. translated αa, the β2 with a of in translated and were utilized as of in Translated αa and β2 proteolysis of AP-2 major protein one N-terminal core domains of the α and β2 subunits associated with and AP17 subunits, and the other consisting of domains to C-terminal of the α and β subunits and proteolysis of the in vitro translated αa or β2 subunits alone of and results were obtained translated proteins were in the of AP-2, as by of and on of the in the αa and the appendage a The of the in that at the of αa is identical to that of appendage from the of the αa M.S. J. Cell Biol. 1989; 108: 833-842Crossref PubMed Scopus (108) Google Scholar), the C-terminal of αa amino is to contain of the of αa, or of the αa with this of the were in the digestion of that the of translated αa by in a to the of an α subunit in the AP-2 proteolysis of translated α and β The of core and appendage domains from the subunit are for of αa and β2 translation were for at with the and by and and and and and and of in vitro translated α of of of the as of with of the appendage as of on limited digestion of in vitro translated αa of the a for translated β2 subunit was more to proteolysis than the αa subunit, as has been by S. Ungewickell E. J. Biol Chem. 1991; 266: Full Text PDF PubMed Google Scholar). The of translated αa, resulting in the of cores and 2, and was to that of αa in an AP-2 complex as by digestion of translated αa in the of AP-2 further digestion of both translated subunits revealed from in The core domains of translated αa, and of β2, were more than of of the translated subunits remained at greater than 2, and AP-2 N-terminal were at in of and the C-terminal of both the translated α and β polypeptides were to AP-2 in their at of 2, and have reported that a the of the α subunit. and J. in of mM acid the of α described of Translated to Clathrin the interaction of translated polypeptides with assembled clathrin, utilized a binding that the of the translation on the of clathrin cages that both translated a for the in vitro and a of to the AP-2 large subunits, for the assembled clathrin cages in the binding 4, As from S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar), translated β2 subunit bind to clathrin cages As in 4, the of translated α with the clathrin cages while a of translated and α was by of translated αa in the of cages, as a of was of the binding vitro translated αa, but or binds to assembled clathrin cages. of translation or or αa were in the and or 2, 4, and of of clathrin cages for at The were and of and 2, and and and and 4, and and were for αa, and were also the in vitro translation with increasing of clathrin cages obtained a of the of the binding the α bound was that of the translated protein was capable of binding clathrin. The binding was by mM a of AP-2 K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar), further that binding was specific and to or of translated αa and as a function of clathrin of translation in vitro translated αa or were with clathrin cages at the for and by The bound was by and of the and and was for The were to of and binding for the αa, and and binding for binding by translated αa subunit was by of AP-2, confirming that the interaction was specific the and the for the AP-2-clathrin interaction of S. Ungewickell E. J. Biol Chem. 1991; 266: Full Text PDF PubMed Google Scholar, J. Cell Biol. 1993; PubMed Scopus Google Scholar), a for the interaction of in with the by of translated αa to clathrin cages is by were with a the of AP-2 in Buffer for by with of translated αa for an additional αa was The were for and are expressed to binding in the of and interactions are by of such as J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, M.S. J. Cell Biol. 1989; 108: 833-842Crossref PubMed Scopus (108) Google Scholar, 15Keen J.H. Willingham M.C. Pastan I.H. Cell. 1979; 16: 303-312Abstract Full Text PDF PubMed Scopus (297) Google Scholar). The α-clathrin interaction was also of the cages, to which translated αa was with mM pH by a released most of the αa into the the αa with the clathrin cages by of the into Buffer and into Buffer A of Translated to Clathrin on Sepharose CL-4B used to the of α and β subunits to bind clathrin Sepharose CL-4B bound of of the translation The translated β2 bound tightly to the of its to bind clathrin as as of the αa was also although it was as as that of the β2 these while β2 bound with sensitivity to αa binding to clathrin was more to increasing pH the of the binding of in vitro translated αa and β2 to of translation of αa or β2 were for with of and or Sepharose and which been with A to the pH were with of the and the bound protein with of Buffer with All were with and of and were by and 2, and pH and pH and pH and pH B, from A for each are and as the of binding for each was at pH and was for αa and for of Translated studies have that the large core of AP-2 an to bind to clathrin J.H. Beck K.A. Biochem. Biophys. Res. Commun. 1989; 158: 17-23Crossref PubMed Scopus (18) Google Scholar). The C-terminal to interact with clathrin cages. We to the of domains of α subunit and its individual We that αa bound clathrin cages in the and remained predominantly in the to the of the N-terminal of translated αa to bind to preformed clathrin cages, also a by transcription-translation This protein bind in a to clathrin cages, although the binding was that of the protein of the protein was capable of suggesting that a greater of the translated protein was or that a binding been to the of core or appendage of translated and αa to cages with bound αa were with the core were by the cages while the appendage was released into the A has been obtained with native W. Kirchhausen T. Biochemistry. 1990; 29: 10791-10798Crossref PubMed Scopus (80) Google Scholar). on more proteolysis of the translated αa a fragment and was in the of clathrin cages 2, with This fragment likely to the clathrin-binding of the α of in vitro translated αa subunit bound to clathrin cages a unique ≈29-kDa Clathrin cages bound in vitro translated αa in Buffer A were for at in the and or and of and released αa were by and and 2, and 4, is capable of binding clathrin with an interaction an in the coat assembly with urea or guanidinium the AP-2 complex has been by gel or purified α, and subunits. the large α and β2 subunits of AP-2 were for clathrin coat assembly in K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar). β2 subunits from such preparations were to bind to clathrin cages but alone clathrin S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar). studies using recombinant protein have reported that β2 alone is capable of clathrin A. Kirchhausen T. EMBO J. 1993; 12: 5237-5244Crossref PubMed Scopus (124) Google Scholar), and has been implicated in clathrin in the S. Ungewickell E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). work from this that α subunits bind to clathrin and S. K. Keen J.H. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). these from in that the α of β and an of clathrin binding to the α further the of clathrin-binding subunits, have translated the αa and β2 subunits of AP-2 in vitro in a rabbit reticulocyte lysate system to their clathrin binding The approach of in vitro translation has important The individual subunits are to the that it difficult to be that the native has been the translation system polypeptides in a with the folding more the intracellular polypeptides are that can be used at in This is a major in the study of AP-2 structure and function the protein and its subunits are to and K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4437-4441Abstract Full Text PDF PubMed Google Scholar) at protein or the study of individual polypeptides of proteins by in vitro transcription-translation J. Biol Chem. 1991; 266: Full Text PDF PubMed Google Scholar) may be for the these proteins have been to function as is that the functions of the AP-2 complex as an of the W. J. Biol Chem. Full Text PDF PubMed Google Scholar, J. Biol Chem. 1993; Full Text PDF PubMed Google Scholar). The structural and of the α and β polypeptides reported here and previously S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar, A. Kirchhausen T. EMBO J. 1993; 12: 5237-5244Crossref PubMed Scopus (124) Google Scholar) that have results that of AP-2 αa and β2 polypeptides can be expressed in a form in to the and by folding into the core and appendage domains that are of the AP-2 The obtained on limited proteolysis to from the large subunits of AP-2, are the C-terminal appendage fragment is to further the core to be more and This is consistent with the structure of J.H. Beck K.A. Biochem. Biophys. Res. Commun. 1989; 158: 17-23Crossref PubMed Scopus (18) Google Scholar, S. Keen J.H. J. Cell Biol. PubMed Scopus Google Scholar). The α and β2 C-terminal with other subunits of AP-2 and likely function as and in native AP-2 protein the N-terminal core domains of α and β are in to each other and to the and AP17 These interactions with the translated the in vitro translated subunits may be and more to the results in proteolysis of αa, the appendage are while the core is retained. is the of a fragment αa is The of this with the of the core suggesting that further digestion is by and by clathrin. this fragment may be more in the of clathrin, of this are It likely that this fragment of a clathrin-binding the α to the of the core and fragment the protein is and core to binding interact with This may that the appendage C-terminal of αa are to the core in a in which it is to interact with clathrin. the appendage or may interact with clathrin that both αa and β2 subunits of AP-2 have clathrin-binding sites the that coat assembly by bivalent binding and of clathrin in a that to formation, the cross-linking model J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar). this to other proteins such as AP-3/AP180, S. Ungewickell E. J. Cell Biol. 1990; PubMed Scopus Google Scholar, Ungewickell E. Biochemistry. 1991; 30: PubMed Scopus (18) Google Scholar) and a K. E. Biol. Cell. 1993; Scholar) that have been reported to promote clathrin assembly in vitro remains to be the of proteins capable of promoting polymerization that coat assembly may be by a of in both αa and β2 subunits bind to assembled clathrin αa subunit binding to clathrin is more to pH in the than is the β subunit. This to be a of of the α in as in the proteolysis or of translated α subunit this pH The of the α subunit for clathrin with pH with the of AP-2 to coat with S. Keen J.H. J. Cell Biol. PubMed Scopus Google Scholar), for a role of the α subunit in lattice assembly. cytoplasmic to pH also results in of clathrin with J. J. Cell Biol. 1989; 108: PubMed Scopus Google Scholar) K. S. J. Cell Biol. 1987; 105: PubMed Scopus Google Scholar, S. Keen J.H. J. Cell Biol. PubMed Scopus Google Scholar, J. J. J. Cell Biol. 1989; 108: PubMed Scopus Google Scholar, K. J. Cell Biol. 1993; PubMed Scopus Google Scholar). These that the α-clathrin interaction may also be in lattice and or that this binding clathrin may other α functions. INTRODUCTIONReceptor-mediated endocytosis is a multi-step process involving membrane invagination, coated pit formation, and budding of these pits to form coated vesicles(2Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar). 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(1Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, 3Brodsky F.M. Science. 1988; 242: 1396-1402Crossref PubMed Scopus (205) Google Scholar). These coated membranes also contain additional protein components that have been referred to as assembly, adaptor, or associated proteins (APs). 1The abbreviations used are: APsassociated proteinsMES4-morpholineethanesulfonic acidPAGEpolyacrylamide gel electrophoresis. 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 γ, β1, AP47, and AP19 polypeptides; AP-2, a plasma membrane-associated heterotetramer of α, β2, AP50, and AP17 polypeptides; and AP-3/AP180, a neuronspecific monomer(2Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar, 4Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (535) Google Scholar). This study concerns the AP-2 complex and focuses on the interactions of its α subunit with clathrin. Two genetically distinct isoforms of α subunit exist: αa, an isoform which is expressed ubiquitously, and αa, an isoform believed to be expressed primarily in neurons. The isoforms are 84% identical and differ predominantly in their C-terminal portions. The αa isoform contains a unique 42 amino acid insert beginning at position 704(6Robinson M.S. J. Cell Biol. 1989; 108: 833-842Crossref PubMed Scopus (108) Google Scholar).Although AP-2-clathrin interactions have been studied in detail(1Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, 7Ahle S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar, 8Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4437-4441Abstract Full Text PDF PubMed Google Scholar, 9Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar, 10Keen J.H. Beck K.A. Biochem. Biophys. Res. Commun. 1989; 158: 17-23Crossref PubMed Scopus (18) Google Scholar, 11Keen J.H. Beck K.A. Kirchhausen T. Jarrett T. J. Biol Chem. 1991; 266: 7950-7956Abstract Full Text PDF PubMed Google Scholar, 12Matsui W. Kirchhausen T. Biochemistry. 1990; 29: 10791-10798Crossref PubMed Scopus (80) Google Scholar, 13Prasad K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar), 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 α and β2 subunits alone were necessary and sufficient for coat assembly activity(13Prasad K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar). Ahle and Ungewickell(7Ahle S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar), using mild denaturation to purify β2 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 (14Gallusser A. Kirchhausen T. EMBO J. 1993; 12: 5237-5244Crossref PubMed Scopus (124) Google Scholar) who demonstrated that recombinant β2 subunit purified by denaturation-renaturation from Escherichia coli inclusion bodies was capable of promoting clathrin assembly. Collectively, these results support the hypothesis that the β2 subunit plays an important role in AP-2-driven clathrin assembly in vivo, but the role of the α subunit remains undefined.We have previously reported that an α/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 α subunit, also recognizes and binds clathrin(13Prasad K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar). 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 α subunit can bind tightly to clathrin, consistent with a role in coat assembly or other coat-associated functions.