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• W2146282045 abstract "Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623–680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo. Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623–680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo. Clathrin-coated vesicles mediate protein and lipid transport between intracellular compartments in the regulated secretory and endocytic pathways of all eukaryotic cells (reviewed in Ref. 1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1294) Google Scholar). The outermost layer of a clathrin-coated vesicle consists of clathrin that has been polymerized into an icosahedral cage (2Pearse B.M. J. Mol. Biol. 1975; 97: 93-98Crossref PubMed Scopus (347) Google Scholar). Between the outer clathrin shell and the vesicle membrane lie the clathrin assembly proteins (3Vigers G.P. Crowther R.A. Pearse B.M. EMBO J. 1986; 5: 2079-2085Crossref PubMed Scopus (104) Google Scholar). Clathrin assembly proteins belong to one of two gene families, the tetrameric AP 1The abbreviations used are: AP, assembly protein; CaMKII, calcium/calmodulin-dependent protein kinase II; CKII, casein kinase II; GST, glutathione S-transferase; GST-AP180, GST fused with full length of AP180 (amino acids 1–901); GST-C16, GST fused with amino acids 745–901 of AP180; GST-C58, GST fused with amino acids 305–901 of AP180; GST-M42, GST fused with amino acids 305–744 of AP180; GST-N33, GST fused with amino acids 1–304 of AP180; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; CCV, clathrin-coated vesicle 1The abbreviations used are: AP, assembly protein; CaMKII, calcium/calmodulin-dependent protein kinase II; CKII, casein kinase II; GST, glutathione S-transferase; GST-AP180, GST fused with full length of AP180 (amino acids 1–901); GST-C16, GST fused with amino acids 745–901 of AP180; GST-C58, GST fused with amino acids 305–901 of AP180; GST-M42, GST fused with amino acids 305–744 of AP180; GST-N33, GST fused with amino acids 1–304 of AP180; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; CCV, clathrin-coated vesiclefamily and the monomeric AP family. Three tetrameric APs have been described and designated AP-1, AP-2, and AP-3. AP-1 and AP-2 were first characterized in 1987 as major clathrin-coated vesicle coat proteins (4Manfredi J.J. Bazari W.L. J. Biol. Chem. 1987; 262: 12182-12188Abstract Full Text PDF PubMed Google Scholar, 5Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar). AP-3 has only recently been identified (6Dell'Angelica E.C. Ooi C.E. Bonifacino J.S. J. Biol. Chem. 1997; 272: 15078-15084Crossref PubMed Scopus (117) Google Scholar, 7Dell'Angelica E.C. Ohno H. Ooi C.E. Rabinovich E. Roche K.W. Bonifacino J.S. EMBO J. 1997; 16: 917-928Crossref PubMed Scopus (327) Google Scholar, 8Simpson F. Peden A.A. Christopoulou L. Robinson M.S. J. Cell Biol. 1997; 137: 835-845Crossref PubMed Scopus (304) Google Scholar, 9Odorizzi G. Cowles C.R. Emr S.D. Trends Cell Biol. 1998; 8: 282-288Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and also appears to be associated with clathrin-coated vesicles (10Dell'Angelica E.C. Klumperman J. Stoorvogel W. Bonifacino J.S. Science. 1998; 280: 431-434Crossref PubMed Scopus (308) Google Scholar). Two members of the monomeric AP family have been described: AP180 and CALM. AP180 was independently discovered in a number of laboratories and was originally referred to as AP180 (11Ahle S. Ungewickell E. EMBO J. 1986; 5: 3143-3149Crossref PubMed Scopus (144) Google Scholar, 12Morris S.A. Schroder S. Plessmann U. Weber K. Ungewickell E. EMBO J. 1993; 12: 667-675Crossref PubMed Scopus (92) Google Scholar), pp155 (13Keen J.H. Black M.M. J. Cell Biol. 1986; 102: 1325-1333Crossref PubMed Scopus (45) Google Scholar), NP185 (14Kohtz D.S. Puszkin S. J. Biol. Chem. 1988; 263: 7418-7425Abstract Full Text PDF PubMed Google Scholar), or F1–20 (15Sousa R. Tannery N.H. Lafer E.M. Neuroscience. 1990; 34: 403-410Crossref PubMed Scopus (11) Google Scholar,16Zhou S. Sousa R. Tannery N.H. Lafer E.M. J. Neurosci. 1992; 12: 2144-2155Crossref PubMed Google Scholar). AP180, pp155, NP185, and F1–20 were found to be identical (17Murphy J.E. Pleasure I.T. Puszkin S. Prasad K. Keen J.H. J. Biol. Chem. 1991; 266: 4401-4408Abstract Full Text PDF PubMed Google Scholar,18Zhou S. Tannery N.H. Yang J. Puszkin S. Lafer E.M. J. Biol. Chem. 1993; 268: 12655-12662Abstract Full Text PDF PubMed Google Scholar) and given the name AP-3. However, in view of the decision to designate the newest member of the tetrameric AP family AP-3 (7Dell'Angelica E.C. Ohno H. Ooi C.E. Rabinovich E. Roche K.W. Bonifacino J.S. EMBO J. 1997; 16: 917-928Crossref PubMed Scopus (327) Google Scholar, 8Simpson F. Peden A.A. Christopoulou L. Robinson M.S. J. Cell Biol. 1997; 137: 835-845Crossref PubMed Scopus (304) Google Scholar), we refer to the monomeric AP-3 as AP180. AP180 localizes to synapses (19Perry D.G. Hanson V. Benuck M.L. Puszkin S. J. Histochem. Cytochem. 1991; 39: 1461-1470Crossref PubMed Scopus (16) Google Scholar, 20Sousa R. Tannery N.H. Zhou S. Lafer E.M. J. Neurosci. 1992; 12: 2130-2143Crossref PubMed Google Scholar, 21Perry D.G. Li S. Hanson V. Puszkin S. J. Neurosci. Res. 1992; 33: 408-417Crossref PubMed Scopus (10) Google Scholar), while CALM is expressed ubiquitously (22Dreyling M.H. Martinez-Climent J.A. Zheng M. Mao J. Rowley J.D. Bohlander S.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4804-4809Crossref PubMed Scopus (248) Google Scholar).AP-1, AP-2, and AP180 localize to clathrin-coated vesicles in situ and potentiate clathrin assembly in vitro (5Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, 11Ahle S. Ungewickell E. EMBO J. 1986; 5: 3143-3149Crossref PubMed Scopus (144) Google Scholar,23Zaremba S. Keen J.H. J. Cell Biol. 1983; 97: 1339-1347Crossref PubMed Scopus (153) Google Scholar, 24Prasad K. Lippoldt R.E. Biochemistry. 1988; 27: 6098-6104Crossref PubMed Scopus (34) Google Scholar). A comparative quantitative study revealed that AP180 is approximately 4-fold more efficient at promoting clathrin assembly than AP-1 or AP-2 (25Lindner R. Ungewickell E. J. Biol. Chem. 1992; 267: 16567-16573Abstract Full Text PDF PubMed Google Scholar). AP-1 localizes to clathrin-coated vesicles budding from Golgi membranes, while AP-2 localizes to clathrin-coated vesicles budding from plasma membranes (26Robinson M.S. J. Cell Biol. 1987; 104: 887-895Crossref PubMed Scopus (154) Google Scholar, 27Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO J. 1988; 7: 919-929Crossref PubMed Scopus (251) Google Scholar). AP180 localizes to clathrin-coated vesicles budding from presynaptic plasma membranes (28Takei K. Mundigl O. Daniell L. De Camilli P. J. Cell Biol. 1996; 133: 1237-1250Crossref PubMed Scopus (328) Google Scholar). Both AP-2 and AP180 also contain high affinity binding sites for inositides, the binding of which inhibits their ability to promote clathrin assembly (29Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar, 30Voglmaier S.M. Keen J.H. Murphy J.E. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar, 31Timerman A.P. Mayrleitner M.M. Lukas T.J. Chadwick C.C. Saito A. Watterson D.M. Schindler H. Fleischer S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8976-8980Crossref PubMed Scopus (52) Google Scholar, 32Norris F.A. Ungewickell E. Majerus P.W. J. Biol. Chem. 1995; 270: 214-217Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 33Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 34Gaidarov I. Chen Q. Falck J.R. Reddy K.K. Keen J.H. J. Biol. Chem. 1996; 271: 20922-20929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 35Hao W. Tan Z. Prasad K. Reddy K.K. Chen J. Prestwich G.D. Falck J.R. Shears S.B. Lafer E.M. J. Biol. Chem. 1997; 272: 6393-6398Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar).AP-2 and AP180 co-localize to budding coated vesicles in neuronal cells arrested with GTPγS (28Takei K. Mundigl O. Daniell L. De Camilli P. J. Cell Biol. 1996; 133: 1237-1250Crossref PubMed Scopus (328) Google Scholar). Furthermore, AP-2 and AP180 co-immunoprecipitate from coated vesicle extracts (14Kohtz D.S. Puszkin S. J. Biol. Chem. 1988; 263: 7418-7425Abstract Full Text PDF PubMed Google Scholar) and co-purify from various cell extracts (36Wang L.-H. Sudhof T.C. Anderson R.G. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 37Zhang J.Z. Davletov B.A. Sudhof T.C. Anderson R.G. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (431) Google Scholar). However, since AP-2 and AP180 also both bind to clathrin, it is unclear if their co-immunoprecipitation and co-purification reflect an indirect interaction with clathrin in a ternary complex or a direct interaction between these two proteins. We show here that AP-2 and AP180 interact directly, and we map the binding site for AP-2 on AP180 to amino acids 623–680. Both the α and β subunits of AP-2 bind to this same region of AP180.We also find that AP180 and AP-2 together are more efficient at assembling clathrin than is either AP180 or AP-2 alone. The assembly activity of the two proteins together is significantly greater than can be accounted for by simply adding the assembly activities of each protein alone. Further, we show that disruption of the AP180/AP-2 interaction by a recombinant fragment of AP180 containing the AP-2 binding site inhibits the enhanced assembly activity of the AP180 plus AP-2 combination. This indicates that formation of the AP180-AP-2 complex is required to obtain the supra-additive effects on clathrin assembly observed when both of these proteins are added to an assembly reaction.Coated vesicles also contain an associated CKII, and clathrin light chain β has been shown to be a good substrate for this enzyme (38Bar-Zvi D. Branton D. J. Biol. Chem. 1986; 261: 9614-9621Abstract Full Text PDF PubMed Google Scholar). AP180 is known to be phosphorylated in vivo, from both labeling studies of cultured neurons (13Keen J.H. Black M.M. J. Cell Biol. 1986; 102: 1325-1333Crossref PubMed Scopus (45) Google Scholar) and phosphatase studies of mouse brain extracts (16Zhou S. Sousa R. Tannery N.H. Lafer E.M. J. Neurosci. 1992; 12: 2144-2155Crossref PubMed Google Scholar). Furthermore, AP180 has been shown to be phosphorylated in an assembly protein fraction under conditions in which CKII is stimulated (39Morris S.A. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar). To extend these studies, we carried out experiments with purified proteins to determine if AP180 is a substrate for CKII. We find that there are three CKII phosphorylation sites in AP180, all within the central 42-kDa domain of the protein. Since this domain also contains residues important for clathrin assembly and AP-2 binding, we evaluated the effects of phosphorylation on these activities. While phosphorylation has no effect on the clathrin assembly activity of AP180 alone, it weakens the interaction between AP180 and AP-2. Apparently as a consequence of this, phosphorylation also reduces the assembly activity of the AP180/AP-2 combination by an amount consistent with its effect on AP180-AP-2 affinity.We propose a model in which AP180 and AP-2 associate to form a clathrin assembling complex. Phosphorylation, by modulating the affinity of AP180 for AP-2, may influence formation of this complex and thereby contribute to the regulation of clathrin assembly.EXPERIMENTAL PROCEDURESMaterialsGlutathione-Sepharose 4B was obtained from Amersham Pharmacia Biotech. Monoclonal antibodies against α or β subunits of AP-2, monoclonal antibodies against the γ subunit of AP-1, anti-IgG, ATP, bovine α-casein, polylysine, calf intestinal alkaline phosphatase agarose, trypsin, trypsin inhibitor, and the catalytic subunit of PKA were from Sigma. [γ-32P]ATP was from DuPont. CaMKII and CKII were from New England BioLabs. PKC was from Roche Molecular Biochemicals.Clathrin, AP-1, and AP-2 were purified from bovine brain clathrin-coated vesicles according to published procedures (5Keen J.H. J. Cell Biol. 1987; 105: 1989-1998Crossref PubMed Scopus (124) Google Scholar, 40Pearse B.M. Robinson M.S. EMBO J. 1984; 3: 1951-1957Crossref PubMed Scopus (144) Google Scholar, 41Prasad K. Keen J.H. Biochemistry. 1991; 30: 5590-5597Crossref PubMed Scopus (28) Google Scholar, 42Ungewickell E. Plessmann U. Weber K. Eur. J. Biochem. 1994; 222: 33-40Crossref PubMed Scopus (9) Google Scholar). Bacterially expressed mouse GST-AP180, GST-N33, GST-M42, GST-C58, GST-C16, and GST were expressed and purified as described previously (43Ye W. Lafer E.M. J. Biol. Chem. 1995; 270: 10933-10939Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The expression vector for the β2 subunit of AP-2 was kindly provided by Dr. Tomas Kirchhausen. Recombinant β2 was expressed and purified according to the published procedure (44Gallusser A. Kirchhausen T. EMBO J. 1993; 12: 5237-5244Crossref PubMed Scopus (124) Google Scholar). Monoclonal antibody F1–20 against AP180 was prepared from the hybridoma cell line F1–20 (15Sousa R. Tannery N.H. Lafer E.M. Neuroscience. 1990; 34: 403-410Crossref PubMed Scopus (11) Google Scholar).MethodsPhosphorylation of AP180 and Its DomainsProteins (GST-AP180, GST-N33, GST-M42, GST-C58, GST-C16, and GST) were all dialyzed overnight into buffer A (25 mm Tris-HCl, pH 7.4, 6 mm MgCl2, 1 mm EGTA, 1 mm EDTA, 1 mm dithiothreitol) at 4 °C and were clarified by centrifuging at 400,000 × g for 10 min at 4 °C.For all the reactions, 0.6 μm protein, 25 μm ATP, and 1 μCi of [γ-32P]ATP in buffer A were used in a reaction volume of 50 μl. For phosphorylation by CKII, 100 μg/ml polylysine, 130 mm KCl, and 30 units/μl CKII in buffer A was added (except for the experiment shown in Fig. 10 B, where the amount of enzyme is indicated on the displayed plot). For phosphorylation by CaMKII, 2.4 μmcalmodulin, 2 mm CaCl2, and 15 units/μl CaMKII in buffer A were added. For phosphorylation by PKA, 10 units/μl PKA in buffer A was added. For phosphorylation by PKC, 2 mm CaCl2, 100 μg/ml phosphatidylserine, 20 μg/ml diacylglycerol, and 0.25 milliunits/ml PKC in buffer A were added. In all cases, the reaction was carried out by adding kinases and incubating for 1 h at 30 °C. The reaction was stopped by adding 5× SDS sample buffer, followed by boiling for 5 min. The samples were electrophoresed on 10% SDS-polyacrylamide gels, followed by staining with Coomassie Blue. Protein amounts were determined by densitometric analysis relative to GST and bovine AP180 standards (Molecular Dynamics Personal Densitometer SI). The radioactivity in the labeled proteins was determined using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).Figure 10There are three CKII phosphorylation sites in the middle 42-kDa domain of AP180. A, phosphorylation of GST, GST-AP180, GST-N33, GST-M42, GST-C58, or GST-16 in the presence (+) or absence (−) of CKII was carried out. A representative autoradiogram is displayed. B, a quantitative analysis of the phosphorylation of GST-AP180 (closed circle), GST-M42 (open circle), and GST-C58 (cross) as a function of CKII concentration is plotted.View Large Image Figure ViewerDownload (PPT)Construction and Expression of Deletion Mutants of AP180A series of progressive deletion constructs expressing the carboxyl-terminal portion of AP180 were made by polymerase chain reaction using one of the following sense primers, 5′-AGCTTCGGTCGACTCGCATGCTCAGGAAATGAC-3′ (C42), 5′-AGCTTCGGTCGACTCGCCCCTCCAGTTCCCGCA-3′ (C38), 5′-AGCTTCGGTCGACTCGCATCCACTGCCTCTCCT-3′ (C27), 5′-AGCTTCGGTCGACTCACGCCAGTGACTCCAG-3′ (C22), and a common antisense primer, 5′-GCTGAAATGCGGCCGCTCTTACAAGAAATCCTT-3′. To construct M36 of AP180, two primers, 5′-AGCTTCGGTCGACTCTCTCCAGCCACAACTGTT-3′ and 5′-GCTGAAATGCGGCCGCTCTGTAGAAGGGGCCAT-3′, were used. To construct M11 of AP180, two primers, 5′-AGCTTCGGTCGACTCGCATCCACTGCCTCTCCT-3′ and 5′-GCTGAAATGCGGCCGCTCTGCAGGACTGGGTGG-3′, were used. To construct M5 of AP180, two primers, 5′-AGCTTCGGTCGACTCGCATCCACTGCCTCTCCT-3′ and 5′-GCTGAAATGCGGCCGCTCTGTAGAAGGGGCCAT-3′, were used. A restriction site of SalI was designed in all sense primers, and aNotI site was designed in all the antisense primers. In all the polymerase chain reactions, plasmid pGEX3X-F1–20 (AS15−) was used as the template (39Morris S.A. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar). The PCR products were digested with SalI/NotI, and the digestion products were gel-purified, followed by subcloning into pGEX4T-1 expression vector at SalI/NotI sites. The constructs were first transformed into Escherichia coliJM109 and then introduced into E. coli BL21 for protein expression. The recombinant proteins were expressed and purified under the same conditions as described previously for GST-AP180 (39Morris S.A. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar), except that the optimal induction time for GST-C22 is 2 h.Characterization of the AP180 and AP-2/AP-1 InteractionAll of the procedures were performed at 4 °C or on ice unless otherwise indicated. Bovine AP-2 was dialyzed into buffer B (50 mmHEPES, pH 7.5, 150 mm NaCl, 1 mmphenylmethylsulfonyl fluoride) overnight and clarified by centrifugation at 400,000 × g for 10 min. 100 μl of glutathione-Sepharose beads coupled with a 10 μmconcentration of either GST-AP180 or other GST-AP180 deletion fragments was mixed with 0.63 μm (final concentration) bovine AP-2 in buffer C (0.05% Tween 20, 0.1% gelatin, and 2 mm EGTA in buffer B) in a final volume of 200 μl. The mixture was incubated for 1 h with gentle rocking before the beads were pelleted in a microcentrifuge by centrifugation at 1,300 × g for 5 min. The supernatant was removed, and the beads were washed three times with 500 μl of buffer C. Pelleted beads were resuspended in 100 μl of 2× SDS sample buffer, followed by boiling for 5 min. The samples were analyzed by SDS-PAGE, followed by Western blot analysis using a monoclonal antibody to the α subunit of AP-2 and detected by ECL (NEN Life Science Products). Data were quantitated using the Personal Densitometer SI (Molecular Dynamics) to determine the percentage of AP-2 bound. GST served as the negative control. The interaction between GST-AP180 and AP-1 was measured utilizing the same assay. The interactions between the GST-AP180 fusion proteins and the β2 subunit of AP-2 or the α-ear domains of AP-2 were measured utilizing the same assay, except that 20 μmfusion proteins were coupled to the beads, and the final concentration of β2 subunit or the α-ear domains in the reaction was 4 μm. The inhibition of the AP180/AP-2 interaction by M11 was measured similarly, but the indicated amounts of M11 were preincubated with bovine AP-2 for 30 min before the mixture was added to the fusion protein-coupled beads.Preparation of Phosphorylated and Dephosphorylated Fusion Proteins for either AP180-AP-2 Binding or Clathrin Assembly Assays2.4 μm protein, 160 μg/ml polylysine, 100 μm ATP, and 32 units/μl CKII were mixed in buffer A in a final volume of 300 μl, and the reaction was incubated for 80 min at 30 °C. For proteins used in the AP180-AP-2 binding assays, the phosphorylated fusion proteins were incubated with 50 μl of pelleted glutathione-Sepharose beads by rocking for 4 h at 4 °C, followed by three washes each of 500 μl of buffer B and used immediately. In each experiment, it was verified that the same amount of phosphorylated and unphosphorylated proteins had bound to the beads. For proteins used in the clathrin assembly assays, the phosphorylated fusion proteins were incubated with 200 μl of pelleted glutathione-Sepharose beads by rocking for 2 h at 4 °C, followed by three washes each of 500 μl of buffer D (50 mm Tris-HCl, 1 mm MgCl2, pH 8.0) and elution with 400 μl of buffer E (20 mm glutathione, 50 mm Tris-HCl, 1 mm MgCl2, pH 8.0). Half of the eluate (phosphorylated GST-AP180) was dialyzed into 10 mm Tris-HCl, pH 8.5. The other half of the eluate was dialyzed into 50 mm Tris-HCl, 1 mmMgCl2, pH 8.0, for 4 h and was added to insoluble calf intestinal alkaline phosphatase-agarose at a ratio of 0.35 μg of protein/unit of phosphatase. The dephosphorylation was carried out at room temperature by rocking for 1 h, and the reaction was stopped by gentle spinning to remove the phosphatase agarose. The dephosphorylated GST-AP180 was subsequently dialyzed into 10 mm Tris-HCl, pH 8.5, for use in a clathrin assembly assay.As a control, a parallel phosphorylation reaction with the addition of 4 μCi of [γ-32P]ATP was carried out under the same conditions in a volume of 50 μl to ensure that the stoichiometry of phosphorylation was similar to that described in Fig. 10. The mock phosphorylated control proteins were incubated under identical conditions but without the addition of CKII.Preparation of CytosolAll of the procedures were performed at 4 °C. Fresh bovine brain tissue was mixed at a weight ratio of 1:2 with buffer B and blended for 3 × 20 s, followed by centrifugation at 14,500 × g for 20 min. The supernatant was further clarified by centrifugation at 125,000 ×g for 1 h and concentrated 5 times using 50% saturated ammonium sulfate in 0.5 m Tris-HCl, pH 7.0. It was then suspended in buffer B and used at 7 mg/ml (final concentration) under the same conditions as used for pure AP-2 in the characterization of the AP180/AP-2 interaction.Partial Proteolysis of AP-2Partial digestion of AP-2 was carried out by mixing AP-2 and trypsin at a weight ratio of 1:500 in 10 mm Tris-HCl, pH 8.0, for 40 min at room temperature. The digestion was stopped by the addition of soybean trypsin inhibitor at a weight ratio of 5:1 relative to trypsin. 110 volume of 1m MES, pH 6.2 was added to the reaction mixture, and the mixture was incubated on ice for 2 h. Under these conditions the “trunk” of AP-2 aggregated, leaving the “ear” or appendage domains in a soluble form that was easily separated by centrifugation at 400,000 × g for 15 min at 4 °C (45Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4437-4441Abstract Full Text PDF PubMed Google Scholar).Co-immunoprecipitation of AP-2 and AP180 with AP180 Monoclonal AntibodyClathrin-coated vesicles (4–5 mg), prepared according to Nandi et al. (46Nandi P.K. Prasad K. Lippoldt R.E. Alfsen A. Edelhoch H. Biochemistry. 1982; 21: 6434-6440Crossref PubMed Scopus (16) Google Scholar) were stripped of clathrin by incubation in 10 mm Tris-HCl, pH 8.5, followed by sedimentation at 400,000 × g for 15 min at 4 °C. The partially decoated vesicles, which still contained APs were suspended in 5 ml of 4% Triton X-100 in 10 mm Tris-HCl, pH 8.5, and incubated on ice for 15 min. The suspension was centrifuged at 400,000 ×g at 4 °C for 15 min, and the supernatant was retained. The supernatant was applied to either an affinity column at room temperature made with affinity-purified monoclonal antibodies against AP180 (5 mg of mAb F1–20/ml of CNBr-activated Sepharose 4B) or applied to a control Sepharose 4B column. Unbound protein was washed from the columns with 10 column volumes of phosphate-buffered saline, and bound protein was eluted from the columns with 50 mm glycine, 150 mm NaCl, pH 2.3. The samples were electrophoresed on 4–20% SDS-polyacrylamide gels, followed by Western blot analysis with antibodies against AP180 and AP-2.Clathrin Assembly AssayFor assembly reactions carried out at pH 6.7, mock-phosphorylated GST-AP180, phosphorylated GST-AP180 (P-GST-AP180), M11, and clathrin were dialyzed overnight into 10 mm Tris-HCl, pH 8, 2 mm dithiothreitol at 4 °C, followed by clarification at 400,000 × g and 4 °C for 10 min. The indicated assembly protein was mixed with clathrin, and the assembly was initiated by adding 110 volume of 1 m MES-NaOH, pH 6.7. The final conditions in the reaction were 0.5 μm clathrin, 0.1 mMES-NaOH, 9 mm Tris-HCl, pH 6.7, and the indicated amounts of fusion proteins in a final volume of 200 μl. The mixture was incubated on ice for 45 min and then was centrifuged at 400,000 ×g and 4 °C for 6 min. The upper 80% of the supernatant was removed and analyzed by SDS-PAGE and Coomassie Blue staining. The percentage of clathrin assembly was determined by the relative depletion of clathrin from the solution before and after centrifugation and quantitated by densitometry of Coomassie Blue-stained gels (Molecular Dynamics Personal Densitometer SI). In the absence of GST-AP180, 3% of the clathrin was depleted by centrifugation.For assembly reactions carried out at pH 7.0, either GST-AP180, AP-2, or AP-2 combined with either GST-AP180, mock-phosphorylated GST-AP180, CKII-phosphorylated-GST-AP180, or dephosphorylated-phosphorylated GST-AP180 (alkaline phosphatase-treated CKII-phosphorylated GST-AP180) were mixed at a 1:1 molar ratio of AP180 to AP-2 for 10 min, and the assembly was initiated by adding clathrin and 110 volume of 1m MES-NaOH, pH 7.0. The final conditions in all the experiments were 0.2 μm clathrin, 0.1 mMES-NaOH, 9 mm Tris-HCl, pH 7.0, and the indicated amounts of assembly proteins in a final volume of 200 μl. For experiments involving the inhibition of clathrin assembly by M11, the indicated amounts of M11 were mixed with bovine AP-2, followed by the addition of GST-AP180. The final concentrations of both AP-2 and GST-AP180 were 0.3 μm.DISCUSSIONThe finding that AP180 and AP-2 co-localize to the same budding CCV (28Takei K. Mundigl O. Daniell L. De Camilli P. J. Cell Biol. 1996; 133: 1237-1250Crossref PubMed Scopus (328) Google Scholar) raises the question of why there needs to be two clathrin assembly proteins in the same CCV. It has long been argued that the cell uses APs to promote clathrin assembly to ensure uniformly sized cages and to ensure that the assembly reaction is efficient under physiological conditions (23Zaremba S. Keen J.H. J. Cell Biol. 1983; 97: 1339-1347Crossref PubMed Scopus (153) Google Scholar, 49Ye W. Lafer E.M. J. Neurosci. Res. 1995; 41: 15-26Crossref PubMed Scopus (70) Google Scholar). Clathrin, in the absence of an assembly protein, will form irregularly sized cages but only under conditions of low pH and high calcium (23Zaremba S. Keen J.H. J. Cell Biol. 1983; 97: 1339-1347Crossref PubMed S" @default.
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• W2146282045 title "AP180 and AP-2 Interact Directly in a Complex That Cooperatively Assembles Clathrin" @default.
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