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• W2029046578 abstract "The assembly of clathrin-coated buds on the Golgi requires the recruitment of the heterotetrameric AP-1 adaptor complex, which is dependent on both guanine nucleotides and the small GTP-binding protein ADP-ribosylation factor (ARF). Here, we have investigated the structural domains of the AP-1 complex necessary for ARF-mediated translocation of the adaptor complex onto Golgi membranes and the subsequent recruitment of clathrin onto the membrane. Controlled proteolysis of purified AP-1, derived from bovine adrenal coated vesicles, was used to generate AP-1 core fragments composed of the amino-terminal trunk regions of the β1 and γ subunits and associated μ1 and σ1 subunits, and lacking either the β1 subunit carboxyl-terminal appendage or both β1 and γ subunit appendages. On addition of these truncated fragments to AP-1-depleted adrenal cytosol, both types of core fragments were efficiently recruited onto Golgi membranes in the presence of GTPγS. Recruitment of both core fragments was inhibited by the fungal metabolite brefeldin A, indicative of an ARF-dependent process. Limited tryptic digestion of recruited, intact cytosolic AP-1 resulted in the quantitative release of the globular carboxyl-terminal appendage domains of the β1 and γ subunits. The adaptor core complex remained associated with the Golgi membranes. Recruitment of cytosolic clathrin onto the Golgi membranes was strictly dependent on the presence of intact AP-1. Tryptic removal of the β1 subunit appendage prevented subsequent clathrin recruitment. We conclude that the structural determinants required for the ARF-mediated binding of cytosolic AP-1 onto Golgi membranes are contained within the adaptor core, and that the carboxyl-terminal appendage domains of the β1 and γ subunits do not play any role in this process. Subsequent recruitment of cytosolic clathrin, however, requires an intact β1 subunit. The assembly of clathrin-coated buds on the Golgi requires the recruitment of the heterotetrameric AP-1 adaptor complex, which is dependent on both guanine nucleotides and the small GTP-binding protein ADP-ribosylation factor (ARF). Here, we have investigated the structural domains of the AP-1 complex necessary for ARF-mediated translocation of the adaptor complex onto Golgi membranes and the subsequent recruitment of clathrin onto the membrane. Controlled proteolysis of purified AP-1, derived from bovine adrenal coated vesicles, was used to generate AP-1 core fragments composed of the amino-terminal trunk regions of the β1 and γ subunits and associated μ1 and σ1 subunits, and lacking either the β1 subunit carboxyl-terminal appendage or both β1 and γ subunit appendages. On addition of these truncated fragments to AP-1-depleted adrenal cytosol, both types of core fragments were efficiently recruited onto Golgi membranes in the presence of GTPγS. Recruitment of both core fragments was inhibited by the fungal metabolite brefeldin A, indicative of an ARF-dependent process. Limited tryptic digestion of recruited, intact cytosolic AP-1 resulted in the quantitative release of the globular carboxyl-terminal appendage domains of the β1 and γ subunits. The adaptor core complex remained associated with the Golgi membranes. Recruitment of cytosolic clathrin onto the Golgi membranes was strictly dependent on the presence of intact AP-1. Tryptic removal of the β1 subunit appendage prevented subsequent clathrin recruitment. We conclude that the structural determinants required for the ARF-mediated binding of cytosolic AP-1 onto Golgi membranes are contained within the adaptor core, and that the carboxyl-terminal appendage domains of the β1 and γ subunits do not play any role in this process. Subsequent recruitment of cytosolic clathrin, however, requires an intact β1 subunit. The clathrin-coated vesicle exhibits a well defined structural organization, and the major protein components have been purified and characterized(1Brodsky F.M. Science. 1988; 242: 1396-1402Crossref PubMed Scopus (205) Google Scholar, 2Morris S.A. Ahle S. Ungewickell E. Curr. Opin. Cell Biol. 1989; 1: 684-690Crossref PubMed Scopus (32) Google Scholar, 3Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-442Crossref PubMed Scopus (170) Google Scholar, 4Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (536) Google Scholar). In addition to the principal coat protein clathrin, two major adaptor complexes have been identified. One type is restricted to the trans-Golgi network (TGN) 1The abbreviations used are: TGNtrans-Golgi networkAP-1Golgi-specific heterotetrameric adaptor complexAP-2plasma membrane-specific heterotetrameric adaptor complexARFADP-ribosylation factorBFAbrefeldin AGTPγSguanosine 5′-O-(thiotriphosphate). and Golgi-derived coated vesicles and termed AP-1. The second, localized to the plasma membrane and endocytic clathrin-coated vesicles, is termed AP-2. Adaptors are heterotetrameric complexes, composed of one related and one unique ∼100-kDa subunit, and two additional components, the ∼50-kDa μ and ∼20-kDa σ subunits. Thus, the Golgi AP-1 adaptor complex is composed of γ, β1, μ1, and σ1 subunits, and the related AP-2 is composed of α, β2, μ2, and σ2 subunits. The β1 and β2 subunits are very similar in structure(5Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 919-929Crossref PubMed Scopus (252) Google Scholar, 6Kirchhausen T. Nathanson K.L. Matsui W. Vaisberg A. Chow E.P. Burne C. Keen J.H. Davis A.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2612-2616Crossref PubMed Scopus (89) Google Scholar, 7Ponnambalam S. Robinson M.S. Jackson A.P. Peiperl L. Parham P. J. Biol. Chem. 1990; 265: 4814-4820Abstract Full Text PDF PubMed Google Scholar, 8Ungewickell E. Plessmann E. Weber K. Eur. J. Biochem. 1994; 222: 33-40Crossref PubMed Scopus (9) Google Scholar), while the α and γ subunits are the most distantly related adaptor subunits(4Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (536) Google Scholar, 9Robinson M.S. J. Cell Biol. 1990; 111: 2319-2326Crossref PubMed Scopus (102) Google Scholar). Electron microscopy has revealed that the AP-2 adaptor has a large globular core domain and two exposed globular head or appendage domains 2The nomenclature that we have used to distinguish between the different domains and subunits of the AP-1 adaptor is as follows. The appendage refers to the globular carboxyl-terminal head domain and the hinge region of either the β1 or γ subunit. The hinge connects the appendage to the adaptor core, consisting of the 60-70-kDa amino-terminal trunks of both the β1 and γ subunits and the intact μ1 and σ1 subunits. (10Heuser J.E. Keen J. J. Cell Biol. 1988; 107: 877-886Crossref PubMed Scopus (114) Google Scholar). Controlled proteolysis of both AP-1 and AP-2 (11Zaremba S. Keen J.H. J. Cell. Biochem. 1985; 28: 47-58Crossref PubMed Scopus (30) Google Scholar, 12Keen J.H. Beck K.A. Biochem. Biophys. Res. Commun. 1989; 158: 17-23Crossref PubMed Scopus (19) Google Scholar, 13Matsui W. Kirchhausen T. Biochemistry. 1990; 29: 10791-10798Crossref PubMed Scopus (80) Google Scholar, 14Schröder S. Ungewickell E. J. Biol. Chem. 1991; 266: 7910-7918Abstract Full Text PDF PubMed Google Scholar) has shown that the core is composed of the amino-terminal trunk regions of either the α and β2 or the β1 and γ subunits as well as the intact μ and σ subunits, while the appendages represent the 30-40-kDa carboxyl-terminal regions of the large subunits. trans-Golgi network Golgi-specific heterotetrameric adaptor complex plasma membrane-specific heterotetrameric adaptor complex ADP-ribosylation factor brefeldin A guanosine 5′-O-(thiotriphosphate). Adaptors are believed to facilitate clathrin-coated vesicle formation by combining a clathrin binding domain and a membrane association domain within an oligomeric protein complex. When it became apparent that the β1 and β2 subunits were highly related, it was suggested that these subunits might contain a common domain capable of binding to clathrin(5Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 919-929Crossref PubMed Scopus (252) Google Scholar). Indeed, the purified β2 subunit interacts directly with preformed clathrin cages(15Ahle S. Ungewickell E. J. Biol. Chem. 1989; 264: 20089-20093Abstract Full Text PDF PubMed Google Scholar), and, more recently, it has been shown that recombinant β-type subunits alone can induce clathrin polymerization(16Gallusser A. Kirchhausen T. EMBO (Eur. Mol. Biol. Organ.) J. 1993; 12: 5237-5244Crossref PubMed Scopus (124) Google Scholar). Further attempts to define the clathrin binding site more precisely indicated that proteolytically generated cores can bind to preformed clathrin cages(12Keen J.H. Beck K.A. Biochem. Biophys. Res. Commun. 1989; 158: 17-23Crossref PubMed Scopus (19) Google Scholar), suggesting that the clathrin binding site is located in the amino-terminal trunk domain. However, in buffer conditions that prevent adaptor self-association, tryptic removal of the β subunit appendage results in the release of both appendage and core fragments from clathrin-coated vesicles(14Schröder S. Ungewickell E. J. Biol. Chem. 1991; 266: 7910-7918Abstract Full Text PDF PubMed Google Scholar). This suggests that both the trunk and appendage domains of the β-type subunits are required for high affinity clathrin binding(14Schröder S. Ungewickell E. J. Biol. Chem. 1991; 266: 7910-7918Abstract Full Text PDF PubMed Google Scholar, 16Gallusser A. Kirchhausen T. EMBO (Eur. Mol. Biol. Organ.) J. 1993; 12: 5237-5244Crossref PubMed Scopus (124) Google Scholar). Before clathrin binding and polymerization can occur, however, the adaptors must associate stably with the appropriate intracellular membrane compartment. According to the simplest model for the interaction of adaptors with membranes(1Brodsky F.M. Science. 1988; 242: 1396-1402Crossref PubMed Scopus (205) Google Scholar, 3Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-442Crossref PubMed Scopus (170) Google Scholar, 4Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (536) Google Scholar, 17Pearse B.M.F. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 3331-3336Crossref PubMed Scopus (233) Google Scholar, 18Glickman J.N. Conibear E. Pearse B.M.F. EMBO (Eur. Mol. Biol. Organ.) J. 1989; 8: 1041-1047Crossref PubMed Scopus (207) Google Scholar), it was assumed that they attach directly to the cytoplasmic portions of selected receptors. Indeed, there is evidence that adaptors can interact directly, albeit weakly, with the cytoplasmic regions of certain proteins sorted into clathrin-coated vesicles(17Pearse B.M.F. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 3331-3336Crossref PubMed Scopus (233) Google Scholar, 18Glickman J.N. Conibear E. Pearse B.M.F. EMBO (Eur. Mol. Biol. Organ.) J. 1989; 8: 1041-1047Crossref PubMed Scopus (207) Google Scholar, 19Beltzer J.P. Spiess M. EMBO (Eur. Mol. Biol. Organ.) J. 1991; 10: 3739-3742Google Scholar, 20Chang M.P. Mallet W.G. Mostov K.E. Brodsky F.M. EMBO (Eur. Mol. Biol. Organ.) J. 1993; 12: 2169-2180Crossref PubMed Scopus (81) Google Scholar, 21LeBorgne R. Schmidt A. Mauxion F. Griffiths G. Hoflack B. J. Biol. Chem. 1993; 268: 22552-22556Abstract Full Text PDF PubMed Google Scholar, 22Sosa M.A. Schmidt B. von Figura K. Hille-Rehfeld A. J. Biol. Chem. 1993; 268: 12537-12543Abstract Full Text PDF PubMed Google Scholar). This simple model, while attractive, failed to explain the restricted localization of AP-1 within the cell and why AP-1 does not bind to cytoplasmically oriented trafficking motifs in receptors transiting through other intracellular compartments(2Morris S.A. Ahle S. Ungewickell E. Curr. Opin. Cell Biol. 1989; 1: 684-690Crossref PubMed Scopus (32) Google Scholar, 3Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-442Crossref PubMed Scopus (170) Google Scholar, 4Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (536) Google Scholar). Furthermore, the dramatic effect of brefeldin A (BFA) on the intracellular localization of AP-1 also suggested that clathrin coat formation is highly regulated (23Robinson M.S. Kreis T.E. Cell. 1992; 69: 129-142Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 24Wong D.H. Brodsky F.M. J. Cell Biol. 1992; 117: 1171-1179Crossref PubMed Scopus (84) Google Scholar). Recently, the recruitment of cytosolic AP-1 onto purified Golgi membranes was reconstituted in vitro(25Stamnes M.A. Rothman J.E. Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). Adaptor binding was found to be dependent on GTP and antagonized by BFA. ARF, a small GTP-binding protein, was identified as the GTP-requiring component(25Stamnes M.A. Rothman J.E. Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). Coatomer-coated vesicle formation also proceeds by the initial recruitment of ARF(27Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 28Palmer D.J. Helms J.B. Beckers C.J.M. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar), explaining the sensitivity of both clathrin- and coatomer-coated vesicle formation to BFA. However, since no clear ARF specificity has yet been discerned(25Stamnes M.A. Rothman J.E. Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar), we proposed that ARF may be required to ensure the selective interaction of AP-1 with a specific docking protein in the context of the TGN membrane. While our model overcomes the limitations of the direct association model, the structural determinants on the AP-1 complex that are recognized by the putative docking protein remain to be identified. The role of the α and γ subunit appendages has been probed using a chimera-based approach(29Robinson M.S. J. Cell Biol. 1993; 123: 67-77Crossref PubMed Scopus (59) Google Scholar). Swapping the α and γ subunit head and/or hinge regions did not appear to affect either the assembly or targeting of the AP-1 or AP-2 chimeric complexes. Here, we have taken a different approach to directly assess the structural features of the AP-1 heterotetramer required for Golgi membrane association and subsequent clathrin recruitment. Defined tryptic fragments were produced by controlled proteolysis(8Ungewickell E. Plessmann E. Weber K. Eur. J. Biochem. 1994; 222: 33-40Crossref PubMed Scopus (9) Google Scholar, 14Schröder S. Ungewickell E. J. Biol. Chem. 1991; 266: 7910-7918Abstract Full Text PDF PubMed Google Scholar), and this has enabled us to follow the ARF-dependent recruitment of the different fragments onto Golgi membranes. We have found the translocation of the AP-1 core complex to be indistinguishable from intact AP-1. This precludes the globular carboxyl terminus and hinge regions from controlling this process. In contrast, clathrin binding to Golgi membranes was found to be strictly dependent on an intact β1 subunit. [α-32P]GTP (>3,000 Ci/mmol) was obtained from ICN. GTPγS was purchased from Boehringer Mannheim. Aprotinin, ATP, benzamidine, BFA, dithiothreitol, leupeptin, soybean trypsin inhibitor, Triton X-100, and Tween 20 were from Sigma. BFA was stored as a 5 mg/ml stock solution in ethanol at −20°C. Sprague-Dawley rats were purchased from SASCO, and the frozen bovine adrenal glands were obtained from PelFreez. BA 83 nitrocellulose membranes were obtained from Schleicher and Schuell, and Sepharose 4B, CNBr-activated Sepharose 4B, and the Mr markers for electrophoresis were obtained from Pharmacia Biotech Inc. Tosylphenylalanyl chloromethyl ketone-treated trypsin was purchased either from Cooper Biomedical or Worthington. The ECL reagents for chemiluminescent detection were obtained from Amersham. All other reagents were the highest grade available. The anti-β subunit antibody mAb 100/1(5Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 919-929Crossref PubMed Scopus (252) Google Scholar), the anti-α subunit antibodies mAb 100/2 (5Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 919-929Crossref PubMed Scopus (252) Google Scholar) and mAb AP.6(30Chin D.J. Straubinger R.M. Acton S. Nathke I. Brodsky F.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9289-9293Crossref PubMed Scopus (80) Google Scholar), the anti-γ subunit antibody mAb 100/3(5Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 919-929Crossref PubMed Scopus (252) Google Scholar), the polyclonal anti-clathrin light chain antibody R461(5Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 919-929Crossref PubMed Scopus (252) Google Scholar), the anti-clathrin heavy chain antibody mAb TD.1(31Nathke I.S. Heuser J. Lupas A. Stock J. Turck C.W. Brodsky F.M. Cell. 1992; 68: 899-910Abstract Full Text PDF PubMed Scopus (133) Google Scholar), and the polyclonal anti-α-mannosidase II serum (32Moremen K.W. Touster O. J. Biol. Chem. 1986; 261: 10945-10951Abstract Full Text PDF PubMed Google Scholar) were prepared and used as described previously(26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). Antiserum AE/1 was raised against a synthetic peptide corresponding to the carboxyl-terminal dodecapeptide of the murine γ subunit of AP-1(9Robinson M.S. J. Cell Biol. 1990; 111: 2319-2326Crossref PubMed Scopus (102) Google Scholar). Peptide conjugation, immunization, and screening was as detailed previously(33Traub L.M. Sagi-Eisenberg R. J. Biol. Chem. 1991; 266: 24642-24649Abstract Full Text PDF PubMed Google Scholar). AE/1 was further affinity-purified from immune serum on a column of the peptide (AEVNNFPPQSWQ) coupled to CNBr-activated Sepharose 4B. Peptide-specific antibodies were eluted with 100 mM glycine HCl, pH 2.5, followed by 100 mM triethylamine, pH 11.5(34Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Laboratory Harbor Press, Cold Spring Harbor, NY1988Google Scholar). Antiserum GD/1 was similarly raised against a dodecapeptide (GDLLNLDLGPPV), a conserved sequence in the hinge region of all known mammalian β-type subunits. Affinity-purified GD/1 was prepared similarly on a peptide-Sepharose 4B column. The rhodamine-conjugated anti-rabbit IgG and fluorescein-conjugated anti-mouse IgG secondary antibodies were purchased from Dakopatts. Rat liver Golgi membranes and cytosol were prepared as described previously(26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). For the preparation of bovine adrenal cytosol, frozen adrenal glands were rapidly thawed at 37°C in a homogenization buffer of 25 mM Hepes-KOH, pH 7.4, 250 mM sucrose, 1 mM EDTA supplemented with 5 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. Once thawed, the tissue was immediately transferred to homogenization buffer on ice, finely minced, and homogenized in a Potter-Elvehjem homogenizer containing approximately 2 volumes of homogenization buffer with 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.1 trypsin inhibitory unit/ml aprotinin, and 5 μg/ml leupeptin. This and all subsequent procedures were performed at 0-4°C. The crude homogenate was centrifuged sequentially at 3,000 × g for 10 min, 10,000 × g for 20 min, and 100,000 × g for 60 min to yield a high speed cytosolic supernatant fraction. The cytosol was stored in aliquots at −80°C after rapid freezing in dry ice. Adrenal or rat liver cytosol was depleted of clathrin by gel filtration on Sepharose 4B. 50-60 ml of cytosol was rapidly thawed, centrifuged at 150,000 × g for 60 min to remove aggregated material, and loaded at 1 ml/min onto a Sepharose 4B column (5.0 × 70 cm) equilibrated in 25 mM Hepes-KOH, pH 7.3, 125 mM potassium acetate, 5 mM magnesium acetate, and 0.05% sodium azide. Fractions of 20 ml were collected, and the elution positions of clathrin and AP-1 and AP-2 adaptors was determined on immunoblots using mAb TD.1 and mAb 100/1. The clathrin triskelia eluted well ahead of the adaptors, and fractions after the clathrin peak were pooled, concentrated by precipitation with 60% ammonium sulfate, and dialyzed against 25 mM Hepes-KOH, pH 7.0, 125 mM potassium acetate, 5 mM magnesium acetate, and 1 mM dithiothreitol (Buffer C). After centrifugation at 12,000 × g for 15 min, the clathrin-depleted cytosol was aliquotted, quick frozen in dry ice, and stored at −80°C. The clathrin-containing fractions were pooled separately and concentrated approximately 10-fold by ultrafiltration using an Amicon YM 30 filter. After centrifugation at 12,000 × g for 15 min, the clathrin-enriched fraction was stored at 4°C. Endogenous AP-1 was removed from the clathrin-depleted adrenal cytosol by immunodepletion(26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). Briefly, cytosol was passed over an immobilized mAb 100/3 column equilibrated in buffer C on ice, and the loading effluent was reapplied to the column several times, before similar passages over a second mAb 100/3 column equilibrated in buffer C on ice. The resulting AP-1-depleted cytosol was analyzed on immunoblots using affinity-purified AE/1, and the extent of depletion was more than 95%. Clathrin-coated vesicles and coated vesicle-derived AP-1 were purified from frozen bovine adrenal glands as described in detail previously(8Ungewickell E. Plessmann E. Weber K. Eur. J. Biochem. 1994; 222: 33-40Crossref PubMed Scopus (9) Google Scholar). Protein concentrations were estimated using either the Coomassie Blue method (35Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar) with bovine serum albumin as a standard or, for the purified AP-1 preparations, by A280, using an E1cm1% value of 6.0(14Schröder S. Ungewickell E. J. Biol. Chem. 1991; 266: 7910-7918Abstract Full Text PDF PubMed Google Scholar). The assay was a modification of our binding assay utilizing rat liver cytosol(26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). For most assays, bovine adrenal cytosol replaced the rat cytosol, and Golgi membranes and GTPγS were added to final concentrations of 50 μg/ml and 100 μM, respectively. Reactions were terminated by chilling on ice followed by centrifugation, and the Golgi membrane-containing pellets were prepared for immunoblotting as described(26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). Routinely, pellets were resuspended in 20 μl of 1 × SDS sample buffer, and 10 μl was loaded per lane. When aliquots of the assay supernatant were also examined, an aliquot corresponding to 1/80 of each supernatant was loaded. For the two-stage clathrin binding assay, the first stage incubation contained clathrin-depleted rat liver cytosol, Golgi membranes, and GTPγS. Membranes were recovered by centrifugation and digested with trypsin as described below. The trypsinized membranes were then added to a second stage incubation containing 0.1 mg/ml clathrin-enriched Sepharose 4B pool and 20 μg/ml BFA in buffer C. After incubation at 37°C for 15 min, the tubes were returned to ice, and the pellets were prepared for analysis as outlined above. NIH 3T3 fibroblasts were grown at 37°C on round polylysine-coated 1.2-cm glass coverslips in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For permeabilization, the cells were washed first in phosphate-buffered saline, then twice in 25 mM Hepes-KOH, pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mg/ml glucose (buffer E) and then frozen on dry ice. The cells were thawed, and the endogenous cytosol was removed by dipping the coverslip into a beaker of buffer E 10 times at room temperature. Gel-filtered whole rat liver cytosol, at a concentration of ∼5 mg/ml in buffer D, was supplemented with 100 μM GTPγS and coated vesicle-derived adrenal AP-1 or proteolytic fragments to give a final adaptor concentration of 50-70 μg/ml. After addition of the cytosol, the cells were warmed to 37°C for 15 min and then washed by dipping the coverslip into a beaker of buffer E 10 times. The cells were then fixed for 5 min at room temperature with 3.7% formaldehyde in PBS and double-labeled with R461 and mAb 100/3 as described in detail previously(5Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO (Eur. Mol. Biol. Organ.) J. 1988; 7: 919-929Crossref PubMed Scopus (252) Google Scholar). Tryptic digestion of Golgi membranes was carried out in buffer C at a membrane concentration of 1 mg/ml. Triton X-100 and trypsin were added on ice as indicated in the figures, and digestion was performed at 37°C for 10 min, followed by chilling and addition of excess soybean trypsin inhibitor. For some experiments, the membranes were then collected by centrifugation at 15,000 × g for 10 min at 4°C, the supernatant fractions were concentrated by precipitation with methanol/chloroform(36Wessel D. Flugge U.I. Anal. Biochem. 1984; 142: 141-143Crossref Scopus (3191) Google Scholar), and both supernatant and pellet fractions were analyzed by immunoblotting. Proteolysis of the purified adrenal AP-1 was performed in 25 mM Hepes-KOH, pH 7.0, 125 mM potassium acetate for 1 h at 30°C using a protein/trypsin ratio of 1:30 for the quantitative removal of the β1 subunit appendage. The reaction was stopped by adding excess soybean trypsin inhibitor, and the sample was used for the binding studies without further purification. For the preparation of appendageless AP-1 cores, the digestion was carried out at 30°C using a trypsin to substrate ratio of 1:20. Residual AP-1 containing an intact γ subunit, which we have found difficult to cleave quantitatively with trypsin(14Schröder S. Ungewickell E. J. Biol. Chem. 1991; 266: 7910-7918Abstract Full Text PDF PubMed Google Scholar), was removed by immunoadsorption on a mAb 100/3 column. The unbound material was concentrated to approximately 0.5-1.0 mg/ml in a Centricon 30 before use in the binding assays. The polypeptide composition of all the fragments used in this study was verified on Coomassie Blue-stained gels. Discontinuous SDS-polyacrylamide gel electrophoresis was carried out as described previously(26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). Proteins were transferred onto nitrocellulose membranes at 110 V for 70 min in a buffer of 15.6 mM Tris, 120 mM glycine, pH ∼8.3, equilibrated to 4°C. Following transfer, the membranes were blocked and probed with antibodies as described previously(26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). GTP-binding proteins immobilized on nitrocellulose were preincubated for 60 min at room temperature in 50 mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol, and 0.3% Tween 20 (buffer D), supplemented with 200 μM ATP and 2 μCi/ml [α-32P]GTP, and then incubated for an additional 60 min, followed by four washes in buffer D. The GTP-binding proteins were visualized by autoradiography at −80°C using an intensifying screen. The goal of this study was to identify the structural regions of the heterotetrameric AP-1 adaptor complex which are involved in the ARF-dependent recruitment onto Golgi membranes and the subsequent recruitment of cytosolic clathrin. To distinguish between the endogenous rat liver Golgi-associated AP-1 and exogenously added cytosolic AP-1, using a species-specific mAb directed against the γ subunit, we had to modify our existing AP-1 binding assay by substituting bovine adrenal gland cytosol for rat liver cytosol. To limit our analysis to the initial events in clathrin-coated vesicle assembly, clathrin-depleted adrenal cytosol (26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar) has been used for several of the experiments described below. Recruitment of AP-1 onto Golgi membranes is preceded by the recruitment of ARF(25Stamnes M.A. Rothman J.E. Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar). While only very low levels of ARF are found on purified Golgi membranes (37Serafini T. Orci L. Amherdt M. Brunner M. Kahn R.A. Rothman J.E. Cell. 1991; 67: 239-253Abstract Full Text PDF PubMed Scopus (452) Google Scholar, 38Taylor T.C. Kanstein M. Weidman P. Melançon P. Mol. Biol. Cell. 1994; 5: 237-252Crossref PubMed Scopus (44) Google Scholar) (Fig. 1, lane a), the level increased markedly upon incubation with clathrin-depleted adrenal cytosol in the presence of GTPγS (lane c). Several other uncharacterized low molecular weight GTP-binding proteins were also recruited onto the Golgi membrane. The accumulation of ARF was accompanied by recruitment of cytosolic AP-1 onto the Golgi membrane fraction. When recruitment was followed after pretreatment with BFA, decreased levels of both ARF and AP-1 were observed in the membranes, and the extent of recruitment varied according to the time of addition of BFA (Fig. 1, lanes d-g). These results are analogous to those obtained using rat liver cytosol (26Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993;" @default.
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