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BAP, a Mammalian BiP-associated Protein, Is a Nucleotide Exchange Factor That Regulates the ATPase Activity of BiP

2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês

10.1074/jbc.m208377200

1083-351X

Kyung Tae Chung, Ying Shen, Linda M. Hendershot,

Toxin Mechanisms and Immunotoxins

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an ∼54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER. We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an ∼54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER. The Hsp70 family of molecular chaperones are highly homologous and consist of two distinct domains: a highly conserved N-terminal ATPase domain and a less conserved C-terminal polypeptide-binding domain (1Boorstein W.R. Ziegelhoffer T. Craig E.A. J. Mol. Evol. 1994; 38: 1-17Google Scholar). The chaperone activity of Hsp70 proteins is controlled by the ATPase domain that undergoes a reaction cycle comprised of ATP binding, hydrolysis, and nucleotide exchange, which is regulated by co-chaperones and co-factors. In bacteria, DnaJ accelerates ATP hydrolysis, whereas GrpE promotes nucleotide exchange of ADP to ATP (2Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Google Scholar, 3Szabo A. Langer T. Schroder H. Flanagan J. Bukau B. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10345-10349Google Scholar). The mammalian cytosolic Hsc70 is similarly regulated by Hsp40, a homologue of DnaJ (4Minami Y. Hohfeld J. Ohtsuka K. Hartl F.U. J. Biol. Chem. 1996; 271: 19617-19624Google Scholar), and a number of both positive and negative regulators of nucleotide exchange have been identified. BAG-1 can stimulate the ATPase activity of Hsc70, presumably by facilitating nucleotide exchange, although the precise function of BAG-1 is still somewhat controversial (5Hohfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Google Scholar, 6Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Google Scholar, 7Gassler C.S. Wiederkehr T. Brehmer D. Bukau B. Mayer M.P. J. Biol. Chem. 2001; 276: 32538-32544Google Scholar). A negative regulator of the ATPase activity of Hsc70, HspBP1, has been identified (8Raynes D.A. Guerriero Jr., V. J. Biol. Chem. 1998; 273: 32883-32888Google Scholar). Additional Hsc70-interacting proteins such as Hip (9Hohfeld J. Minami Y. Hartl F.U. Cell. 1995; 83: 589-598Google Scholar), Hop (10Chen S. Smith D.F. J. Biol. Chem. 1998; 273: 35194-35200Google Scholar,11Johnson B.D. Schumacher R.J. Ross E.D. Toft D.O. J. Biol. Chem. 1998; 273: 3679-3686Google Scholar), and CHIP (12Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Google Scholar) have been identified that further contribute to the regulation of the Hsc70 ATPase cycle and, as such, serve to control the chaperone function of Hsc70. BiP (also known as GRP78) is a mammalian endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; EST, expressed sequence tag; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; Endo H, endoglycosidase H; DSP, dithiobis[succinimidyl propionate] 1The abbreviations used are: ER, endoplasmic reticulum; EST, expressed sequence tag; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; Endo H, endoglycosidase H; DSP, dithiobis[succinimidyl propionate] homologue of the Hsp70 family. The ER is the site of synthesis, folding, and assembly of secretory pathway proteins that include resident proteins of the endocytic and exocytic organelles, as well as surface and secreted proteins. ER molecular chaperones and folding enzymes associate with the newly synthesized proteins to prevent their aggregation and help them fold and assemble correctly. Through a process called ER quality control, proteins that do not mature properly are retained in the ER and eventually targeted for ER-associated degradation through the action of the chaperones (13Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Google Scholar, 14Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Google Scholar). BiP was first identified bound to nonsecreted free heavy chains in pre-B lymphomas (15Haas I.G. Wabl M. Nature. 1983; 306: 387-389Google Scholar) and was consequently shown to interact with a number of other secretory pathway proteins to prevent their premature transport from the ER and to promote their proper folding and assembly (16Gething M.-J. Sambrook J. Nature. 1992; 355: 33-45Google Scholar). As such, BiP was the first ER chaperone and component of the ER quality control apparatus to be identified. In addition, BiP plays an essential role in maintaining the permeability barrier of the ER translocon during early stages of protein translocation (17Hamman B.D. Hendershot L.M. Johnson A.E. Cell. 1998; 92: 747-758Google Scholar), targeting misfolded proteins for proteasomal degradation (18Brodsky J.L. Werner E.D. Dubas M.E. Goeckeler J.L. Kruse K.B. McCracken A.A. J. Biol. Chem. 1999; 274: 3453-3460Google Scholar), serving as a sensor for ER stress (19Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Nat. Cell Biol. 2000; 2: 326-332Google Scholar, 20Shen J. Chen X. Hendershot L. Prywes R. Dev. Cell. 2002; 3: 99-111Google Scholar), and contributing to ER calcium stores. The ATPase activity of BiP is required for at least some of these roles; thus, it is reasonable to speculate that the ATPase activity of BiP should be regulated in a way similar to that of other members of the Hsp70 family. A total of four mammalian ER DnaJ homologues have been identified (21Brightman S.E. Blatch G.L. Zetter B.R. Gene (Amst.). 1995; 153: 249-254Google Scholar, 22Skowronek M.H. Rotter M. Haas I.G. Biol. Chem. 1999; 380: 1133-1138Google Scholar, 23Bies C. Guth S. Janoschek K. Nastainczyk W. Volkmer J. Zimmermann R. Biol. Chem. 1999; 380: 1175-1182Google Scholar, 24Yu M. Haslam R.H. Haslam D.B. J. Biol. Chem. 2000; 275: 24984-24992Google Scholar, 25Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Google Scholar), and it has been proposed that they be referred to as ERdj1–4. In vitro biochemical studies show that the J domains of ERdj1, ERdj3, and ERdj4 can stimulate the ATPase activity of BiP (24Yu M. Haslam R.H. Haslam D.B. J. Biol. Chem. 2000; 275: 24984-24992Google Scholar, 25Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Google Scholar, 26Chevalier M. Rhee H. Elguindi E.C. Blond S.Y. J. Biol. Chem. 2000; 275: 19620-19627Google Scholar), and both ERdj3 and 4 can bind to BiP in vivo(25Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Google Scholar). 2K. T. Chung, Y. Shen, and L. M. Hendershot, unpublished data.2K. T. Chung, Y. Shen, and L. M. Hendershot, unpublished data. Recently, a yeast ER protein (Sls1p/Sil1) was isolated from two different genera that interacts with the ATPase domain of Kar2p, the yeast homologue of BiP (27Kabani M. Beckerich J.M. Gaillardin C. Mol. Cell. Biol. 2000; 20: 6923-6934Google Scholar, 28Tyson J.R. Stirling C.J. EMBO J. 2000; 19: 6440-6452Google Scholar). Defects in the SLS1 gene are not lethal but affect protein translocation into the ER, especially associated withLHS1 mutations. Sls1p is proposed to be a GrpE-like protein based on its preference for the ADP-bound conformation of Kar2p and its ability to enhance the ATPase activity of Kar2p in the presence of the J domain of Sec63p, a yeast ER transmembrane DnaJ homologue. However, nucleotide exchange activity for Sls1p has not been directly demonstrated. The existence of potential mammalian and invertebrate homologues of Sls1p was reported (28Tyson J.R. Stirling C.J. EMBO J. 2000; 19: 6440-6452Google Scholar), but no data are available on their activity. We attempted to identify potential mammalian regulators of the ATPase activity of BiP using the ATPase domain of a BiP mutant as the bait protein in a yeast two-hybrid screen. A BiP-interacting protein was identified by screening a human liver cDNA library. We designated the protein, which bears low sequence homology with both yeast ER Sls1p, a positive regulator of the ATPase activity of Kar2p, and mammalian cytosolic HspBP1, a negative regulator of the ATPase activity of Hsc70, as BAP for BiP-associatedprotein. In this study, we demonstrated that BAP is a resident ER glycoprotein that interacts with the ATPase domain of BiP and that BAP binding is affected by nucleotide-induced conformational changes in the ATPase domain of BiP. In vitro assays demonstrated that BAP functioned as a nucleotide exchanger for BiP and consequently enhanced the positive effect of ERdj4 on the ATPase activity of BiP. Thus, BAP represents the first mammalian ER nucleotide exchange factor for BiP and reveals a conservation in the regulatory machinery for the ATPase activity of the Hsp70 chaperones found in other eukaryotic cellular compartments and in bacteria. The procedure for yeast two-hybrid screening was performed as published (29Bai C. Elledge S.J. Methods Enzymol. 1996; 273: 331-347Google Scholar). DNA encoding the ATPase domain of the hamster BiPT229G mutant without the ER targeting signal sequence (30Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Google Scholar, 31Wei J. Gaut J.R. Hendershot L.M. J. Biol. Chem. 1995; 270: 26677-26682Google Scholar) was cloned into theNdeI and BamHI sites of the pAS1 vector. The resulting construct, pAS1(T229G) was transformed into the yeast HF7c strain. Trp+ transformants were isolated and subsequently transformed with a human liver cDNA library that contained ∼3.5 × 106 independent clones in theLEU2 marker plasmid pACT2 (Clontech Laboratory Inc., Palo Alto, CA). Transformants were plated on SD-Trp/-Leu/-His medium containing 20 mm 3-aminotriazole, and colonies that grew on this medium were assayed for β-galactosidase activity. Target plasmids from the positive colonies were isolated, sequenced, and used to search for homologous proteins in the data base. Four independent clones of varying lengths of a single cDNA were identified. Interaction between the target DNA product and the ATPase domain of BiP was confirmed by back-transforming yeast with target and bait DNAs. We obtained an EST clone (identification number 5102998) from Incyte Genomics Systems Inc. (St. Louis, MO) encoding the complete cDNA of the positive target gene. The sequence of the inserted cDNA of the EST was verified by DNA sequencing. The BAP-HA tagged construct was produced by inserting full-length BAP without a stop codon into theEcoRI and XhoI sites of the 3HA DSL vector (a kind gift from Dr. Daesik Lim of St. Jude Children's Research Hospital). The resulting construct, pDSL(BAP-HA), contained the HA tag at its C terminus. Full-length BAP with a stop codon was inserted into the EcoRI and XhoI sites of pCDNA3.0 to produce pCDNA(BAP) for expression in mammalian cells. The sequence encoding BAP without the ER targeting signal was cloned into the SalI and HindIII sites of pQE10 and expressed in the M15 Escherichia coli strain (Qiagen). The recombinant His6-BAP was induced with 0.1 mmisopropyl-β-d-thiogalactopyranoside for 18 h at 17 °C. The cells were sonicated in lysing buffer (50 mmNa2HPO4, 500 mm NaCl, 10 mm imidazole, 1% Triton X-100, and 10% glycerol). His6-BAP was purified from Ni2+-nitrilotriacetic acid-agarose column using a stepwise gradient of 20–300 mm imidazole after extensively washing the column with lysing buffer that did not include 1% Triton X-100. The 100–300 mm eluent fractions were pooled and desalted by passing over a PD-10 column (Amersham Biosciences) that had been equilibrated with PBS containing 10% glycerol. The isolated His6-BAP was fairly pure as judged by Coomassie staining of SDS-PAGE gels and was used for in vitro assays and to produce a polyclonal antiserum in rabbits. The resulting antiserum was affinity-purified using His6-BAP immobilized on CNBr-activated agarose. BiP and the J domain of ERdj4 were purified as described before (25Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Google Scholar, 30Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Google Scholar). A Northern blot of multiple human tissue mRNA was obtained (Clontech Laboratory Inc., Palo Alto, CA) and probed as previously described (32Brewer J.W. Cleveland J.L. Hendershot L.M. EMBO J. 1997; 16: 7207-7216Google Scholar). The human BAP probe was prepared from the coding sequence (1–1386 bp) of theBAP gene using a Prime-it II kit and a NucTrap column (Stratagene, La Jolla, CA). The 1,308-bp coding segment of mouse BAP homologue was produced by PCR from a mouse B cell cDNA library. The forward primer was 5′-AAGCGTCGACTCTCAGCTGTCAGAACTCAAAT-3′ and the reverse primer was 5′-CTGGGAAGCTTTCATCTTAGTTCCTTCATCAA-3′. This PCR product was used to prepare the mouse BAP probe as above. BiP and β-actin probes were prepared as described (32Brewer J.W. Cleveland J.L. Hendershot L.M. EMBO J. 1997; 16: 7207-7216Google Scholar). pDSL(BAP-HA) was used to transfect COS-1 cells using the FuGENE 6 reagent (Roche Molecular Biochemicals). 18 h after transfection, the cells were fixed with 5% glacial acetic acid, 95% ethanol and incubated overnight with a mouse anti-HA tag monoclonal antibody (kindly provided by Dr. Al Reynolds, Vanderbilt University) and with a rabbit anti-GRP94 polyclonal antibody produced in our lab. The cells were then washed with PBS and incubated with TRITC-labeled goat anti-mouse antibody and fluorescein isothiocyanate-labeled goat anti-rabbit antibody for 2 h. The slides were examined on a confocal microscope (Leica TCS NT with Leica DMIRBE). The images were processed by Adobe Photoshop 5.0. HepG2 cells were treated with Me2SO or tunicamycin (final concentration, 2.5 μg/ml) in RPMI1640 containing [35S]-Translabel (ICN) for 16 h. The labeled cells were washed with PBS three times, lysed in Nonidet P-40 lysing buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% deoxycholic acid, and 0.5% Nonidet P-40), and centrifuged for 10 min at 14,000 rpm. The clarified lysate was immunoprecipitated with affinity-purified rabbit anti-BAP or preimmune IgG and protein A-Sepharose for 2 h at 4 °C. For endoglycosidase H (Endo H) digestion, BAP was immunoprecipitated from untreated HepG2 cell lysate, resuspended in 0.1 m sodium acetate buffer, pH 5.5, and digested with 0.5 unit of Endo H (Roche Molecular Biochemicals) for 18 h at 37 °C. The precipitated proteins were analyzed by SDS-PAGE. An aliquot of the total cell lysate of HepG2 was analyzed by Western blotting with anti-BiP and anti-BAP antisera. In addition, 293T cells were transfected with pCDNA(BAP) using the FuGENE 6 reagent (Roche Molecular Biochemicals). 24 h after transfection, the cells were treated with Me2SO or tunicamycin (final concentration, 2.5 μg/ml) in Dulbecco's modified Eagle's medium containing [35S]-Translabel (ICN) for 4 h. Immunoprecipitation of 293T cell lysates was done as described for HepG2 cells. The interaction between BAP and BiP was investigated in COS-1 cells by transiently co-expressing the two proteins. COS-1 cells were co-transfected with BAP-HA and either BiP, BiPT229G, BiPG227D, or BiPT37G (30Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Google Scholar, 31Wei J. Gaut J.R. Hendershot L.M. J. Biol. Chem. 1995; 270: 26677-26682Google Scholar) using the DEAE-dextran method. 30 h after transfection, the cells were metabolically labeled for 18 h, trypsinized, and resuspended in 1 ml of PBS. Resuspended cells were treated with 30 μl of dithiobis[succinimidyl propionate] (DSP, 5 mg/ml in Me2SO) for 1 h on ice. DSP was quenched by incubating with 100 μl of 1 m glycine for an additional 15 min on ice. The cells were washed with PBS and lysed in Nonidet P-40 lysing buffer. Each cell lysate was immunoprecipitated with the polyclonal anti-rodent BiP antiserum. The isolated proteins were then separated on a reducing SDS-PAGE gel. One-tenth of each cell lysate was blotted to determine the expression level of the two proteins in each sample. BAP and BiP were identified by the anti-HA tag and anti-rodent BiP antiserum, respectively. The ATPase activity of BiP was assayed as described previously with minor modifications (2Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Google Scholar, 4Minami Y. Hohfeld J. Ohtsuka K. Hartl F.U. J. Biol. Chem. 1996; 271: 19617-19624Google Scholar). Briefly, 0.5 μm recombinant BiP was incubated with or without 1 μm recombinant BAP and/or 2 μmrecombinant J domain from ERdj4 in ATPase assay buffer (20 mm HEPES, pH 7.2, 50 mm KCl, 5 mmMgCl2, and 10 mm dithiothreitol) at a total volume of 50 μl. Each reaction mixture contained 100 μmATP and 1 μCi of [γ-32P]ATP (3000Ci/mmol; AmershamBiosciences). At various time points, 2-μl aliquots were removed and analyzed by thin layer chromatography on polyethyleneimine cellulose sheets (Sigma) using 0.5 m formic acid, 0.5 mLiCl. BAP did not show ATPase activity by itself or bind to nucleotides. Natural hydrolysis of ATP in the presence of bovine serum albumin was routinely subtracted from experimental groups. The amount of [γ-32P]ATP hydrolyzed was quantified by a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The data were obtained from three independent experiments. The percentage of ADP of total nucleotide was determined, and the standard deviations were calculated. The effect of BAP on nucleotide-bound BiP was examined under both excess and limited nucleotide conditions as previously described (2Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Google Scholar, 4Minami Y. Hohfeld J. Ohtsuka K. Hartl F.U. J. Biol. Chem. 1996; 271: 19617-19624Google Scholar, 12Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Google Scholar). Briefly, 2.5 μm BiP was incubated with 50 μm of [α-32P]ATP (3000 Ci/mmol; Amersham Biosciences) for 10 min at room temperature and applied to MicroSpin G-50 columns (AmershamBiosciences) to separate the [α-32P]ATP-BiP complex from free nucleotide. Approximately 0.5 μm[α-32P]ATP-BiP complex was then transferred to the nucleotide exchange assay mixture containing 100 μm cold ATP and when indicated, 1 μm J domain or 1 μm J domain together with 1 μm BAP. The reactions were further incubated at room temperature for the indicated times, and the aliquots were removed and applied to a second MicroSpin G-50 column to separate ATP bound to BiP from free nucleotides. For nucleotide release under limited conditions, 0.5 μm BiP was incubated with 1 μCi of [α-32P]ATP for 30 min at 30 °C in the presence of 1 μm BAP alone, 1 μm J domain alone, or 1 μm J domain and 1 μm BAP. The reaction mixtures were applied to MicroSpin G-50 columns and analyzed as above. To identify BiP interacting proteins, a yeast two-hybrid screen was performed using the ATPase domain of a BiP mutant, BiPT229G, as the bait protein and a human liver cDNA library as the target. The BiPT229G binds ATP but cannot hydrolyze it (30Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Google Scholar, 31Wei J. Gaut J.R. Hendershot L.M. J. Biol. Chem. 1995; 270: 26677-26682Google Scholar). We isolated four independent positive clones that carried target genes encoding putative BiP-interacting proteins. DNA sequencing data revealed that all four clones represented the same gene but had various amounts of the 5′ coding region missing. We obtained an EST clone (identification number 5102998) that contained the complete open reading frame of the BiP-associated protein, which we named BAP. The BAP cDNA encoded a 461-amino acid protein with a potential N-terminal ER targeting sequence, two possible N-linked glycosylation sites, and an ER retention tetra-peptide (KELR) at the C terminus (Fig. 1 A). Interestingly, two polyadenylation signal sequences were identified in the human BAP mRNA. Both may be used, because EST sequences corresponding to both were found in the data base. However, our Northern blot analyses of primary human tissues revealed only a single BAP transcript (Fig. 2 A). BAP shared low sequence homology with a recently identified yeast ER protein Sls1p (Fig. 1 B), which interacts with the ATPase domain of yeast BiP and enhances the Sec63p-mediated increase in the ATPase activity of BiP (27Kabani M. Beckerich J.M. Gaillardin C. Mol. Cell. Biol. 2000; 20: 6923-6934Google Scholar) and was identical to an EST-derived sequence described as a potential human homologue of Sls1p (28Tyson J.R. Stirling C.J. EMBO J. 2000; 19: 6440-6452Google Scholar). BAP also shared 29% homology with a cytosolic Hsp70-binding protein, HspBP1, which in contrast to Sls1p, appears to inhibit the ATPase activity of Hsp70 (8Raynes D.A. Guerriero Jr., V. J. Biol. Chem. 1998; 273: 32883-32888Google Scholar).Figure 2Tissue distribution and cellular localization of human BAP. A, a human multiple tissue Northern blot was hybridized with probes corresponding to the coding regions of the human BAP, hamster BiP, and human β-actin genes. Lane 1, peripheral blood leukocytes; lane 2, lung; lane 3, placenta; lane 4, small intestine; lane 5, liver; lane 6, kidney; lane 7, spleen;lane 8, thymus; lane 9, colon; lane 10, skeletal muscle; lane 11, heart; lane 12, brain. B, COS-1 cells were transfected with pDSL(BAP-HA). At 18 h post-transfection, the cells were immunostained with a monoclonal anti-HA tag antibody and a polyclonal anti-GRP94 antibody followed by TRITC- and fluorescein isothiocyanate-conjugated secondary antibodies, respectively. The images were visualized on a confocal microscope and processed with Adobe Photoshop 5.0. The merged image reveals co-localization of these two proteins (yellow).View Large Image Figure ViewerDownload (PPT) A cDNA probe corresponding to the mature form of BAP was hybridized to a human multiple-tissue blot to determine the expression pattern of BAP. An ∼1.8-kb transcript was detected in all of the tissues examined, but BAP was expressed most highly in secretory tissues such as liver, placenta, and kidney (Fig. 2 A). The expression pattern of BAP on the multiple-tissue blot was almost identical to that of BiP and two recently identified ER DnaJ proteins, ERdj4 and ERdj3 (not shown), suggesting that they may function together. To determine the subcellular localization of BAP, COS-1 cells were transfected with a cDNA encoding a HA-tagged version of BAP. Immunofluorescence staining revealed that BAP co-localized with endogenous GRP94 in the ER of COS-1 cells (Fig. 2 B). To further analyze BAP, a polyclonal antiserum was raised against full-length recombinant BAP protein and affinity-purified using the same recombinant protein immobilized on CNBr-activated Sepharose beads. The affinity-purified antiserum precipitated an ∼54-kDa protein from HepG2 cells, which is in keeping with the deduced cDNA sequence, whereas the preimmune serum failed to react with a protein in this region. Several additional high molecular weight bands were detected with the anti-BAP antiserum. It is not clear at this time whether these represent proteins that co-precipitate with BAP or whether they are proteins that cross-react with the antiserum. Labeling cells in the presence of tunicamycin or treating immunoprecipitated material with Endo H produced a protein that migrated more rapidly on SDS gels, demonstrating that BAP is aN-linked glycoprotein and therefore must enter the ER (Fig.3 A, left panel). cDNAs encoding BAP with and without an an HA tag were transfected into 293T cells, and the 293T cells were examined by similar methods. When full-length BAP without an HA tag was expressed, a 54-kDa protein was precipitated with the anti-BAP antiserum, which migrated more rapidly after labeling in the presence of tunicamycin or Endo H digestion (Fig. 3 A, right panel). Detection of the HA-tagged form with a HA-specific monoclonal antibody showed similar results (data not shown). An aliquot of whole cell lysates from control and tunicamycin-treated HepG2 cells was analyzed by Western blotting with antisera specific for both BAP and BiP. In the control cells, a major band migrating at 54 kDa was observed, which migrated faster after tunicamycin treatment (Fig. 3 B). After 16 h of tunicamycin treatment, none of the glycosylated protein could be detected, suggesting that the half-life of BAP, at least during ER stress, is relatively short. In addition, unlike BiP, the amount of BAP protein present in the tunicamycin-treated cell lysates was actually less than in the untreated cell lysates. This suggested that unlike other ER chaperones, BAP might not be a target of the unfolded protein response. The binding of BAP to BiP was examined in vivo by transiently co-expressing BAP with various BiP mutant proteins in COS-1 cells. We have identified a number of BiP mutants that are impaired in nucleotide binding, in the ATP-dependent conformational change, or in ATP hydrolysis (30Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Google Scholar, 31Wei J. Gaut J.R. Hendershot L.M. J. Biol. Chem. 1995; 270: 26677-26682Google Scholar). Transfected cells were metabolically labeled with [35S]methionine and [35S]cysteine and then treated with the membrane-permeable cross-linking agent DSP to stabilize BAP-BiP complexes within the cell. The complexes were isolated with rodent-specific anti-BiP antiserum and separated on a SDS gel. An aliquot of the total cell lysate was analyzed by Western blotting, which revealed that each of the co-transfected set of cells expressed very similar levels of the two proteins (Fig.4 A). However, the co-precipitation experiments demonstrated that much more BAP was co-precipitated with the various BiP mutants than with wild type BiP. This indicated that the binding of BAP to BiP was significantly affected by the conformation of the ATPase domain of BiP (Fig.4 A). Similar results were observed when we expressed BAP in yeast. The BAP-Gal4 activation domain fusion protein was transformed into yeast together with the Gal4 DNA-binding domain fused to either the wild type or T229G BiP ATPase domains and used to drive Gal4-mediated His3 gene expression. Only the combination of BAP and T229G was able to grow on His− selective medium. Yeast cells expressing BAP with the wild type ATPase domain of BiP failed to do so (Fig. 4 B). Together, these data showed that BAP binds better or more stably to the BiP mutants than to wild type BiP, possibly because of conformational differences in the ATPase domains or in the nucleotide bound state of the domains. Because BAP specifically interacted with the ATPase domain of BiP, we determined whether BAP could modulate the ATPase activity of BiP under conditions of nucleotide excess. In this experiment, aliquots of the ATP hydrolysis assay mixture were removed at 10-min intervals and analyzed on TLC plates. Our recombinant BAP preparation was first tested for nucleotide binding and intrinsic ATPase activity and found to be negative for both (data not shown). When BAP was added to BiP (2:1 molar ratio of BAP:BiP), it increased the ATPase activity of BiP by about 2-fold, which was similar to the increase observed when the J domain of ERdj4 was added to BiP (Fig.5). When both BAP and the J domain were added to BiP, the rate of ATP hydrolysis by BiP was stimulated by about 4-fold over basal levels, indicating that both BAP and ERdj4 positively regulate the ATPase activity of BiP (Fig. 5). DnaJ homologues, including ERdjs, bind to the ATP-bound form of Hsp70 family members and accelerate their intrinsic ATPase activity (33Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Google Scholar). In contrast, binding studies with our BiP mutants suggested that BAP might preferentially interact with the ADP-bound state of BiP. For BAP to stimulate the ATPase activity of BiP, it was conceivable that BAP served as a nucleotide exchange factor for BiP. We examined the ability of BAP to affect nucleotide binding under steady state conditions. BiP alone or BiP with the indicated combinations of BAP and ERdj4 was incubated in the presence of limited quantities of [α-32P]ATP for 30 min. Under these conditions, in the reaction mixture containing BiP alone, BiP remained associated with both ADP and ATP (Fig.6 A). Addition of the J domain to BiP caused increased hydrolysis of labeled ATP to ADP, which remained bound. In contrast, the addition of BAP either alone or together with ERdj4 resulted in the almost complete absence of a signal for labeled nucleotide with BiP (Fig. 6 A). This finding is compatible with BAP inhibiting nucleotide binding, as has been suggested for the function of HspBP1 (8Raynes D.A. Guerriero Jr., V. J. Biol. Chem. 1998; 273: 32883-32888Google Scholar). Conversely, these data could suggest that BAP is serving to accelerate the exchange of labeled nucleotide with unlabeled nucleotide, which is in molar excess in this experiment. Thus, we directly examined the nucleotide exchange function of BAP. BiP was loaded with [α-32P]ATP and separated from unbound nucleotide (Fig. 6 B, first lane). The labeled BiP was then incubated alone, with the J domain from ERdj4, or with the J domain and recombinant BAP for the indicated times and then separated on a second column. In the absence of additional proteins, approximately half of the labeled ADP was exchanged for cold ATP within 5 min. Unlike other members of Hsp70 family, however, BiP hydrolyzed the remaining labeled ATP to ADP during the second spin column process, even though it was carried out at 4 °C (Fig. 6 B, comparefirst and second lanes). The amount of ADP that remained bound to BiP in the presence of the J domain was ∼20% greater than BiP alone, but the rates of exchange were very similar in both cases. Thus, the ER J protein accelerated ATP hydrolysis but did not affect the rate of nucleotide exchange, which is in keeping with studies on the bacterial and cytosolic DnaJ homologues. When both ERdj4 and BAP were added to BiP, the [32P]ADP was quickly replaced with cold ATP. Even after a 1-min incubation, almost all of the labeled ADP was released (Fig. 6 B). Thus, BAP accelerates the rate of ATP hydrolysis of BiP by increasing the rate of nucleotide exchange. All of the Hsp70 family members bind and hydrolyze ATP, and their functions are regulated by the nucleotide-bound state. Hsp70s bind to unfolded substrate proteins when they are in the ATP-bound state. ATP hydrolysis, catalyzed by DnaJ family members, serves to stabilize their binding to these substrates, and exchange of ATP back into the nucleotide-binding cleft allows the Hsp70 protein to release the substrate. For most Hsp70s, co-factors that regulate ATP hydrolysis and nucleotide exchange have been identified. However, until now no regulators of nucleotide exchange for the mammalian ER Hsp70 family orthologue had been identified. In this study, we provide the first description of a resident ER protein that serves as a nucleotide exchange factor for BiP. BAP was isolated as a protein that bound to the ATPase domain of a hamster BiP mutant in a yeast two-hybrid screen. Our characterization of BAP revealed that it possessed an N-terminal ER targeting signal sequence, a C-terminal ER retention motif, andN-linked glycans that remained Endo H-sensitive, implying that BAP is not transported beyond the ER. In addition, BAP was associated with BiP in mammalian cells and interacted with BiP functionally in vitro. The amino acid sequence of BAP shows homology with two groups of proteins that have been implicated in regulating the ATPase cycle of different Hsp70 proteins. The region between amino acids 70 and 108 of BAP is highly homologous to a similar region of Sls1p, which was recently identified as a resident ER protein in both Yarrowia lipolytica and Saccharomyces cerevisiae (27Kabani M. Beckerich J.M. Gaillardin C. Mol. Cell. Biol. 2000; 20: 6923-6934Google Scholar, 28Tyson J.R. Stirling C.J. EMBO J. 2000; 19: 6440-6452Google Scholar). Sls1p binds to the ATPase domain of Kar2p, the yeast homologue of BiP, and functionally interacts with the yeast DnaJ protein Sec63p to increase the ATPase activity of Kar2p (27Kabani M. Beckerich J.M. Gaillardin C. Mol. Cell. Biol. 2000; 20: 6923-6934Google Scholar). SLS1 also interacts genetically with LHS1, another Hsp70 homologue in the yeast ER (28Tyson J.R. Stirling C.J. EMBO J. 2000; 19: 6440-6452Google Scholar). BAP also shares some sequence homology with the cytosolic protein HspBP1 in its more central region, which includes the armadillo repeat domain. HspBP1 interacts with Hsp70 and inhibits the Hsp40-mediated activation of Hsp70s (8Raynes D.A. Guerriero Jr., V. J. Biol. Chem. 1998; 273: 32883-32888Google Scholar), perhaps by preventing nucleotide rebinding (34Kabani M. Beckerich J.M. Brodsky J.L. Mol. Cell. Biol. 2002; 22: 4677-4689Google Scholar). Homologues of HspBP1 can be found in databases for a vast variety of species. 3J. L. Brodsky, personal communication. Thus, BAP shares homology with two functionally different families of nucleotide exchange regulators for Hsp70 proteins. Recombinant BAP protein stimulated the ATPase activity of BiP in vitro and caused a further increase in the presence of recombinant J domain from ERdj4, a recently identified mammalian ER DnaJ homologue (25Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Google Scholar), suggesting that BAP was more likely to be a functional homologue of Sls1p than of HspBP1. When nucleotide exchange assays were performed under conditions of excess cold ATP, BAP caused the rapid release of labeled ADP from BiP. When the exchange assays were performed under conditions where both ADP and ATP were associated with BiP, it appeared that both nucleotides were readily released by BAP. This characteristic is more similar to results obtained with GrpE, the nucleotide exchange factor present in bacteria (2Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Google Scholar) and in organelles like chloroplasts and mitochondria (35Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Google Scholar), which are thought to be of bacterial origin. Co-expression of BAP with either wild type or mutant BiP revealed that more BAP was associated with the ATPase mutants than with wild type BiP. In addition the ATPase domain of BiP mutant interacted with BAP in the two-hybrid screen, whereas the wild type domain did not. These data suggest that BAP may prefer the ADP bound form of BiP, which is in keeping with binding data for both Sls1p (27Kabani M. Beckerich J.M. Gaillardin C. Mol. Cell. Biol. 2000; 20: 6923-6934Google Scholar) and Fes1p, a recently identified yeast homologue of HspBP1 (34Kabani M. Beckerich J.M. Brodsky J.L. Mol. Cell. Biol. 2002; 22: 4677-4689Google Scholar), both of which bind preferentially to their Hsp70 partners when in the ADP-bound state. In the case of BAP and Sls1p, this should result in the preferential exchange of ADP out of the nucleotide-binding cleft, which explains their positive effects on the ATPase activity of their respective Hsp70 proteins. Our nucleotide binding experiments demonstrated that BAP does not directly bind either ATP or ADP, so its ability to act as an exchanger for BiP must occur as a result of conformational changes that occur in the ATPase domain of BiP when BAP binds. Like other Hsp70 proteins, BiP binds to unfolded regions in substrate proteins and prevents them from folding or aggregating (16Gething M.-J. Sambrook J. Nature. 1992; 355: 33-45Google Scholar, 36Haas I.G. Experientia. 1994; 50: 1012-1020Google Scholar).In vivo, it is assumed that ATP must rebind to the nucleotide-binding cleft to allow release of bound proteins at the appropriate time so they can fold. This hypothesis is supported by data obtained with BiP ATP-binding mutants showing that the mutants prevent the folding of bound substrates but keep them in a soluble form (37Hendershot L.M. Wei J.-Y. Gaut J.R. Melnick J. Aviel S. Argon Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5269-5274Google Scholar). The in vitro addition of ATP to complexes induces the release of BiP, which can provide an opportunity for the substrate to fold or in some cases leads to aggregation of the unfolded substrate (38Lee Y.K. Brewer J.W. Hellman R. Hendershot L.M. Mol. Biol. Cell. 1999; 10: 2209-2219Google Scholar, 39Vanhove M. Usherwood Y.-K. Hendershot L.M. Immunity. 2001; 15: 105-114Google Scholar). Thus, it is reasonable to assume that the timing or conditions of release might be important and that overexpression of a nucleotide exchange factor could have either positive or negative effects on protein folding. In keeping with the idea of negative effects on protein folding, the overexpression of the cytosolic nucleotide exchange factor BAG-1 inhibited the ability of Hsp70 to refold luciferase (40Nollen E.A. Brunsting J.F. Song J. Kampinga H.H. Morimoto R.I. Mol. Cell. Biol. 2000; 20: 1083-1088Google Scholar) and suppressed the positive effect of Hip on Hsp70 chaperone activity (41Nollen E.A. Kabakov A.E. Brunsting J.F. Kanon B. Hohfeld J. Kampinga H.H. J. Biol. Chem. 2001; 276: 4677-4682Google Scholar). In support of the idea that BAP positively regulates protein folding in the mammalian ER, we found that BAP is expressed most highly in tissues like the liver, kidney, and placenta, which produce large amounts of secreted proteins. These tissues also show high levels of BiP, ERdj3, and ERdj4 (25Shen Y. Meunier L. Hendershot L.M. J. Biol. Chem. 2002; 277: 15947-15956Google Scholar), which was shown here to interact with BAP. The role of BAP in regulating protein folding by BiP is currently under investigation using in vitro and in vivoexperimental models. In conclusion, we have identified the first mammalian ER nucleotide exchange factor for BiP, which appears to be a homologue of yeast Sls1p. However, although BAP is highly expressed in secretory tissues, unlike Sls1p, BAP is regulated independently of ER chaperones during ER stress. This suggests that mammalian cells have the ability to inhibit the release of BiP from substrate proteins under conditions that are not conducive to proper folding or assembly. We thank Dr. Daesik Lim (St. Jude Children's Research Hospital) for helpful discussions and providing reagents for the yeast two-hybrid screen and Isaac Estrada for technical assistance.