Binding to Chaperones Allows Import of a Purified Mitochondrial Precursor into Mitochondria
2002; Elsevier BV; Volume: 277; Issue: 28 Linguagem: Inglês
10.1074/jbc.m203474200
ISSN1083-351X
AutoresAntonio Artigues, Ana Iriarte, Marino Martinez‐Carrion,
Tópico(s)Enzyme Structure and Function
ResumoRefolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852–16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Coimmunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate. Refolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852–16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Coimmunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate. precursor to mitochondrial aspartate aminotransferase mitochondrial aspartate aminotransferase presequence binding factor mitochondrial import stimulation factor rabbit reticulocyte lysate nickel-nitrilotriacetic acid 5-iodoacetamidofluorescein N-tosyl-l-phenylalanyl chloromethyl ketone 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid acetamidofluorescein The majority of mitochondrial proteins are encoded by the nuclear genome and synthesized on cytoplasmic ribosomes, most of them as precursor forms with N-terminal targeting sequences that are usually removed by a specific peptidase in the matrix. Import through the outer and inner membrane translocation complexes requires energy in the form of ATP hydrolysis in the matrix and an electrochemical potential across the inner mitochondrial membrane, as well as the participation of several mitochondrial chaperones including the import motor, mitochondrial Hsp70 (reviewed in Refs. 1Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar and 2Voos W. Martin H. Krimmer T. Pfanner N. Biochim. Biophys. Acta. 1999; 1422: 235-254Crossref PubMed Scopus (129) Google Scholar). Although some proteins might be imported cotranslationally (3Verner K. Trends Biochem. Sci. 1993; 18: 366-371Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 4Lithgow T. FEBS Lett. 2000; 476: 22-26Crossref PubMed Scopus (108) Google Scholar), for the majority of proteins import can take place posttranslationally, both in vitro and in vivo (1Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar, 5Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (922) Google Scholar). In addition, it is well known that mitochondrial precursors are not imported into mitochondria in their native state (6Eilers M. Schatz G. Nature. 1986; 322: 228-232Crossref PubMed Scopus (467) Google Scholar, 7Wienhues U. Becker K. Schleyer M. Guiard B. Tropschug M. Horwich A.L. Pfanner N. Neupert W. J. Cell Biol. 1991; 115: 1601-1609Crossref PubMed Scopus (97) Google Scholar). The degree of folding of the import substrate compatible with translocation varies among precursors. Some proteins can be presented to mitochondria in their native conformation, and mitochondria are able to unfold them during translocation (8Matouscheck A. Pfanner N. Voos W. EMBO Rep. 2000; 1: 404-410Crossref PubMed Scopus (130) Google Scholar). These proteins usually do not require ATP hydrolysis outside the mitochondria for translocation. The import of many other mitochondrial precursors, including the precursor for rat liver aspartate aminotransferase (pmAAT)1used in this work, requires extramitochondrial ATP (9Mattingly J.R., Jr. Youseff J. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1993; 268: 3925-3937Abstract Full Text PDF PubMed Google Scholar, 10Wachter C. Schatz G. Glick B.S. Mol. Biol. Cell. 1994; 5: 465-474Crossref PubMed Scopus (107) Google Scholar), probably for their release from complexes with cytosolic chaperones. It is likely that these mitochondrial precursors never fold in the cytosol and engage the translocation machinery when still in a partially unfolded state. In fact, the folding of pmAAT synthesized in a cell-free extract is inhibited by cytosolic factors, and once the protein is folded mitochondria can no longer import it (9Mattingly J.R., Jr. Youseff J. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1993; 268: 3925-3937Abstract Full Text PDF PubMed Google Scholar). The interaction with cytosolic chaperones might prevent not only premature folding but also aggregation and proteolysis of the incompletely folded polypeptides. The requirement for cytosolic proteins may not be universal because some precursor proteins can be imported in vitro into mitochondria in the absence of cytosolic extracts (6Eilers M. Schatz G. Nature. 1986; 322: 228-232Crossref PubMed Scopus (467) Google Scholar,11Becker K. Guiard B. Rassow J. Sölner T. Pfanner N. J. Biol. Chem. 1992; 267: 5637-5643Abstract Full Text PDF PubMed Google Scholar). It is clear that the requirements for the delivery of newly synthesized precursors to the outer mitochondrial membrane differ among precursor proteins and may depend on factors such as overall hydrophobicity or oligomeric state of the native mature protein. Cytosolic components reportedly performing this chaperone function for mitochondrial precursors include members of the Hsp70 and Hsp40 (DnaJ-related) family of molecular chaperones (1Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar). The constitutively expressed cytosolic member of the Hsp70 family of molecular chaperones (Hsc70) is involved in a broad spectrum of cellular processes (12McKay D.B. Adv. Protein Chem. 1993; 44: 67-98Crossref PubMed Google Scholar, 13Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 14Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Crossref PubMed Scopus (944) Google Scholar). Its chaperone activity is regulated by a number of co-chaperones, including members of the Hsp40 family that stimulate its ATPase activity and promote substrate association with Hsp70 (14Frydman J. Annu. Rev. 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Depletion of Hsp70 proteins in yeast mutants interfered with the biogenesis of mitochondrial proteins (20Deshaires R.J. Koch B.D. Werner-Washburne M. Craig E.A. Chekman R. Nature. 1988; 332: 800-805Crossref PubMed Scopus (1005) Google Scholar), and the yeast DnaJ homologue Ydj1 was initially isolated as MAS5, a mutation that causes defects in mitochondrial import in yeast (21Atencio D.P. Yaffe M.P. Mol. Cell. Biol. 1992; 12: 283-291Crossref PubMed Scopus (122) Google Scholar). Furthermore, both Hsc70 and the DnaJ homologues dj2 or dj3 appear to be required for import of preornithine decarboxylase translated in vitro into mammalian mitochondria (22Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (97) Google Scholar, 23Terada K. Ohtsuka K. Imamoto N. Yoneda Y. Mori M. Mol. Cell. Biol. 1995; 15: 3708-3713Crossref PubMed Scopus (50) Google Scholar, 24Terada K. Mori M. J. Biol. 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Biochemistry. 1992; 31: 7325-7329Crossref PubMed Scopus (40) Google Scholar) described the isolation of a fraction from rabbit reticulocyte lysate (RRL) containing Hsp90 and Hsp70, which both stimulated import into mitochondria of a hybrid precursor protein and mediated formation of Hsp90-steroid receptor heterocomplexes. Yet the involvement of Hsp90 on protein translocation remains obscure. We have shown previously that cytosolic Hsc70 binds to pmAAT both during translation in a cell-free extract (32Lain B. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1994; 269: 15588-15596Abstract Full Text PDF PubMed Google Scholar) and in vitrorefolding of the acid-unfolded protein (33Artigues A. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1997; 272: 16852-16861Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Subsequently we identified seven putative Hsc70-binding sites in the pmAAT polypeptide (34Artigues A. Crawford D.L. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1998; 273: 33130-33134Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) which included the cleavable presequence peptide in addition to several peptide regions in the mature portion of the protein. The complexes obtained at low molar excess of Hsc70 over purified unfolded pmAAT were insoluble and formed aggregates containing equal amounts of the two proteins (33Artigues A. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1997; 272: 16852-16861Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Thus, a question remained as to whether Hsc70 was able to bind simultaneously to several of the binding sites in pmAAT. Here we report that in the presence of high concentrations of Hsc70, purified pmAAT forms soluble complexes containing multiple Hsc70 molecules bound to a single pmAAT polypeptide. Soluble complexes can also be assembled with purified yeast YDJ1 and mammalian Hsp90 and unfolded pmAAT. The import competence of these complexes is chaperone-specific. Mitochondria were unable to import Hsp90-bound pmAAT, whereas import from the complexes with Hsc70 or Ydj1 requires the presence of reticulocyte lysate. This contrasts previous reports on the import of another matrix protein, preadrenodoxin, from preassembled complexes with Hsp70 or MSF which could take place in the absence of a cytosolic extract or even ATP (35Komiya T. Sakaguchi M. Mihara K. EMBO J. 1996; 15: 399-407Crossref PubMed Scopus (94) Google Scholar). To our knowledge this is the first report of in vitro import of a mitochondrial protein from a preassembled Hsp40-precursor complex. The expression in Escherichia coli and purification of the histidine-tagged protein pmAAT(His6)Tyr, a protein construct containing a tag of six histidines and a tyrosine residue fused to the C-terminal end of pmAAT, was carried out as described previously (36Mattingly J.R., Jr. Torella C. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1998; 273: 23191-23202Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) using metal chelation chromatography in a Ni2+-nitrilotriacetic acid (Ni-NTA)-agarose column (Qiagen). The mature form of the protein, mAAT(His6)Tyr, was obtained by incubating the precursor with trypsin (1:100 trypsin/pmAAT(His6)Tyr molar ratio) to remove the N-terminal presequence peptide, followed by chromatography on a CM-Sephadex column. Protein concentration was estimated from the absorbance at 280 nm using the calculated molar absorption coefficient of 55,610 m−1 cm−1 (36Mattingly J.R., Jr. Torella C. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1998; 273: 23191-23202Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) andM r = 48,540 and 45,824 for the precursor and mature protein, respectively. Because all the experiments reported in this work were performed using this tagged protein construct, for brevity and clarity of exposure we will omit the suffix (His6)Tyr and we will refer to these proteins as pmAAT for the precursor or mAAT for the mature form. Hsc70 was purified from bovine brain following published procedures (37Welch W.J. Feramisco J.R. Mol. Cell. Biol. 1985; 5: 1229-1237Crossref PubMed Scopus (260) Google Scholar). Protein concentration was estimated using the published ε (280 nm) = 47,800m−1 cm−1 andM r = 70,000 (38Palleros D.R. Welch W.J. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5719-5723Crossref PubMed Scopus (281) Google Scholar). Hsp90 was purified from bovine brain as described previously (39Welch W.J. Feramisco J.R. J. Biol. Chem. 1982; 257: 14949-14959Abstract Full Text PDF PubMed Google Scholar). Ydj1 was expressed in E. coli and purified according to Cyr et al. (40Cyr D.M., Lu, X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar). Hsp90 and Ydj1 protein concentrations were determined from the intensity of Coomassie Blue-stained SDS-PAGE gels, using mAAT as standard. The purified proteins were stored at 4 °C. Radiolabeled pmAAT was prepared by incubating the protein (200 μg) with 2 μmNa125I (1 μCi) in 100 μl of refolding buffer (100 mm Hepes, 0.1 mm EDTA, 1 μmdithiothreitol) in an IODO-GEN-coated tube (Sigma) for 30 min at room temperature. The reaction was quenched by addition of 5 μl of 100 mm NaI and 5 μl of 4 mm hydroxymethyl acetate, followed by extensive dialysis in refolding buffer. The final specific radioactivity of the labeled protein was 300,000 dpm/μg. Syncatalytic modification of pmAAT with 5-iodoacetamidofluorescein (5-IAF) was achieved by incubation of pmAAT (1 mg/ml) in refolding buffer with 2 mm 5-IAF in the presence of 50 mmof the substrate analogue α-methyl aspartate. After incubation for 4 h at room temperature, the excess of 5-IAF and α-methyl aspartate was removed by ultrafiltration through a Centricon 30 (Amicon). The resulting AF-labeled pmAAT (AF-pmAAT) contains a strong absorbance reporting group (ε492 nm = 75,000m−1 cm−1) suitable to follow the sedimentation profiles of pmAAT and its complex with Hsc70 during analytical ultracentrifugation. The concentration of the modified proteins was estimated from the Coomassie Blue-stained SDS-PAGE gels by comparing the intensity of the protein bands with standards of known concentrations of unlabeled mAAT. Acid unfolding of 125I- or 5-AF-labeled pmAAT was performed by incubation in 2 mm Tris acidified to pH 2.0 with HCl for 90 min at room temperature. The final protein concentration was calculated from the specific radioactivity of the125I-labeled protein. Binding of pmAAT to Hsc70, Hsp90, or Ydj1 was accomplished by diluting the acid-denatured protein with 10 volumes of ice-cold refolding buffer containing a given chaperone followed by incubation for 90 min at 10 °C. The final concentration of pmAAT monomer in the reaction was 2.5 × 10−3μm monomer, and the concentration of each molecular chaperone was 1.8 μm, unless indicated otherwise. Trypsin digestion was performed at 4 °C by addition of a concentrated stock solution of TPCK-trypsin in 1 mm HCl to give a final Hsc70/trypsin molar ratio of 200:1. In control experiments using Hsc70 alone, we found that with this trypsin/Hsc70 ratio about 90% of Hsc70 remains intact even after a 1-h incubation at 4 °C. To evaluate the extent of proteolysis of both chaperone and pmAAT, samples were analyzed by SDS-PAGE and visualized either in a Molecular Dynamics PhosphorImagerTM(125I-pmAAT) or by staining with Coomassie Blue (Hsc70). Immunoprecipitation reactions of Hsc70-125I-pmAAT complexes (60 μl) were performed by addition of an equal volume of rabbit anti-Hsc70 polyclonal antibodies diluted 1:1 in PBS buffer, 5 mm EDTA, and 0.1% Triton X-100. The same amount of preimmune serum was used as a control. Following incubation for 4 h at 10 °C, 60 μl of protein A-agarose (Repligen) was added to each reaction, and the samples were incubated for an additional hour at 4 °C with gentle shaking. The suspensions were centrifuged at maximum speed on a microcentrifuge. The supernatants were diluted in SDS sample buffer, and the pellets, following an additional wash in phosphate-buffered saline, 0.1% Triton X-100, 5 mm EDTA, were resuspended in SDS sample buffer and heated at 60 °C for 20 min. The agarose beads were pelleted by centrifugation, and the supernatants were analyzed by SDS-PAGE and PhosphorImaging. Determination of the sedimentation coefficient of the AF-labeled pmAAT alone or in complex with Hsc70 was done on a Beckman XL-A analytical ultracentrifuge, using an AN-60 Ti analytical rotor. Samples were centrifuged at 30,000 or 15,000 rpm for AF-pmAAT alone or Hsc70-AF-pmAAT complexes, respectively. Sedimentation coefficients were estimated from the absorbance boundary profiles at 492 nm (the λmax of fluorescein) using the software provided with the instrument. Native Hsc70 (1.8 μm) and pmAAT (2.5 × 10−3μm) as well as samples of Hsc70-pmAAT complex formation reactions were layered on top of a 5-ml 10–30% glycerol gradient in refolding buffer. The gradients were preformed on a Biocomp model 106 gradient maker following the manufacturer's instructions. Samples were centrifuged at 55,000 rpm for 4 h on a TLX Beckman ultracentrifuge using a TLX-L rotor. Following fractionation of the gradients on a Retriever II (Isco), the fractions were analyzed for the presence of125I-pmAAT by counting aliquots on a Beckman Gamma 5500 counter and for the presence of Hsc70 by SDS-PAGE followed by staining the gels with Coomassie Blue. Mitochondria were isolated from male Wistar rat liver as described previously (41Parsons D.F. Williams G.R. Chance B. Ann. N. Y. Acad. Sci. 1966; 137: 643-666Crossref PubMed Scopus (233) Google Scholar) and resuspended in MESH buffer (220 mm mannitol, 0.1 mm EDTA, 70 mmsucrose, 20 mm HEPPS, pH 7.4). The import reaction was performed by mixing 40 μl of Hsc70-125I-pmAAT complexes with 40 μl of freshly isolated mitochondria (4 mg/ml final concentration) and 40 μl of RRL or MESH buffer containing ATP and an ATP-regenerating system (200 μm MgATP, 2 mg/ml creatine kinase, 500 μm creatine phosphate, 400 mmμm malate, 400 μm succinate, 200 μm NADH, in MESH buffer, pH 7.4). The reaction mixture was incubated for 30 min at 30 °C. After stopping import by chilling on ice, an aliquot was taken and mixed with an equal volume of SDS-PAGE sample buffer. The remaining of the reaction was centrifuged at 16,000 × g for 4 min, and the pellet containing the reisolated mitochondria was washed twice with 200 μl of MESH buffer and finally resuspended in an adequate volume of MESH buffer. Aliquots of this mitochondrial fraction were either left untreated and mixed directly with SDS-PAGE sample buffer (sample 1) or treated with 20 μg/ml l-tosylamido-2-phenylethylchloromethylketone (TPCK)-trypsin for 30 min on ice either in the absence (sample 2) or presence (sample 3) of 0.1% Triton X-100. The trypsin digestion was terminated by addition of 0.5 μl of phenylmethylsulfonyl fluoride, and the samples were mixed with an equal volume of SDS-PAGE sample buffer. All samples were subsequently analyzed by SDS-PAGE and exposure to a PhosphorImager screen. The amount of protein bound to mitochondria (sample 1), imported into mitochondria (sample 2), or imported and properly folded (sample 3) was estimated from the intensity of the corresponding radiolabeled protein band and is expressed as percentage relative to the intensity of the pmAAT band in the unfractionated import reaction. The protein construct used in this work contains an LEHHHHHHY tag fused to the C-terminal end of pmAAT. The addition of this C-terminal tail does not alter the overall stability, catalytic properties, or refolding ability of the enzyme (data not shown), but it provides a convenient tool for the purification and detection of the protein. The histidine tag allows for rapid purification of the protein using metal affinity resins, whereas the C-terminal tyrosine can be easily iodinated for radiolabeling purposes. Hsc70 binds refolding intermediates of acid-denatured pmAAT and prevents further folding and reactivation of the enzyme (33Artigues A. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1997; 272: 16852-16861Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The nature of the complexes formed depends on the concentration of Hsc70 in the refolding reaction. At a low molar excess of Hsc70, the resulting complexes contain stoichiometric amounts of pmAAT and Hsc70 and form insoluble aggregates that can be removed by centrifugation (Fig.1). However, the fraction of pmAAT recovered in the supernatant increases with increasing concentrations of Hsc70 until all of the pmAAT remains in solution at molar ratios of Hsc70 over pmAAT of 200 or higher (Fig. 1). Yet no transaminase activity is detected in the supernatant, which indicates that the soluble pmAAT is not completely folded. The inactive soluble pmAAT could be immunoprecipitated using Hsc70 polyclonal antibodies (Fig.2), which confirms that the pmAAT in the supernatant is indeed bound to Hsc70. The solubility of these complexes is marginal, and they readily precipitate when the sample is concentrated slightly after preparation (data not shown). Native pmAAT, either alone or in the presence of an excess of Hsc70, did not precipitate with anti-Hsc70 antibodies (data not shown). Similar concentration-dependent formation of insoluble or soluble complexes was observed with two other chaperones, Hsp90 and Ydj1 (Fig.1).Figure 2Coimmunoprecipitation of pmAAT with Hsc70 antibodies. Immunoprecipitation reactions of Hsc70-125pmAAT soluble complexes were performed as indicated under “Experimental Procedures” using rabbit preimmune or anti-Hsc70 whole antiserum. Both supernatants (Sn) and pellets (P) were resuspended in SDS sample buffer to the same final volume and analyzed by SDS-PAGE. The dried gels were exposed overnight to a PhosphorImager screen, and the radiolabeled protein bands were detected on a Molecular Dynamics PhosphorImagerTM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A possible explanation for the different behavior of the complexes formed at low and high concentrations of Hsc70 is that the soluble complexes contain multiple Hsc70 molecules associated with a single pmAAT polypeptide. To determine the stoichiometry of the complexes, we first isolated the complex from the large excess of Hsc70 present in the complex formation reaction by metal chelation chromatography in a Ni-NTA column taking advantage of the presence of the histidine tag at the C-terminal end of pmAAT. In control experiments we established that Hsc70 alone does not bind to the column and elutes with the loading buffer. On the other hand, histidine-tagged native pmAAT binds very tightly but can be eluted with 110 mm imidazole. When Hsc70-pmAAT complexes were loaded on the column, the majority of Hsc70 eluted with the loading buffer, as expected considering the large excess of Hsc70 present in the mixture. SDS-PAGE analysis of the material bound to the resin showed the presence of Hsc70 together with pmAAT (data not shown). Following visualization either by Coomassie Blue staining (Hsc70) or autoradiography (125I-pmAAT), the amount of each protein bound to the Ni-NTA resin was estimated from the intensity of the corresponding electrophoretic bands, using known concentrations of each protein as standards (see “Experimental Procedures” for details). According to these measurements, we found that on average between 5 and 7 molecules of Hsc70 are bound to each pmAAT polypeptide chain. This figure represents only a rough approximation because several factors such as dissociation of Hsc70 from low affinity binding sites during purification of the complex could influence the estimate. In any case, it is clear that several Hsc70 molecules must bind to a single pmAAT polypeptide in order to prevent aggregation and subsequent precipitation of the complex. Moreover, this stoichiometry is in good agreement with the number of Hsc70-binding sites (seven) identified in the pmAAT sequence by screening a synthetic peptide library (34Artigues A. Crawford D.L. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1998; 273: 33130-33134Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Interestingly, attempts at eluting the proteins from the Ni-NTA resin with high concentrations of imidazole were unsuccessful. Only solubilization with SDS-PAGE sample buffer released the bound proteins. A possible explanation for these findings is that the tight binding of the Hsc70-pmAAT complexes to the resin involves not only specific interactions of the histidine tag with the Ni2+ immobilized in the Ni-NTA resin but also nonspecific interactions of nonnative pmAAT with the solid matrix. This conclusion is supported by the observation that in vitro translated wild type pmAAT, which is also partially unfolded, binds not only to Ni-NTA resins but also to several other agarose-based solid supports. 2C. Tanase and A. Iriarte, unpublished observations. The hydrodynamic properties of these soluble complexes were further characterized using ultracentrifugation methods. To follow the distribution of pmAAT during boundary sedimentation velocity experiments in the presence of high concentrations of Hsc70, pmAAT was selectively labeled with 5-IAF at Cys-166 (42Berezov A. Iriarte A. Martinez-Carrion M. J. Biol. Chem. 1994; 269: 22222-22229Abstract Full Text PDF PubMed Google Scholar). Upon labeling, native pmAAT loses ∼50–60% of the original activity. This chromophore was selected because of its high molar extinction coefficient (75,000m−1 cm−1) at 492 nm which allowed the detection of small amounts of pmAAT during ultracentrifugation by absorption optics. When native AF-pmAAT is analyzed, it sed
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