Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit
2011; Elsevier BV; Volume: 286; Issue: 25 Linguagem: Inglês
10.1074/jbc.m111.237966
ISSN1083-351X
AutoresMichael Taylor, Tuhina Banerjee, Supriyo Ray, Suren A. Tatulian, Ken Teter,
Tópico(s)Transgenic Plants and Applications
ResumoProtein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin. Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin. IntroductionCholera toxin (CT) 2The abbreviations used are: CTcholera toxinCDcircular dichroismERendoplasmic reticulumERADER-associated degradationFTIRFourier transform infraredPTpertussis toxinPDIprotein-disulfide isomeraseSPRsurface plasmon resonance. is an AB5 protein toxin that consists of a catalytic A moiety and a cell-binding B moiety (1De Haan L. Hirst T.R. Mol. Membr. Biol. 2004; 21: 77-92Crossref PubMed Scopus (173) Google Scholar, 2Sánchez J. Holmgren J. Cell Mol. Life Sci. 2008; 65: 1347-1360Crossref PubMed Scopus (167) Google Scholar). The B subunit is pentameric ring-like structure that adheres to GM1 gangliosides on the plasma membrane of a target cell. The A subunit is initially synthesized as a 26 kDa protein that undergoes proteolytic nicking to generate a disulfide-linked A1/A2 heterodimer. The 21 kDa CTA1 polypeptide is an ADP-ribosyltransferase that modifies and activates Gsα in the host cell cytosol. CTA1 can be divided into three subdomains: the A11 subdomain contains the catalytic core of the toxin; the A12 subdomain is a short extended linker that connects the A11 and A13 subdomains; and the A13 subdomain is a globular structure with many hydrophobic residues as well as a cysteine residue involved with the single disulfide bridge between CTA1 and CTA2 (3Zhang R.G. Scott D.L. Westbrook M.L. Nance S. Spangler B.D. Shipley G.G. Westbrook E.M. J. Mol. Biol. 1995; 251: 563-573Crossref PubMed Scopus (297) Google Scholar). The 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the central pore of the B pentamer and thereby anchors CTA1 to CTB5. A ribbon diagram of the CT holotoxin which highlights the subdomain structure of CTA1 is provided in supplemental Fig. S1.To reach its cytosolic Gsα target, CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular traffic (4Wernick N.L. Chinnapen D.J. Cho J.A. Lencer W.I. Toxins. 2010; 2: 310-325Crossref PubMed Scopus (135) Google Scholar). A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4Wernick N.L. Chinnapen D.J. Cho J.A. Lencer W.I. Toxins. 2010; 2: 310-325Crossref PubMed Scopus (135) Google Scholar, 5Lencer W.I. Constable C. Moe S. Jobling M.G. Webb H.M. Ruston S. Madara J.L. Hirst T.R. Holmes R.K. J. Cell Biol. 1995; 131: 951-962Crossref PubMed Scopus (182) Google Scholar). Conditions in the ER lead to reductive cleavage of the CTA1/CTA2 disulfide bond and chaperone-assisted dissociation of CTA1 from CTA2/CTB5 (6Lencer W.I. de Almeida J.B. Moe S. Stow J.L. Ausiello D.A. Madara J.L. J. Clin. Invest. 1993; 92: 2941-2951Crossref PubMed Scopus (81) Google Scholar, 7Orlandi P.A. J. Biol. Chem. 1997; 272: 4591-4599Abstract Full Text Full Text PDF PubMed Google Scholar, 8Majoul I. Ferrari D. Söling H.D. FEBS Lett. 1997; 401: 104-108Crossref PubMed Scopus (67) Google Scholar, 9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Unfolding of the free A1 subunit then activates the quality control system of ER-associated degradation (ERAD), thereby promoting CTA1 translocation to the cytosol (10Teter K. Holmes R.K. Infect. Immun. 2002; 70: 6172-6179Crossref PubMed Scopus (69) Google Scholar, 11Teter K. Jobling M.G. Holmes R.K. Traffic. 2003; 4: 232-242Crossref PubMed Scopus (36) Google Scholar). Most exported ERAD substrates are degraded by the ubiquitin-proteasome system, but it was hypothesized that CTA1 and the A chains of other ER-translocating toxins avoid this fate because they lack substantial numbers of lysine residues for ubiquitin conjugation (12Hazes B. Read R.J. Biochemistry. 1997; 36: 11051-11054Crossref PubMed Scopus (277) Google Scholar). Subsequent experimental studies verified the paucity of lysine residues protects CTA1 and other toxin A chains from ubiquitin-dependent proteasomal degradation (13Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 14Deeks E.D. Cook J.P. Day P.J. Smith D.C. Roberts L.M. Lord J.M. Biochemistry. 2002; 41: 3405-3413Crossref PubMed Scopus (116) Google Scholar, 15Worthington Z.E. Carbonetti N.H. Infect Immun. 2007; 75: 2946-2953Crossref PubMed Scopus (36) Google Scholar). The translocated pool of CTA1 instead interacts with ADP-ribosylation factors and possibly other host factors to regain an active, folded conformation in the cytoplasm (16Murayama T. Tsai S.C. Adamik R. Moss J. Vaughan M. Biochemistry. 1993; 32: 561-566Crossref PubMed Scopus (40) Google Scholar, 17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 18Ampapathi R.S. Creath A.L. Lou D.I. Craft Jr., J.W. Blanke S.R. Legge G.B. J. Mol. Biol. 2008; 377: 748-760Crossref PubMed Scopus (36) Google Scholar).The reduced form of protein-disulfide isomerase (PDI), an ER-localized oxidoreductase and molecular chaperone (19Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (437) Google Scholar, 20Hatahet F. Ruddock L.W. FEBS J. 2007; 274: 5223-5234Crossref PubMed Scopus (123) Google Scholar), was originally proposed to unfold the holotoxin-associated CTA1 subunit and to thereby promote the separation of CTA1 from CTA2/CTB5 (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). It was further posited that unfolding of the CTA1 polypeptide was directly coupled with toxin delivery to the Derlin-1 pore (21Bernardi K.M. Forster M.L. Lencer W.I. Tsai B. Mol. Biol. Cell. 2008; 19: 877-884Crossref PubMed Scopus (85) Google Scholar, 22Moore P. Bernardi K.M. Tsai B. Mol. Biol. Cell. 2010; 21: 1305-1313Crossref PubMed Google Scholar). Release of CTA1 from PDI in preparation for translocation through the Derlin-1 pore was thought to involve the oxidation of PDI by Ero1p (23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar). Thus, PDI is viewed as a redox-dependent chaperone that binds to holotoxin-associated CTA1 in a reduced state, actively unfolds the toxin, and then releases the dissociated CTA1 subunit upon its oxidation by Ero1p.Many aspects of PDI-CTA1 interactions remain controversial. Lumb and Bulleid demonstrated that PDI does not act in a redox-dependent fashion when assisting the folding of other polypeptide chains (24Lumb R.A. Bulleid N.J. EMBO J. 2002; 21: 6763-6770Crossref PubMed Scopus (81) Google Scholar). Recent experiments using zebrafish and mammalian cells have questioned the role of Derlin-1 in CTA1 translocation (25Saslowsky D.E. Cho J.A. Chinnapen H. Massol R.H. Chinnapen D.J. Wagner J.S. De Luca H.E. Kam W. Paw B.H. Lencer W.I. J. Clin. Invest. 2010; 120: 4399-4409Crossref PubMed Scopus (62) Google Scholar). Our structural studies have indicated that CTA1 will unfold spontaneously after dissociation from the holotoxin at physiological temperature (17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar, 27Banerjee T. Pande A. Jobling M.G. Taylor M. Massey S. Holmes R.K. Tatulian S.A. Teter K. Biochemistry. 2010; 49: 8839-8846Crossref PubMed Scopus (23) Google Scholar). It is therefore possible that, in terms of cholera intoxication, the primary function of PDI is to simply dislodge CTA1 from CTA2/CTB5. The dissociated CTA1 polypeptide would then spontaneously unfold and consequently trigger the ERAD translocation mechanism.In this work we evaluated the role of PDI in disassembly of the CT holotoxin and unfolding of the CTA1 subunit. The prevailing model of PDI-toxin interactions is largely based upon the results of a protease sensitivity assay which demonstrated that CTA1 shifts from a protease-resistant conformation to a protease-sensitive conformation in the presence of reduced PDI (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 13Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar, 28Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (99) Google Scholar, 29Forster M.L. Mahn J.J. Tsai B. J. Biol. Chem. 2009; 284: 13045-13056Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). As folded proteins are generally more resistant to proteolysis than unfolded variants of the same protein, this shift was interpreted to represent the PDI-induced unfolding of CTA1 and the PDI-induced displacement of CTA1 from CTA2/CTB5. Yet this assay, which was typically performed at 30 °C, only provides an indirect measure of protein folding. Here, we developed a surface plasmon resonance (SPR) assay to directly monitor in real time the disassembly of the CT holotoxin. Biophysical methods were used to directly examine the conformation of CTA1 in the presence or absence of PDI. Cell-based assays further probed the role of PDI in CT intoxication and CTA1 translocation. Our collective data indicate that PDI does not unfold the CTA1 polypeptide and that PDI can displace CTA1 from CTA2/CTB5 in a process which does not involve substantial alterations to the structure of CTA1. In further contrast with the current model of PDI-toxin interactions where PDI acts as an unfoldase, we found that the PDI-induced shift of CTA1 to a protease-sensitive conformation does not correlate to the disassembly of the CT holotoxin. Moreover, the release of PDI from CTA1 does not require Ero1p but instead results from the spontaneous unfolding of CTA1 which occurs after its dissociation from the holotoxin. The unstable nature of the isolated CTA1 polypeptide at physiological temperature thus plays a central role in toxin translocation and leads to a new model of CT disassembly in the ER.DISCUSSIONFor CTA1 translocation, PDI has been proposed to act as a redox-dependent chaperone that actively unfolds the holotoxin-associated CTA1 subunit to separate CTA1 from the rest of the toxin (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). The unfoldase activity of PDI in this process is, to the best of our knowledge, a unique property that has not been reported for PDI interactions with any other substrate. Furthermore, the proposed unfoldase activity of PDI is based upon a protease sensitivity assay that only serves as an indirect measure of protein folding (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 13Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar, 28Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (99) Google Scholar, 29Forster M.L. Mahn J.J. Tsai B. J. Biol. Chem. 2009; 284: 13045-13056Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In this work, we demonstrated that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit. CTA1 unfolds spontaneously after its dissociation from CTA2/CTB5 at physiological temperature (17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar). PDI-mediated disassembly of the CT holotoxin thus leads to the spontaneous unfolding of dissociated CTA1, but PDI itself does not function as an unfoldase to actively unfold CTA1.Using SPR, we were able to monitor the PDI-mediated disassembly of the CT holotoxin in real time. The kinetics of holotoxin disassembly were linked to the available concentration of PDI (supplemental Fig. S2). Holotoxin disassembly required the reduced form of PDI and did not occur in the presence of reducing agent alone (Fig. 1). These results were consistent with previous reports (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 36Mekalanos J.J. Collier R.J. Romig W.R. J. Biol. Chem. 1979; 254: 5855-5861Abstract Full Text PDF PubMed Google Scholar), as was the observed retention of CTA2 with CTB5 after release of CTA1 from the holotoxin (Fig. 1B) (46Wernick N.L. De Luca H. Kam W.R. Lencer W.I. J. Biol. Chem. 2010; 285: 6145-6152Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We could thus faithfully reconstitute the process of CT disassembly on an SPR sensor slide.Conditions that block CTA1 unfolding in vitro also block the ER-to-cytosol export of CTA1 in vivo (26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar, 27Banerjee T. Pande A. Jobling M.G. Taylor M. Massey S. Holmes R.K. Tatulian S.A. Teter K. Biochemistry. 2010; 49: 8839-8846Crossref PubMed Scopus (23) Google Scholar). However, conditions that block CTA1 unfolding do not block CTA1 dissociation from the CT holotoxin: reduced PDI could separate CTA1 from CTA2/CTB5 in the presence of acidic pH (Fig. 4A) or 10% glycerol (26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar). Secretion of the free A1 subunit from glycerol-treated cells (26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar) further suggested that in vivo unfolding of CTA1 is unnecessary for toxin disassembly: only folded proteins exit the ER for secretory transport, so the secreted pool of CTA1 must have been in a folded conformation. CT disassembly could also occur at 4 °C (supplemental Fig. S4), a temperature that maintains CTA1 in a folded conformation and prevents the PDI-induced shift of CTA1 to a protease-resistant conformation (29Forster M.L. Mahn J.J. Tsai B. J. Biol. Chem. 2009; 284: 13045-13056Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Collectively, these results strongly suggested that CTA1 unfolding is not required for CTA1 dissociation from the rest of the toxin. In further support of this interpretation, we demonstrated that the thermal denaturation of reduced but holotoxin-associated CTA1 did not displace CTA1 from its non-covalent assembly in the CT holotoxin (supplemental Fig. S5). A similar observation was previously made by Goins and Friere with the technique of differential scanning calorimetry (37Goins B. Freire E. Biochemistry. 1988; 27: 2046-2052Crossref PubMed Scopus (67) Google Scholar). Thus, PDI apparently removes CTA1 from CTA2/CTB5 by a mechanism that does not involve unfolding of the holotoxin-associated CTA1 subunit.To directly examine the putative unfoldase activity of PDI, we employed the structural technique of isotope-edited FTIR spectroscopy. Experiments were performed under three conditions that facilitate PDI-CTA1 interactions and support PDI-mediated disassembly of the CT holotoxin: 10 °C, 30 °C, and 37 °C at pH 6.5. For each condition, deconvolution of the conformation-sensitive amide I bands demonstrated that PDI did not substantially alter the percentage of irregular structure in CTA1 and, thus, did not unfold the CTA1 polypeptide (Table 1). In contrast, heating CTA1 alone at neutral pH shifted the percentage of its irregular structure from 8% at 10 °C to 53% at 37 °C at the expense of both α-helix and β-sheet structures (Table 1). CT disassembly therefore occurs under conditions that do not involve substantial PDI-induced disordering of the CTA1 polypeptide. These results, which represent the first direct examination of the impact of PDI binding on CTA1 structure, demonstrated that CTA1 is unfolded by physiological temperature (due to its intrinsic thermal instability) but not by PDI.The unfoldase activity of PDI has only been described with a biochemical protease sensitivity assay. Using this method, the folding state of a protein is inferred from its susceptibility to proteolysis: a folded protein is generally more resistant to proteolysis than an unfolded variant of the same protein. The resistance of CTA1 to trypsin-mediated proteolysis, combined with the sensitivity of PDI-treated CTA1 to proteolysis, thus yielded the conclusion that PDI actively unfolds the CTA1 subunit. Unfortunately, interpretation of these experiments is complicated by the conformational shift in CTA1 that occurs upon its separation from the holotoxin and by the unstable, heat-labile nature of the free A1 subunit (17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar). Protease concentration can also affect the outcome of the experiment, as can the temperature of the experiment (17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar, 28Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (99) Google Scholar). It should be noted that most experiments to monitor the "unfoldase" activity of PDI have been performed at 30 °C (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 13Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar, 29Forster M.L. Mahn J.J. Tsai B. J. Biol. Chem. 2009; 284: 13045-13056Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), but incubation of CTA1 alone at 37 °C will induce the toxin to assume a protease-sensitive conformation (17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar) (supplemental Fig. S10). Furthermore, the sensitivity of PDI-treated CTA1 to proteolysis depends upon the protease used: the toxin is more sensitive to trypsin (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar) than to the thermolysin protease which cleaves bulky and aromatic residues (supplemental Fig. S10). These issues suggest that protease sensitivity is a poor, indirect measure of the purported conformational change required for CTA1 dissociation from the CT holotoxin. More important, however, is the disconnect between the PDI-induced shift of CTA1 to a trypsin-sensitive conformation and the PDI-mediated disassembly of the CT holotoxin. PDI does not induce CTA1 to assume a protease-sensitive conformation at 4 °C (29Forster M.L. Mahn J.J. Tsai B. J. Biol. Chem. 2009; 284: 13045-13056Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), yet PDI can still displace CTA1 from CTA2/CTB5 at 4 °C (supplemental Fig. S4). These results demonstrate that the PDI-induced shift of CTA1 to a protease-sensitive conformation, which has been interpreted as an unfolding event (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 13Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar, 28Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (99) Google Scholar, 29Forster M.L. Mahn J.J. Tsai B. J. Biol. Chem. 2009; 284: 13045-13056Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), does not correlate to the PDI-induced separation of CTA1 from CTA2/CTB5.PDI binds to folded but not unfolded conformations of CTA1 (FIGURE 1, FIGURE 2, FIGURE 3). The spontaneous unfolding of CTA1 that occurs upon holotoxin disassembly at 37 °C thus displaces the toxin-bound PDI (FIGURE 3, FIGURE 5). This observation is consistent with a previous report that concluded PDI-substrate interactions can be disrupted by changes to the structure of the substrate (24Lumb R.A. Bulleid N.J. EMBO J. 2002; 21: 6763-6770Crossref PubMed Scopus (81) Google Scholar). However, our results conflict with the model of CTA1-PDI interactions that proposes PDI oxidation by Ero1p is responsible for displacing toxin-bound PDI (23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar). That model is based upon studies performed at temperatures below 37 °C. While Ero1p clearly regulates the redox status of PDI (22Moore P. Bernardi K.M. Tsai B. Mol. Biol. Cell. 2010; 21: 1305-1313Crossref PubMed Google Scholar, 23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar, 47Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 48Inaba K. Masui S. Iida H. Vavassori S. Sitia R. Suzuki M. EMBO J. 2010; 29: 3330-3343Crossref PubMed Scopus (97) Google Scholar), our results strongly suggest that Ero1p function is not required to release PDI from CTA1 at physiological temperature.As demonstrated with cell-based assays, CTA1 translocation does not require PDI function after disassembly of the holotoxin. PDI-deficient cell lines were completely resistant to exogenously applied CT, as expected from its essential role in separating CTA1 from CTA2/CTB5. However, the same PDI-deficient cells displayed wild-type sensitivity to a CTA1 construct that was directly expressed in the ER of transfected cells. This experimental condition, which mimicked the status of ER-localized CTA1 after its dissociation from the holotoxin, demonstrated that PDI is not required for the ER-to-cytosol export of free CTA1. Direct monitoring of the CTA1 translocation event provided further support for this conclusion.Based on the available biophysical, biochemical, and cell biological data, we propose the following revised model of CTA1 translocation. CT travels from the cell surface to the ER as an intact holotoxin (4Wernick N.L. Chinnapen D.J. Cho J.A. Lencer W.I. Toxins. 2010; 2: 310-325Crossref PubMed Scopus (135) Google Scholar). The disulfide bond linking CTA1 to CTA2/CTB5 is reduced at the resident redox state of the ER (8Majoul I. Ferrari D. Söling H.D. FEBS Lett. 1997; 401: 104-108Crossref PubMed Scopus (67) Google Scholar). However, at this point CTA1 remains anchored to the holotoxin through non-covalent interactions (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 36Mekalanos J.J. Collier R.J. Romig W.R. J. Biol. Chem. 1979; 254: 5855-5861Abstract Full Text PDF PubMed Google Scholar). Reduced PDI then binds to a region in the A11 subdomain of CTA1 and physically displaces CTA1 from the holotoxin. Released from the structural constraints of its non-covalent interactions with CTA2/CTB5, the dissociated CTA1 subunit undergoes a thermal transition to a partially unfolded conformation (17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar). This unfolding event, which occurs spontaneously at 37 °C, displaces the toxin-bound PDI and identifies free CTA1 as a misfolded protein for processing by the ERAD system. CTA1 is consequently delivered to the Sec61p and/or Derlin-1 pores for Hsp90-mediated extraction to the cytosol (21Bernardi K.M. Forster M.L. Lencer W.I. Tsai B. Mol. Biol. Cell. 2008; 19: 877-884Crossref PubMed Scopus (85) Google Scholar, 25Saslowsky D.E. Cho J.A. Chinnapen H. Massol R.H. Chinnapen D.J. Wagner J.S. De Luca H.E. Kam W. Paw B.H. Lencer W.I. J. Clin. Invest. 2010; 120: 4399-4409Crossref PubMed Scopus (62) Google Scholar, 34Taylor M. Navarro-Garcia F. Huerta J. Burress H. Massey S. Ireton K. Teter K. J. Biol. Chem. 2010; 285: 31261-31267Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 49Schmitz A. Herrgen H. Winkeler A. Herzog V. J. Cell Biol. 2000; 148: 1203-1212Crossref PubMed Scopus (174) Google Scholar, 50Dixit G. Mikoryak C. Hayslett T. Bhat A. Draper R.K. Exp. Biol. Med. 2008; 233: 163-175Crossref PubMed Scopus (48) Google Scholar). An interaction with ARF and possibly other host factors then allows the cytosolic pool of CTA1 to regain an active conformation for the ADP-ribosylation of its Gsα target (16Murayama T. Tsai S.C. Adamik R. Moss J. Vaughan M. Biochemistry. 1993; 32: 561-566Crossref PubMed Scopus (40) Google Scholar, 17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 18Ampapathi R.S. Creath A.L. Lou D.I. Craft Jr., J.W. Blanke S.R. Legge G.B. J. Mol. Biol. 2008; 377: 748-760Crossref PubMed Scopus (36) Google Scholar).Toxin-ERAD interactions involving pertussis toxin (PT) and ricin may follow a similar pattern to CT. Like CTA1, the catalytic PTS1 subunit shifts to a disordered conformation after separation from its B oligomer at physiological temperature (51Pande A.H. Moe D. Jamnadas M. Tatulian S.A. Teter K. Biochemistry. 2006; 45: 13734-13740Crossref PubMed Scopus (32) Google Scholar). This conformational shift would identify PTS1 as an ERAD substrate and thereby facilitate PTS1 passage into the cytosol. Ricin A chain is more stable than either CTA1 or PTS1 (52Argent R.H. Parrott A.M. Day P.J. Roberts L.M. Stockley P.G. Lord J.M. Radford S.E. J. Biol. Chem. 2000; 275: 9263-9269Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), but an interaction with anionic phospholipids of the ER membrane at 37 °C induces ricin A chain to assume a disordered conformation that would be treated as an ERAD substrate (53Day P.J. Pinheiro T.J. Roberts L.M. Lord J.M. Biochemistry. 2002; 41: 2836-2843Crossref PubMed Scopus (65) Google Scholar, 54Mayerhofer P.U. Cook J.P. Wahlman J. Pinheiro T.T. Moore K.A. Lord J.M. Johnson A.E. Roberts L.M. J. Biol. Chem. 2009; 284: 10232-10242Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Furthermore, the interaction between PDI and ricin mirrors the interaction between PDI and CT: PDI is responsible for disassembly of the ricin holotoxin but does not unfold ricin A chain (55Bellisola G. Fracasso G. Ippoliti R. Menestrina G. Rosén A. Soldà S. Udali S. Tomazzolli R. Tridente G. Colombatti M. Biochem. Pharmacol. 2004; 67: 1721-1731Crossref PubMed Scopus (69) Google Scholar, 56Spooner R.A. Watson P.D. Marsden C.J. Smith D.C. Moore K.A. Cook J.P. Lord J.M. Roberts L.M. Biochem. J. 2004; 383: 285-293Crossref PubMed Scopus (120) Google Scholar). Our results are thus consistent with a general model of toxin translocation in which PDI-mediated toxin disassembly and temperature-induced A chain unfolding are used in similar fashion by multiple ERAD-exploiting toxins. IntroductionCholera toxin (CT) 2The abbreviations used are: CTcholera toxinCDcircular dichroismERendoplasmic reticulumERADER-associated degradationFTIRFourier transform infraredPTpertussis toxinPDIprotein-disulfide isomeraseSPRsurface plasmon resonance. is an AB5 protein toxin that consists of a catalytic A moiety and a cell-binding B moiety (1De Haan L. Hirst T.R. Mol. Membr. Biol. 2004; 21: 77-92Crossref PubMed Scopus (173) Google Scholar, 2Sánchez J. Holmgren J. Cell Mol. Life Sci. 2008; 65: 1347-1360Crossref PubMed Scopus (167) Google Scholar). The B subunit is pentameric ring-like structure that adheres to GM1 gangliosides on the plasma membrane of a target cell. The A subunit is initially synthesized as a 26 kDa protein that undergoes proteolytic nicking to generate a disulfide-linked A1/A2 heterodimer. The 21 kDa CTA1 polypeptide is an ADP-ribosyltransferase that modifies and activates Gsα in the host cell cytosol. CTA1 can be divided into three subdomains: the A11 subdomain contains the catalytic core of the toxin; the A12 subdomain is a short extended linker that connects the A11 and A13 subdomains; and the A13 subdomain is a globular structure with many hydrophobic residues as well as a cysteine residue involved with the single disulfide bridge between CTA1 and CTA2 (3Zhang R.G. Scott D.L. Westbrook M.L. Nance S. Spangler B.D. Shipley G.G. Westbrook E.M. J. Mol. Biol. 1995; 251: 563-573Crossref PubMed Scopus (297) Google Scholar). The 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the central pore of the B pentamer and thereby anchors CTA1 to CTB5. A ribbon diagram of the CT holotoxin which highlights the subdomain structure of CTA1 is provided in supplemental Fig. S1.To reach its cytosolic Gsα target, CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular traffic (4Wernick N.L. Chinnapen D.J. Cho J.A. Lencer W.I. Toxins. 2010; 2: 310-325Crossref PubMed Scopus (135) Google Scholar). A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4Wernick N.L. Chinnapen D.J. Cho J.A. Lencer W.I. Toxins. 2010; 2: 310-325Crossref PubMed Scopus (135) Google Scholar, 5Lencer W.I. Constable C. Moe S. Jobling M.G. Webb H.M. Ruston S. Madara J.L. Hirst T.R. Holmes R.K. J. Cell Biol. 1995; 131: 951-962Crossref PubMed Scopus (182) Google Scholar). Conditions in the ER lead to reductive cleavage of the CTA1/CTA2 disulfide bond and chaperone-assisted dissociation of CTA1 from CTA2/CTB5 (6Lencer W.I. de Almeida J.B. Moe S. Stow J.L. Ausiello D.A. Madara J.L. J. Clin. Invest. 1993; 92: 2941-2951Crossref PubMed Scopus (81) Google Scholar, 7Orlandi P.A. J. Biol. Chem. 1997; 272: 4591-4599Abstract Full Text Full Text PDF PubMed Google Scholar, 8Majoul I. Ferrari D. Söling H.D. FEBS Lett. 1997; 401: 104-108Crossref PubMed Scopus (67) Google Scholar, 9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Unfolding of the free A1 subunit then activates the quality control system of ER-associated degradation (ERAD), thereby promoting CTA1 translocation to the cytosol (10Teter K. Holmes R.K. Infect. Immun. 2002; 70: 6172-6179Crossref PubMed Scopus (69) Google Scholar, 11Teter K. Jobling M.G. Holmes R.K. Traffic. 2003; 4: 232-242Crossref PubMed Scopus (36) Google Scholar). Most exported ERAD substrates are degraded by the ubiquitin-proteasome system, but it was hypothesized that CTA1 and the A chains of other ER-translocating toxins avoid this fate because they lack substantial numbers of lysine residues for ubiquitin conjugation (12Hazes B. Read R.J. Biochemistry. 1997; 36: 11051-11054Crossref PubMed Scopus (277) Google Scholar). Subsequent experimental studies verified the paucity of lysine residues protects CTA1 and other toxin A chains from ubiquitin-dependent proteasomal degradation (13Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 14Deeks E.D. Cook J.P. Day P.J. Smith D.C. Roberts L.M. Lord J.M. Biochemistry. 2002; 41: 3405-3413Crossref PubMed Scopus (116) Google Scholar, 15Worthington Z.E. Carbonetti N.H. Infect Immun. 2007; 75: 2946-2953Crossref PubMed Scopus (36) Google Scholar). The translocated pool of CTA1 instead interacts with ADP-ribosylation factors and possibly other host factors to regain an active, folded conformation in the cytoplasm (16Murayama T. Tsai S.C. Adamik R. Moss J. Vaughan M. Biochemistry. 1993; 32: 561-566Crossref PubMed Scopus (40) Google Scholar, 17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 18Ampapathi R.S. Creath A.L. Lou D.I. Craft Jr., J.W. Blanke S.R. Legge G.B. J. Mol. Biol. 2008; 377: 748-760Crossref PubMed Scopus (36) Google Scholar).The reduced form of protein-disulfide isomerase (PDI), an ER-localized oxidoreductase and molecular chaperone (19Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (437) Google Scholar, 20Hatahet F. Ruddock L.W. FEBS J. 2007; 274: 5223-5234Crossref PubMed Scopus (123) Google Scholar), was originally proposed to unfold the holotoxin-associated CTA1 subunit and to thereby promote the separation of CTA1 from CTA2/CTB5 (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). It was further posited that unfolding of the CTA1 polypeptide was directly coupled with toxin delivery to the Derlin-1 pore (21Bernardi K.M. Forster M.L. Lencer W.I. Tsai B. Mol. Biol. Cell. 2008; 19: 877-884Crossref PubMed Scopus (85) Google Scholar, 22Moore P. Bernardi K.M. Tsai B. Mol. Biol. Cell. 2010; 21: 1305-1313Crossref PubMed Google Scholar). Release of CTA1 from PDI in preparation for translocation through the Derlin-1 pore was thought to involve the oxidation of PDI by Ero1p (23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar). Thus, PDI is viewed as a redox-dependent chaperone that binds to holotoxin-associated CTA1 in a reduced state, actively unfolds the toxin, and then releases the dissociated CTA1 subunit upon its oxidation by Ero1p.Many aspects of PDI-CTA1 interactions remain controversial. Lumb and Bulleid demonstrated that PDI does not act in a redox-dependent fashion when assisting the folding of other polypeptide chains (24Lumb R.A. Bulleid N.J. EMBO J. 2002; 21: 6763-6770Crossref PubMed Scopus (81) Google Scholar). Recent experiments using zebrafish and mammalian cells have questioned the role of Derlin-1 in CTA1 translocation (25Saslowsky D.E. Cho J.A. Chinnapen H. Massol R.H. Chinnapen D.J. Wagner J.S. De Luca H.E. Kam W. Paw B.H. Lencer W.I. J. Clin. Invest. 2010; 120: 4399-4409Crossref PubMed Scopus (62) Google Scholar). Our structural studies have indicated that CTA1 will unfold spontaneously after dissociation from the holotoxin at physiological temperature (17Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 26Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar, 27Banerjee T. Pande A. Jobling M.G. Taylor M. Massey S. Holmes R.K. Tatulian S.A. Teter K. Biochemistry. 2010; 49: 8839-8846Crossref PubMed Scopus (23) Google Scholar). It is therefore possible that, in terms of cholera intoxication, the primary function of PDI is to simply dislodge CTA1 from CTA2/CTB5. The dissociated CTA1 polypeptide would then spontaneously unfold and consequently trigger the ERAD translocation mechanism.In this work we evaluated the role of PDI in disassembly of the CT holotoxin and unfolding of the CTA1 subunit. The prevailing model of PDI-toxin interactions is largely based upon the results of a protease sensitivity assay which demonstrated that CTA1 shifts from a protease-resistant conformation to a protease-sensitive conformation in the presence of reduced PDI (9Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 13Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 23Tsai B. Rapoport T.A. J. Cell Biol. 2002; 159: 207-216Crossref PubMed Scopus (124) Google Scholar, 28Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (99) Google Scholar, 29Forster M.L. Mahn J.J. Tsai B. J. Biol. Chem. 2009; 284: 13045-13056Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). As folded proteins are generally more resistant to proteolysis than unfolded variants of the same protein, this shift was interpreted to represent the PDI-induced unfolding of CTA1 and the PDI-induced displacement of CTA1 from CTA2/CTB5. Yet this assay, which was typically performed at 30 °C, only provides an indirect measure of protein folding. Here, we developed a surface plasmon resonance (SPR) assay to directly monitor in real time the disassembly of the CT holotoxin. Biophysical methods were used to directly examine the conformation of CTA1 in the presence or absence of PDI. Cell-based assays further probed the role of PDI in CT intoxication and CTA1 translocation. Our collective data indicate that PDI does not unfold the CTA1 polypeptide and that PDI can displace CTA1 from CTA2/CTB5 in a process which does not involve substantial alterations to the structure of CTA1. In further contrast with the current model of PDI-toxin interactions where PDI acts as an unfoldase, we found that the PDI-induced shift of CTA1 to a protease-sensitive conformation does not correlate to the disassembly of the CT holotoxin. Moreover, the release of PDI from CTA1 does not require Ero1p but instead results from the spontaneous unfolding of CTA1 which occurs after its dissociation from the holotoxin. The unstable nature of the isolated CTA1 polypeptide at physiological temperature thus plays a central role in toxin translocation and leads to a new model of CT disassembly in the ER.
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