Report |
Address correspondence to Tom A. Rapoport, Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115-6091. Tel.: (617) 432-0637. Fax: (617) 432-1190. E-mail: tom_rapoport{at}hms.harvard.edu
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Abstract |
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Key Words: PDI; Ero1; cholera toxin; retrotranslocation; oxidation
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Introduction |
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Unfolding of the A1 peptide likely represents the first step in retrotranslocation of the toxin. Using a biochemical fractionation approach that made no assumptions about the nature of this unfolding activity, we previously identified the ER oxido-reductase protein disulfide isomerase (PDI) as the major activity that disassembles the toxin and unfolds the A1 chain (Tsai et al., 2001). More detailed analysis demonstrated that PDI acts as a redox-dependent chaperone; in its reduced state, PDI binds and unfolds the toxin, whereas in its oxidized state, PDI releases it. Release of the A1 chain from PDI upon oxidation must occur prior to its retrotranslocation across the ER membrane. When oxidation is induced with oxidized glutathione (GSSG), an unphysiologically high concentration was required to induce release (Tsai et al., 2001). Therefore, we hypothesized that this process must normally be catalyzed by an enzyme, i.e., an oxidase of PDI.
Here we identify the enzyme responsible for the release reaction, provide a mechanism for the release, and describe an additional step in retrotranslocation of the toxin. Our data demonstrate that the ER oxidase Ero1 is responsible for inducing release of the toxin from reduced PDI through oxidation of the COOH-terminal disulfide bond in PDI. Furthermore, we show that the complex of PDI and unfolded toxin is targeted to a protein on the lumenal side of the ER membrane. Subsequently, the toxin is released from PDI by the action of Ero1, presumably committing the toxin to retrotranslocation across the ER membrane.
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Results and discussion |
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Using this assay, we found that a PDIA chain complex can be seen with purified PDI, but not with ER extract or BSA (Fig. 1 B, compare lane 3 with lanes 2 and 1; Tsai et al., 2001). Together with the unfolding assay, these results suggest that the toxin must be undergoing cycles of binding and release from PDI when incubated with ER extract, thereby preventing the formation of a stable PDItoxin complex that can be captured by the crosslinker.
To directly test the possibility that a release activity exists in the ER extract, we first generated the PDIA chain complex by incubating PDI and purified toxin together under reducing conditions. BSA or ER extract were then added to the preformed complex to induce toxin release, and the dissociation of the complex was probed with a bifunctional carbodiimide crosslinker. Indeed, addition of ER extract, but not BSA, resulted in loss of the crosslinked product (Fig. 1 C, compare lane 2 with lane 1). This result confirms the presence of an activity in the ER extract that induces the release of the A1 chain from PDI. Because addition of high concentrations of the oxidant GSSG can also induce toxin release (Tsai et al., 2001), the release activity is likely to be an oxidase of PDI.
Ero1 induces the release of unfolded toxin from reduced PDI
Using two different genetic screens in Saccharomyces cerevisiae, Ero1 was identified as an enzyme that functions as a PDI oxidase in the ER lumen (Frand and Kaiser, 1998, 1999; Pollard et al., 1998). Ero1 contains the cofactor FAD and may use molecular oxygen to reoxidize itself (Tu et al., 2000). We asked whether the mammalian homologue of Ero1 may be responsible for the release activity in the ER extract. In mammals, two different Ero1 isoforms have been identified, Ero1 (Cabibbo et al., 2000) and Ero1ß (Pagani et al., 2000). To test whether both isoforms are present in dog microsomes, three different mammalian Ero1 antibodies were used, one that recognizes both mammalian Ero1 isoforms (
/ß, antibody 194), one that only recognizes the Ero1
isoform, and one that only recognizes the Ero1ß isoform. The Ero1
/ß antibody recognized three distinct bands in the microsomes, with the top band corresponding to Ero1
, the middle band corresponding to Ero1ß, and the bottom band corresponding possibly to a novel Ero1 isoform or a degradation product (Fig. 2 A, lane 1). The slower mobility of Ero1
compared to Ero1ß is consistent with previous results (Pagani et al., 2000). Treatment of microsomes with endoglycosidase H (Endo H) increased the mobility of all three bands, confirming previous findings that Ero1
and Ero1ß are glycoproteins (Fig. 2 A, lane 2; Pagani et al., 2000). The Ero1
- and Ero1ß-specific antibodies each recognized only one Endo Hsensitive band in the microsomes (Fig. 2 A, lanes 3 and 5, respectively), which correspond to the positions of the top and middle bands identified using the Ero1
/ß antibody, respectively. An additional Endo Hresistant band was observed using the Ero1
-specific antibody (Fig. 2 A, lane 4), which may represent an uncharacterized glycosylated form of Ero1
.
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We next used the antibodies to directly test whether the release activity observed in the crude ER extract can be attributed to Ero1. Both isoforms of Ero1 were present in the extract obtained with low detergent concentrations. To deplete Ero1 and Ero1ß we used two different antibodies directed against both proteins (antibodies 193 and 194; Fig. 2 B, compare lanes 2 and 3 with lane 1; lanes 5 and 6 with lane 4; and lanes 8 and 9 with lane 7). Although addition of ER extract to the preformed PDI-toxin complex induced loss of the crosslinked product, addition of either of the two Ero1-depleted extracts did not (Fig. 2 B, compare lane 11 with lanes 12 and 13). In fact, with one antibody the crosslinked band was more intense than in the control, suggesting that in the absence of Ero1, the extract is more reducing than 1 mM GSH, perhaps due to the presence of a PDI-reductase. These results demonstrate that Ero1 is required for the release activity. As previously reported, addition of a high concentration of GSSG (30 mM) also resulted in the loss of the crosslinked product (Fig. 2 B, compare lane 14 with lane 10; Tsai et al., 2001).
We next asked whether Ero1 is sufficient for stimulating toxin release. Addition of purified Ero1, either in substoichiometric (1:10) or stoichiometric (1:1) ratio to PDI, to the preformed PDI-toxin complex resulted in the disappearance of the crosslinked product (Fig. 2 C, compare lanes 2 and 3 with lane 1). The same result was obtained upon addition of purified yeast Ero1 (Fig. 2 C, lane 4), either in substoichiometric (1:10) or near-stoichiometric (1:2) ratio to PDI (Fig. 2 C, compare lanes 6 and 7 with lane 5). We conclude that Ero1 is both necessary and sufficient to induce release of the toxin from PDI.
We also separated the ER extract on an ion exchange column (Q-Sepharose) and tested each fraction for its content of Ero1 and its activity to release the toxin from reduced PDI. Two pools of Ero1 were found using the Ero1/ß antibody, one pool corresponding to fractions 59 (Fig. 3 A, top, lanes 15), identified as Ero1
(Fig. 3 A, middle, lanes 15), and another corresponding to fractions 1721 (Fig. 3 A, top, lanes 1317), identified as Ero1ß (Fig. 3 A, bottom, lanes 1317). Only fractions 59 were active in the release assay (Fig. 3 B, compare lanes 26 with lanes 1418). These data confirm that Ero1
is able to induce toxin release from PDI and raise the possibility that Ero1ß may be inactive in this assay. Interestingly, based on the theoretical pI values of the two isoforms, Ero1ß is expected to elute before Ero1
, in contrast to the observation. It seems therefore likely that interactions with other proteins alter the properties of the isoforms. A novel ER protein, called Erp44, interacts with both Ero1 isoforms and is therefore unlikely to be responsible for the anomalous behavior of the two proteins (Anelli et al., 2002).
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To further establish that Ero1 can oxidize only one of PDI's two disulfide bonds, a tenfold excess of yeast Ero1 was added to reduced PDI. Again, only one of PDI's two disulfide bonds was oxidized (Fig. 4 C, compare lane 2 with lane 1). To determine whether Ero1 oxidizes the disulfide bond in the NH2- or COOH-terminal thioredoxin domain of PDI, we used purified mutants of yeast PDI in which the CxxC motif in either the NH2- or COOH-terminal domain is mutated to AxxA (PDIAxxA-CxxC and PDICxxC-AxxA,, respectively; Fig. 4 D, lanes 1 and 2). Using a tenfold excess of yeast Ero1 over PDI, we found that only the PDIAxxA-CxxC mutant was oxidized by Ero1 (Fig. 4 D, compare lane 6 with lane 8). Thus, Ero1 oxidizes only the disulfide bond in the carboxy-terminal thioredoxin domain.
Interestingly, addition of substoichiometric amounts of Ero1 or yeast Ero1 did not alter the mobility shift of PDI (Fig. 4, A, compare lane 3 with lane 2, B, compare lane 4 with lane 3), indicating that this amount of Ero1 is insufficient to oxidize the bulk of PDI. However, under the same conditions, both Ero1
and yeast Ero1 were able to fully induce release of toxin from PDI (Fig. 2 C, compare lane 2 with lane 1; compare lane 6 with lane 5). One explanation is that Ero1 preferentially binds to and oxidizes the PDItoxin complex. Because the concentration of the toxin is much lower than that of PDI, oxidation of complexed PDI would not be visible in the modification assay. Indeed, when we added a stoichiometric concentration of toxin with respect to PDI, a mobility shift of PDI could be observed even at low concentrations of Ero1 (Fig. 4 E, compare lane 6 with lane 2). The band that runs between the ones corresponding to four and six free thiol groups may perhaps be caused by the formation of a mixed disulfide bond with either the toxin or GSH. Taken together, these data support the idea that Ero1 preferentially acts on the PDI-toxin complex.
PDI-unfolded toxin is transferred to the ER membrane
During retrotranslocation of the A1 chain, the unfolded A1 peptide must be targeted to the ER membrane and ultimately engage the translocation channel in order to be transported to the cytosol. We designed an assay to examine transfer of the unfolded A1 chain to the ER membrane, a process that likely mimics an intermediate step in retrotranslocation of the toxin. Purified A subunit of the toxin was initially incubated with ER extract under reducing conditions to stimulate toxin unfolding. Then proteoliposomes were added which were generated by dissolving microsomes in detergent followed by removal of the detergent with hydrophobic beads. These proteoliposomes contain essentially all ER membrane proteins with their orientations randomized. This should allow the PDItoxin complex to interact with lumenal domains of membrane proteins. To assay for binding of the PDItoxin complex, the proteoliposomes were sedimented and the pellet and supernatant fractions analyzed by immunoblotting with toxin antibodies.
Although addition of ER extract or proteoliposomes alone did not induce transfer of the A1 peptide to the pellet fraction, addition of ER extract followed by proteoliposomes caused a fraction of the A1 peptide (30%) to be transferred to the pellet (Fig. 5 A, compare lanes 1 and 3 with lane 5). Neither liposomes lacking proteins nor intact microsomes (which do not have the lumenal domains of membrane proteins exposed) stimulated toxin transfer (Fig. 5 A, compare lanes 7 and 9 with lane 11), indicating that the unfolded A1 chain does not bind to lipids or irrelevant proteins. This is supported by the fact that proteoliposomes generated from a proteinase K-treated detergent extract did not show binding activity (Fig. 5 A, compare lanes 15 and 13). Thus, it appears that the unfolded A1 peptide specifically binds to a protein at the lumenal side of the ER membrane. An unfolded state of the A1 peptide is insufficient to achieve binding to the ER membrane as demonstrated with A chains that were chemically unfolded with urea. The urea-treated A and A1 peptides were as sensitive to trypsin treatment as the A1 peptide unfolded with an ER extract (Fig. 5 B, compare lanes 6 and 4 with lane 2), but they did not bind to proteoliposomes (Fig. 5 B, compare lane 7 with lane 9). Thus, it appears that the formation of a PDItoxin complex is necessary for transfer to the ER membrane, perhaps because PDI specifically interacts with a membrane protein. To further test this point, we asked whether PDI is required for the transfer reaction by using ER extract immunodepleted of PDI (Fig. 5 C, compare lane 3 with lane 2). Indeed, the PDI-depleted extract did not support toxin transfer to the membrane (Fig. 5 C, compare lane 6 with lane 4). Furthermore, purified PDI was competent in inducing transfer of the A1 chain to proteoliposomes (Fig. 5 C, compare lane 10 with lane 8). Therefore, PDI is both necessary and sufficient for the transfer of unfolded A1 peptide to the ER membrane. These experiments also show that Ero1 in the ER lumen is not required. About 10% of total Ero1 is found in the proteoliposomes, but this population also does not seem to be required for the transfer of the toxin to the ER membrane, because proteoliposomes generated from microsomes of the yeast ero1-1 mutant bound the PDItoxin complex with unreduced efficiency (unpublished data). However, the mutant proteoliposomes were inactive in releasing the toxin from PDI, in contrast to wild-type vesicles (Fig. 5 D, compare lane 4 with lanes 2 and 3). Thus, these results indicate that the complex of toxin and PDI is first transferred to the ER membrane and then dissociated by the action of Ero1.
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Although we have seen release of the toxin from PDI with both Ero1 in the ER detergent extract and with Ero1 present in reconstituted proteoliposomes, in vivo the active species is likely to be membrane-bound Ero1. Because Ero1 lacks a transmembrane domain and a K/HDEL sequence at its COOH terminus, it may be kept in the ER by an association with a membrane protein. Its appearance in the ER extract may be caused by disruption of this interaction by even low concentrations of detergent. Our results show that the -isoform of Ero1 is capable of releasing the A1 chain from PDI, whereas fractions containing Ero1ß were inactive. This raises the possibility that Ero1ß may serve a different function in the cell than Ero1
, perhaps in the refolding of misfolded ER proteins, but we cannot exclude the possibility that Ero1ß was inactivated during sample preparation.
Our data show that Ero1 only oxidizes the COOH-terminal disulfide bond in PDI to cause toxin release. This finding is consistent with previous results showing that the COOH-terminal thioredoxin domain plays a more critical role than its NH2-terminal counterpart during the in vitro refolding of carboxypeptidase Y (CPY), and of bovine pancreatic trypsin inhibitor (Westphal et al., 1999). In addition, the NH2-terminal thioredoxin domain does not appear to play a role in the PDI-mediated retrotranslocation of misfolded prepro- factor (Gillece et al., 1999). However, in vivo the NH2-terminal thioredoxin domain was found to play a more important role than the COOH-terminal domain in the folding of CPY (Holst et al., 1997). The reasons for the differences between the in vitro and in vivo results remain to be clarified. Our results also indicate that Ero1 is more efficient in oxidizing toxin-associated PDI compared to free PDI. Although the molecular mechanism for this preference is unclear, it could prevent futile redox cycles of PDI in the absence of substrate.
Whether the PDI-Ero1driven unfolding mechanism is used to translocate other substrates, such as misfolded ER proteins, remains to be explored. However, PDI and PDI-related proteins have been implicated in the retrotranslocation of misfolded ER substrates (Gillece et al., 1999; Wang and Chang, 1999), suggesting that the proposed pathway may be general.
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Materials and methods |
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Purification of yeast PDI mutants
Plasmids expressing mutant yeast PDICxxC-AxxA and PDIAxxA-CxxC were gifts from J. Weissman (University of California, San Francisco, CA). Purification of the proteins was performed as described in Tu et al. (2000).
Purification of yeast Ero1 and mammalian Ero1
The yeast strain overexpressing yeast Ero1-Myc-His6 (AFY383) was a gift from C. Kaiser. The yeast strain overexpressing the mammalian Ero1-Myc-His6 protein was constructed as follows. The gene encoding Ero1
was excised from pcDNA3.1-Ero1
-Myc-His6, a gift from R. Sitia (Universita Vita-Salute San Raffaele, Milan, Italy) by digesting with XbaI and PmeI. The fragment was ligated between the XbaI and SmaI sites into a yeast overexpression vector (p416-GALL). Yeast cells containing this construct (p416-Ero1
) were grown overnight and then cultured in YEP Raf/Gal. The cells were harvested and homogenized in a french press, followed by centrifugation at 5000g to remove unbroken cells. The supernatant was centrifuged at 13,000 g for 30 min to collect the membrane fraction which was lysed in a buffer containing 1% Triton X-100, 5% glycerol, 50 mM Tris-HCl, pH 7.4, and protease inhibitors. The soluble material was collected by centrifugation at 100,000 g for 40 min. Affinity purification of the proteins was performed by binding to a Ni-NTA column. After washing with 10 mM imidazole, proteins were eluted with 500 mM imidazole in a buffer containing 0.1% Triton X-100, 5% glycerol, 50 mM Tris-HCl, pH 7.4 and protease inhibitors. The eluted sample was dialyzed and bound to a 1 ml Hi-Trap Q-Sepharose column (Amersham Biosciences). Bound material was eluted with a linear 01-M potassium acetate gradient. Fractions of 0.5 ml were collected and Ero1 content analyzed by SDS-PAGE and Coomassie staining.
Preparation of ER extract
An ER extract was prepared by adding 0.2% digitonin to a suspension of 2.2 equivalents/µl canine microsomes followed by centrifugation. The supernatant fraction represents the ER extract.
Immunodepletion of mammalian Ero1 and PDI
100 µl of ER extract derived from dog microsomes were incubated with 10 µl PDI antibodies or 10 µl Ero1/ß antibodies overnight at 4°C, followed by addition of 20 µl protein A Sepharose beads. Mock depletion was performed without antibodies.
Thiol modification with maleimide-PEG5000
Isolated A subunit (70 nM) was incubated with 3 µM PDI in the presence of DTT (100 mM), GSSG (100 mM), or GSH (1 mM) for 30 min at 30°C. To samples incubated in GSH (1 mM), Ero1 was added and incubated for 20 min at 30°C. 1/10 of the samples was precipitated with trichloroacetic acid, washed with acetone, and resuspended in a buffer containing 2% SDS, 50 mM Tris (pH 7.4), and 5 mM maleimide-PEG5000 (Shearwater). After incubation for 60 min at 30°C, the samples were analyzed by nonreducing SDS-PAGE and immunoblotted with an antibody directed against PDI.
Toxin transfer assay
Isolated A subunit (70 nM) was incubated with either ER extract, PDI-depleted extract, or 3 µM PDI for 20 min at 30°C. 10 equivalents of proteoliposomes (prepared as described in Gorlich and Rapoport [1993]) were added for 20 min at 30°C. Samples were sedimented for 20 min at 40,000 rpm in a tabletop ultracentrifuge using a TLA 100.4 rotor. The supernatant and pellet fractions were analyzed in nonreducing SDS-PAGE followed by immunoblotting. For preparation of protease-treated proteoliposomes, proteinase K-agarose beads were first incubated with solubilized microsomes for 30 min at room temperature, followed by removal of the protease by centrifugation. Hydrophobic SM2 beads were subsequently added to the solubilized microsomes to remove the detergent and allow formation of vesicles. Trypsin digestion assay, crosslinking assay, and fractionation of ER extract performed as described in Tsai et al. (2001).
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Footnotes |
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Acknowledgments |
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B. Tsai is supported by a fellowship from the Damon Runyon Cancer Research Fund (DRG1579). T.A. Rapoport is a Howard Hughes Medical Institute Investigator.
Submitted: 23 July 2002
Revised: 13 September 2002
Accepted: 16 September 2002
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References |
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