Differential in Vivo Roles Played by DsbA and DsbC in the Formation of Protein Disulfide Bonds

Several Escherichia coli proteins participate in protein disulfide bond formation. Among them, DsbA is the primary factor that oxidizes target cysteines. Biochemical evidence indicates that DsbC has disulfide isomerization activity. To study intracellular functions of DsbA and DsbC, we used an alkaline phosphatase mutant, PhoA[SCCC], with the most amino-terminal cysteine replaced by serine. It was found that the remaining 3 cysteines in PhoA[SCCC] form a disulfide bond of incorrect as well as correct combinations. An aberrant disulfide bond was preferentially formed in wild-type cells, which was converted slowly to the normal disulfide bond. This conversion did not occur in the dsbC-disrupted cells. Overproduction of DsbC stimulated the formation of the correct disulfide bond. In contrast, the inefficiently formed disulfide bonds in the dsbA-disrupted cells, and the more efficiently formed disulfide bonds in the same strain in the presence of oxidized glutathione were mostly in the correct form. These results suggest that the DsbA-catalyzed reaction can be too rapid for some proteins. DsbA may simply oxidize available pairs of cysteines, which happen to be in an incorrect combination in the case of PhoA[SCCC]. In contrast, DsbC stimulates the formation of correct disulfide bonds and corrects previously introduced aberrant ones. Thus, DsbC acts to isomerize disulfide bonds in vivo. Several Escherichia coli proteins participate in protein disulfide bond formation. Among them, DsbA is the primary factor that oxidizes target cysteines. Biochemical evidence indicates that DsbC has disulfide isomerization activity. To study intracellular functions of DsbA and DsbC, we used an alkaline phosphatase mutant, PhoA[SCCC], with the most amino-terminal cysteine replaced by serine. It was found that the remaining 3 cysteines in PhoA[SCCC] form a disulfide bond of incorrect as well as correct combinations. An aberrant disulfide bond was preferentially formed in wild-type cells, which was converted slowly to the normal disulfide bond. This conversion did not occur in the dsbC-disrupted cells. Overproduction of DsbC stimulated the formation of the correct disulfide bond. In contrast, the inefficiently formed disulfide bonds in the dsbA-disrupted cells, and the more efficiently formed disulfide bonds in the same strain in the presence of oxidized glutathione were mostly in the correct form. These results suggest that the DsbA-catalyzed reaction can be too rapid for some proteins. DsbA may simply oxidize available pairs of cysteines, which happen to be in an incorrect combination in the case of PhoA[SCCC]. In contrast, DsbC stimulates the formation of correct disulfide bonds and corrects previously introduced aberrant ones. Thus, DsbC acts to isomerize disulfide bonds in vivo. Differential in vivo roles played by DsbA and DsbC in the formation of protein disulfide bonds.Journal of Biological ChemistryVol. 273Issue 42PreviewPlasmid pMS002 and its derivatives used in the above two publications proved to contain an additional mutation for a Ser-401 → Cys substitution within PhoA. We traced this mutation back to the phoA plasmid (provided by others) that was used to substitute the amplified segment, as described in the first publication (page 6174, “Experimental Procedures”). Given this fact, we eliminated this unwanted mutation from most of the plasmid constructions and repeated key experiments presented in both publications. Full-Text PDF Open Access Disulfide bonds are found in many extracytosolic proteins in all organisms and contribute to folding and stability of these proteins. While disulfide bond formation is a simple reaction of oxidation of cysteine residues, and it can be reproduced in vitro under appropriate conditions (1Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5338) Google Scholar), recent studies established that it does not occur effectively in vivo without the aid of other proteins (2Bardwell J.C.A. Mol. Microbiol. 1994; 14: 199-205Crossref PubMed Scopus (200) Google Scholar). In Escherichia coli, a periplasmic protein, DsbA, is required for disulfide bond formation in vivo (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (846) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar). It directly oxidizes cysteines on the target proteins in vitro(5Zapun A. Creighton T.E. Biochemistry. 1994; 33: 5202-5211Crossref PubMed Scopus (89) Google Scholar, 6Akiyama Y. Kamitani S. Kusukawa N. Ito K. J. Biol. Chem. 1992; 267: 22440-22445Abstract Full Text PDF PubMed Google Scholar). It has a thioredoxin-like Cys30-X-X-Cys motif characteristically found in disulfide oxidoreductases (7Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). DsbB, an integral membrane protein, is also required for the processes (8Bardwell J.C.A. Lee J.-O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (364) Google Scholar). The role of DsbB is to reoxidize DsbA to enable its catalytic turnover (8Bardwell J.C.A. Lee J.-O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (364) Google Scholar, 9Kishigami S. Kanaya E. Kikuchi M. Ito K. J. Biol. Chem. 1995; 270: 17072-17074Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 10Guilhot C. Jander G. Martin L.N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9895-9899Crossref PubMed Scopus (128) Google Scholar, 11Kishigami S. Ito K. Genes Cells. 1996; 1: 201-208Crossref PubMed Scopus (47) Google Scholar). Genes dsbC and dsbD(dipZ) also encodes factors involved in disulfide bond metabolism (12Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (197) Google Scholar, 13Missiakas D. Schwager F. Raina S. EMBO J. 1995; 14: 3415-3424Crossref PubMed Scopus (171) Google Scholar, 14Shevchik V.E. Condemine G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (135) Google Scholar, 15Crooke H. Cole J. Mol. Microbiol. 1995; 15: 1139-1150Crossref PubMed Scopus (115) Google Scholar). DsbD is a membrane protein with a thioredoxin-like motif in the periplasmic domain, and it may have a regulatory role of conferring reducing power to the periplasm. DsbC is a periplasmic protein with 4 cysteines among which Cys98 and Cys101 forms a thioredoxin-like motif. Creighton and his colleagues (16Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (222) Google Scholar) characterized the redox activity of DsbC using a model substrate. They showed that while DsbA merely oxidized cysteines on the substrate, DsbC efficiently isomerized preformed disulfide bonds. Bacterial alkaline phosphatase, a periplasmic protein, is a dimer of the phoA gene product (PhoA) with two intramolecular disulfide bonds (Cys168-Cys178 and Cys286-Cys336) (17Bradshaw R.A. Cancedda F. Ericsson L.H. Neumann P.E. Piccoli S.P. Schlesinger M.J. Shriefer K. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3473-3477Crossref PubMed Scopus (147) Google Scholar). Disulfide bond formation is essential for the correct folding of this enzyme (3Bardwell J.C.A. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (846) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar, 18Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7722Crossref PubMed Scopus (167) Google Scholar, 19Akiyama Y. Ito K. J. Biol. Chem. 1993; 286: 8146-8150Google Scholar). We found that, of the two disulfide bonds in PhoA, the carboxyl-terminal one (Cys286-Cys336) is required and sufficient for the active conformation of this enzyme (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Thus, a mutant form of PhoA, termed PhoA[SSCC], with the two NH2-terminally located cysteines replaced by serine is as active as the wild-type enzyme, although it is no longer resistant to a protease. Interestingly, the presence of an additional cysteine at residue 178 lowered the enzymatic activity significantly (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). We show here that this mutant PhoA, termed PhoA[SCCC], forms an aberrant disulfide bond among Cys178, Cys286, and Cys336. Using this unique experimental system, we investigated into thein vivo roles played by DsbA and DsbC. It was found that DsbA principally introduced an aberrant disulfide bond into PhoA[SCCC], whereas DsbC stimulated the eventual formation of the correct disulfide bond in vivo. Thus, DsbC functions, in concert with DsbA, as a disulfide isomerase in vivo. Strain MS3 was a ΔphoA strain, KS272 (21Strauch K.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1576-1580Crossref PubMed Scopus (307) Google Scholar), into which F′lacI Q lacPL8 LacZ + Y + A + pro + had been introduced (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). MS4 was a dsbA-33::Tn5 (4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar) transductant of MS3. As a dsbC deletion strain we used W3110tonA ΔdsbC, which was kindly provided by John Joly of Genentech. This strain had been constructed by integration and segregation of a plasmid carrying ΔdsbC, and thedsbC deletion was confirmed by polymerase chain reaction analyses. 1J. Joly, personal communication. An isogenicdsbC + strain, W3110 tonA, was also provided by J. Joly of Genentech. PhoA and its Cys/Ser mutant forms were designated by the four letter notations in brackets, with C for Cys and S for Ser, for residues 168, 178, 286, and 336 in this order (20; numbering according to Ref. 17Bradshaw R.A. Cancedda F. Ericsson L.H. Neumann P.E. Piccoli S.P. Schlesinger M.J. Shriefer K. Walsh K.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3473-3477Crossref PubMed Scopus (147) Google Scholar). They were expressed from the plasmids under the control of thelac operator/promoter (20Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar); pMS002 for wild-type PhoA, pMS003 for PhoA[SCCC], pMS004 for PoA[SSCC], and pMS015 for PhoA[CCSS]. pMS022 was a DsbC-overproducing plasmid. For its construction, a 1.0-kilobase pair SacI-KpnI fragment, containing dsbC (including its own promoter), was excised from pDS30 (14Shevchik V.E. Condemine G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (135) Google Scholar) and cloned into the SmaI site of pSTV28 (a pACYC184-based lac promoter vector; Ref. 22Shimoike T. Taura T. Kihara A. Yoshihisa T. Akiyama Y. Cannon K. Ito K. J. Biol. Chem. 1995; 270: 5519-5526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To examine PhoA molecules at steady states, cells were grown at 30 °C to an exponential phase in L broth (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 1.7 ml of 1 nN NaOH/liter) supplemented with 1 mm IPTG 2The abbreviations used are: IPTG, isopropyl-β-d-thiogalactoside; PAGE, polyacrylamide gel electrophoresis. and appropriate antibiotics. A 200-μl portion was mixed with an equal volume of 10% trichloroacetic acid. Protein precipitates were collected by centrifugation, washed with acetone, and dissolved in SDS/Tris-HCl solution containing iodoacetamide (23Pollitt S. Zalkin H. J. Bacteriol. 1983; 153: 27-32Crossref PubMed Google Scholar). Samples were subjected to 10% SDS-PAGE (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212382) Google Scholar) in the absence of any reducing reagent, and PhoA isoforms were detected by immunoblotting (25Akiyama Y. Ogura T. Ito K. J. Biol. Chem. 1994; 269: 5218-5224Abstract Full Text PDF PubMed Google Scholar) with anti-PhoA serum (obtained from 5 Prime → 3 Prime, Inc., Boulder, CO). To follow the biosynthesis and conversion of different isoforms, cells were grown at 30 °C to an exponential phase in M9 medium (26Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 431Google Scholar) supplemented with 0.4% glycerol, appropriate antibiotics, and 20 μg/ml each of amino acids (except methionine and cysteine). Fifteen minutes after addition of 1 mm IPTG, cells were pulse-labeled with 50 μCi/ml [35S]methionine (1100 Ci/mmol, American Radiolabeled Chemicals) for 30 s, followed by chase with unlabeled l-methionine (200 μg/ml) for indicated periods. Whole cell proteins were immediately precipitated with trichloroacetic acid and dissolved in the SDS/Tris-HCl/iodoacetamide solution as described above. Radioactive PhoA was immunoprecipitated (4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar), electrophoresed, and visualized using a Bioimaging Analyzer BAS2000 (Fuji Film). Disulfide-bonded and reduced forms of PhoA can be separated by SDS-PAGE under nonreducing conditions. PhoA[SSCC] migrated identically with wild-type PhoA (Fig. 1, comparelanes 1 and 3) under nonreducing conditions. In contrast, PhoA[CCSS] migrated at the same position as the reduced PhoA (Fig. 1, lane 2). These results indicate that Cys286-Cys336 disulfide bond mainly contributes to the increased electrophoretic mobility of the oxidized PhoA molecule. We designate this electrophoretic mobility “ox1” (Fig.1). PhoA[SCCC] produced two bands when expressed in wild-type cells (Fig. 1, lane 4). The minor band was at the ox1 position, whereas the major band migrated even faster than ox1. The latter mobility is designated “ox2” (Fig. 1, lane 4). Obviously, the former should represent Cys286-Cys336 disulfide-bonded molecules. The latter species was not due to a proteolytic cleavage, since reduced PhoA[SCCC] migrated as a single band at the position (“red” in Fig. 1) identical to the reduced wild-type PhoA (Fig. 1, lanes 9 and 10). These results indicate that the ox2 form of PhoA[SCCC] contains an aberrant disulfide bond, between Cys178 and either Cys286 or Cys336.