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

(Received for publication, February 4, 1997)

Michio Sone , Yoshinori Akiyama and Koreaki Ito Dagger

From the Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

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 (1), recent studies established that it does not occur effectively in vivo without the aid of other proteins (2). In Escherichia coli, a periplasmic protein, DsbA, is required for disulfide bond formation in vivo (3, 4). It directly oxidizes cysteines on the target proteins in vitro (5, 6). It has a thioredoxin-like Cys30-X-X-Cys motif characteristically found in disulfide oxidoreductases (7).

DsbB, an integral membrane protein, is also required for the processes (8). The role of DsbB is to reoxidize DsbA to enable its catalytic turnover (8-11). Genes dsbC and dsbD (dipZ) also encodes factors involved in disulfide bond metabolism (12-15). 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 (16) 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) (17). Disulfide bond formation is essential for the correct folding of this enzyme (3, 4, 18, 19). 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 (20). 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 (20). 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 the in 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.


EXPERIMENTAL PROCEDURES

E. coli Strains and Plasmids

Strain MS3 was a Delta phoA strain, KS272 (21), into which F'lacIQ lacPL8 LacZ+ Y+ A+ pro+ had been introduced (20). MS4 was a dsbA-33::Tn5 (4) transductant of MS3. As a dsbC deletion strain we used W3110 tonA Delta dsbC, which was kindly provided by John Joly of Genentech. This strain had been constructed by integration and segregation of a plasmid carrying Delta dsbC, and the dsbC deletion was confirmed by polymerase chain reaction analyses.1 An isogenic dsbC+ 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. 17). They were expressed from the plasmids under the control of the lac operator/promoter (20); 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 (14) and cloned into the SmaI site of pSTV28 (a pACYC184-based lac promoter vector; Ref. 22).

Examination of Disulfide Bonds in PhoA

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 IPTG2 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 (23). Samples were subjected to 10% SDS-PAGE (24) in the absence of any reducing reagent, and PhoA isoforms were detected by immunoblotting (25) with anti-PhoA serum (obtained from 5 Prime right-arrow 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 (26) 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 (4), electrophoresed, and visualized using a Bioimaging Analyzer BAS2000 (Fuji Film).


RESULTS

PhoA[SCCC] Forms an Aberrant Disulfide Bond

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, compare lanes 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.


Fig. 1. PhoA[SCCC] forms an aberrant disulfide bond. Cells of MS3 (lanes 1-4 and 6-9) or MS4 (dsbA::Tn5; lanes 5 and 10) were transformed with pMS002 (PhoA[CCCC]; lanes 1 and 6), pMS015 (PhoA[CCSS]; lanes 2 and 7), pMS004 (PhoA[SSCC]; lanes 3 and 8), or pMS003 (PhoA[SCCC]; lanes 4, 5, 9, and 10). They were grown in L medium with IPTG, and whole cell proteins were separated by SDS-PAGE under nonreducing (lanes 1-5) and reducing (lanes 6-10) conditions. PhoA proteins were visualized by immunoblotting with anti-PhoA serum. red, ox1 and ox2 indicate positions of reduced PhoA, oxidized PhoA with the correct disulfide bond between Cys286-Cys336 and oxidized PhoA with an aberrant disulfide bond, respectively.
[View Larger Version of this Image (16K GIF file)]


DsbA-dependent Preferential Introduction of an Aberrant Disulfide Bond to PhoA[SCCC]

PhoA[SCCC] was expressed in dsbA+ cells and dsbA-disrupted and dsbA-disrupted cells growing in broth medium, and its electrophoretic mobilities were examined by immunoblotting. Whereas the ox2 isoform was the major product in the wild-type cells, the ox1 isoform became the major accumulated product in the dsbA-strain (Fig. 1, compare lanes 4 and 5). Apparently, the correct disulfide bond was preferred in the absence of DsbA. The enzymatic activity of PhoA[SCCC] was higher when produced in the dsbA-disrupted cells than in the dsbA+ cells.3

We then studied the synthesis and conversion of the PhoA[SCCC] isoforms by pulse-chase/immunoprecipitation experiments, using cells growing in minimal salt medium. In wild-type cells, the ox1 and ox2 forms of PhoA[SCCC] were initially labeled in about equal intensities. During chase with unlabeled methionine, intensity of the ox2 form decreased with concomitant increase in the ox1 form (Fig. 2, lanes 1-9). This ox2 to ox1 conversion took place over some 1 h.


Fig. 2. Involvement of DsbA and DsbC in the formation and interconversion of disulfide isoforms of PhoA[SCCC]. Cells of MS3 (dsbA+; lanes 1-9), MS4 (dsbA::Tn5; lanes 10-18), and W3110 tonA Delta dsbC (lanes 19-27), each carrying pMS003 (PhoA[SCCC]) were grown in minimal amino acids/glycerol medium at 30 °C, induced for the synthesis of PhoA[SCCC] with IPTG, and pulse-labeled with [35S]methionine for 30 s followed by chase with unlabeled methionine for 1-80 min as indicated. PhoA species were immunoprecipitated and subjected to nonreducing SDS-PAGE as described under "Experimental Procedures." Strain W3110 tonA, the isogenic dsb+ counterpart of W3110 tonA Delta dsbC gave essentially the identical results as MS3 shown in lanes 1-9. WT, wild type.
[View Larger Version of this Image (17K GIF file)]


When PhoA[SCCC] was expressed in the dsbA-disrupted cells, the majority of the newly synthesized molecules were now in the reduced form, which was slowly degraded (Fig. 2, lanes 10-18). A small amount of oxidized form in this strain was in the ox1 form, and no aberrant form (ox2) was detected (Fig. 2, lanes 10-18). The experiments reported in Fig. 1 (lane 4) and Fig. 2 (lanes 10-18) gave different proportions of PhoA[SCCC] isoforms for the dsbA- strain. This may be explained by the presence of some broth components, such as cystine, that may have acted as an oxidant in the former experiment (see below for oxidant effects). In addition, instability of the reduced form may have lowered the detection by immunoblotting.

We examined effects of oxidized glutathione (GSSG) added to the minimal medium (Fig. 3, E and G). In the presence of GSSG, PhoA[SCCC] was almost all in the ox2 form in the wild-type cells (Fig. 3E), whereas it was almost all in the ox1 form in the dsbA- mutant (Fig. 3G). Thus, DsbA introduces an abnormal disulfide bond into Pho[SCCC] molecule in vivo. DsbA-independent disulfide bond formation occurs mostly between the natural combination of cysteines; this was true even when disulfide bond formation was driven by a "nonspecific" oxidant, GSSG (Fig. 3G).


Fig. 3. Effects of DsbC overproduction and oxidized glutathione on the formation of different isoforms of PhoA[SCCC] in the dsbA+ and the dsbA::Tn5 cells. Strains MS3 (dsbA+; A, B, E, and F) and MS4 (dsbA::Tn5; C, D, G, and H) were transformed with two plasmids: one was pMS003 (PhoA[SCCC]) and the other was either pSTV28 (vector; A, C, E, and G) or pMS022 (DsbC; B, D, F, and H). Cells were grown in minimal amino acid/glycerol medium supplement with (E-H) or without (A-D) 10 mM oxidized glutathione (GSSG), induced with IPTG, and subjected to pulse-chase/immunoprecipitation analyses as described in the legend to Fig. 2.
[View Larger Version of this Image (73K GIF file)]


DsbC Is Required for the Conversion of Aberrant to Normal Disulfide Isoforms of PhoA[SCCC]

The results in Fig. 2 (lanes 1-9) demonstrated that the ox2 isoform of PhoA[SCCC] was gradually converted to the ox1 isoform in the wild-type cells. We found that this conversion did not occur in a dsbC deletion strain (Fig. 2, lanes 19-27). The ox2 species in this strain was degraded over the time. These results suggest that the DsbC function is needed for the posttranslational conversion from the aberrant to the correct disulfide isoforms of PhoA[SCCC] in vivo.

DsbC Overproduction Enhances the Production of the Correctly Disulfide-bonded PhoA[SCCC] Molecules

In the presence of a DsbC-overproducing plasmid, even the dsbA+ strain produced preferentially the correctly disulfide-bonded PhoS[SCCC] (Fig. 3B). Control cells with the vector produced mainly the ox2 isoform, which was later converted to the ox1 isoform (Fig. 3A). DsbC overproduction in the dsbA-disrupted strain resulted in almost exclusive production of the reduced form (Fig. 3D), whereas the control cells with the vector produced both the reduced and the ox1 forms (Fig. 3C). Thus, excess DsbC cannot substitute for DsbA with respect to the oxidation of PhoA[SCCC]. Neither can it stimulate the correct disulfide bond formation in the absence of DsbA. Excess DsbC in the absence of DsbA seems to be inhibitory against the background disulfide bond formation.

DsbC Exhibits Different Properties in the Presence of an Excess Oxidant

We repeated these experiments in the presence of supplemented GSSG (Fig. 3, E-H). As already discussed, GSSG stimulated the correct disulfide bond formation in the absence of DsbA (Fig. 3G). Overproduction of DsbC in the presence of both GSSG and DsbA gave only a small stimulation of the formation of the ox1 isoform (Fig. 3, compare E and F). Overproduction of DsbC in the presence of GSSG and in the absence of DsbA resulted in the production of both ox1 and ox2 forms (Fig. 3H). Since GSSG alone (in the absence of DsbA) supported the formation of only the ox1 form (Fig. 3G), the results of Fig. 3H indicate that excess DsbC gained the ability to introduce the incorrect disulfide bond in the presence of GSSG. This is in marked contrast to the oxidation inhibition observed in the dsbA- cells in which DsbC was overproduced in the absence of added GSSG (Fig. 3D).

Thus, in the presence of GSSG, DsbC is transformed to have a DsbA-like ability to introduce disulfide bonds that are not necessarily in the correct combination. Under oxidative conditions, the isomerization activity of DsbC may be suppressed.


DISCUSSION

Artifacts in the determination of in vivo redox states of proteins can be minimized by examining them after acid denaturation (23, 27), the method employed in this study. Of the two disulfide bonds of PhoA, the amino-terminal disulfide (Cys168-Cys178) constrains a loop of only 9 amino acids, while the carboxyl-terminally located disulfide (Cys286-Cys336) constrains a loop of 49 amino acids. The fast migration of the oxidized PhoA in SDS-PAGE can essentially be ascribed to the Cys286-Cys336 disulfide bond (Fig. 1). Since any incorrect disulfide bonds that can be formed in PhoA should connect cysteines that flank at least 107 amino acids (in the case of Cys178-Cys286), they are expected to confer even more increased mobility in gel electrophoresis. The ox2 species we observed in PhoA[SCCC] meets this expectation, although we have not determined whether it contains Cys178-Cys286 or Cys178-Cys336 disulfide bond.

Why does PhoA[SCCC] form an aberrant disulfide bond in the presence of DsbA? Disulfide bond formation is essential for the folding of alkaline phosphatase in vivo (3, 4) and in vitro (19), triggering the subsequent folding and dimerization reactions (19). The fact that PhoA[SSCC] has almost 100% enzymatic activity (20) indicates that the PhoA molecule without the NH2-terminal disulfide bond retains the ability to position the active site residues in proper geometry, but this event is incomplete until the carboxyl-terminal disulfide bond has been formed. In PhoA[SCCC], kinetic competition may occur between different combinations of the 3 cysteines for disulfide bond formation, and it should be modulated by ongoing folding reactions (28). Thus, extremely rapid disulfide bond formation may be more random than slower disulfide bond formation. Furthermore, DsbA may be so potent and efficient that it introduces or initiates to introduce a disulfide while a substrate polypeptide chain is still in the process of membrane translocation; the first translocating cysteine, Cys178, will be committed for disulfide bond formation when it reaches the periplasm and forms a transient disulfide with the reactive Cys30 residue (29-31) of DsbA. In contrast, "spontaneous" or glutathione-driven disulfide bond formation in the absence of DsbA will be less efficient and slower such that the correct combination is preferred due to the local folding properties of the polypeptide chain. DsbA-dependent formation of aberrant disulfide bonds was implicated previously (but not demonstrated) in a system where a foreign protein was expressed in E. coli (32, 33).

We obtained two kinds of results that suggest that DsbC functions to stimulate the formation of the correct disulfide bond. First, slow conversion occurs from the aberrant to the correct disulfide bonds of pulse-labeled PhoA[SCCC] in wild-type cells, but not in the dsbC deletion strain. This observation clearly indicates that DsbC-dependent isomerization of disulfide bond occurs in the cell. Second, overproduction of DsbC in the dsbA+ cells enhanced the rapid formation of the correct disulfide bond. This observation could be interpreted in terms of either very rapid isomerization in the presence of excess DsbC or de novo introduction of the correct disulfide bond in the presence of excess DsbC. In view of the biochemical demonstration of isomerization activity of DsbC (16), we think it reasonable to assume the rapid isomerization. This is consistent with the observation (Fig. 3, D and H) that DsbA is required for DsbC to exhibit the correct disulfide-introducing function.

Recently, Rietsch et al. (34) reported in vivo results that a dsbC disruption had no effect on OmpA (normally with a single disulfide bond), that it resulted in accumulation of a small amount of reduced PhoA4, and that it resulted in greatly diminished production of active urokinase (normally with 12 disulfide bonds). Although these results are consistent with their interpretation that DsbC has a disulfide isomerizing role in vivo, the evidence for the aberrant disulfide bond formation in the absence of DsbC was inconclusive.

Our results provide some additional information about the DsbC functions in vivo. Excess DsbC was inhibitory against the background disulfide bond formation that occurs inefficiently in the absence of DsbA. Probably, the reducing character of DsbC might dominate the air oxidation. It was also found, however, that excess DsbC can exhibit DsbA-like function when GSSG was supplemented to the medium. This is consistent with another finding of Rietsch et al. (34) that the suppression of dsbA null mutation by loss-of-function dsbD (dipZ) mutations requires the functional DsbC. They proposed that DsbD (DipZ) together with thioredoxin normally keeps DsbC in the partially reduced state, and the loss of reducing factor leads to the production of oxidized DsbC, which in turn substitutes for DsbA. This model is consistent with our results; excess DsbC is oxidized by GSSG and then it substitutes for DsbA.

We have now demonstrated that the principal role of DsbC is to facilitate disulfide bonds that are formed between correct pairs of cysteines. This demonstration was made possible by using a unique model protein, PhoA[SCCC]. The results of Rietsch et al. (34) that the formation of active urokinase, a natural (but still foreign to E. coli) protein with multiple disulfide bonds, depends absolutely on DsbC also supports this notion. These in vivo results establish that the cellular role of DsbC lies in the isomerization of disulfide bonds until the protein has been folded into a stable conformation.


FOOTNOTES

*   This work was supported by grants from the Ministry of Education, Science and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Inst. for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. Tel.: 81-75-751-4015; Fax: 81-75-771-5699 or 81-75-761-5626; E-mail: kito{at}virus.kyoto-u.ac.jp.
1   J. Joly, personal communication.
2   The abbreviations used are: IPTG, isopropyl-beta -D-thiogalactoside; PAGE, polyacrylamide gel electrophoresis.
3   M. Sone and K. Ito, unpublished data.
4   Although these authors suggest that it was an aberrant form, we believe that it must have been the reduced form because of the discussion already given about the electrophoretic mobilities of PhoA. That it disappeared when the culture received dithiothreitol before pulse labeling (34) could be explained. For instance, if dithiothreitol can access the periplasm freely but iodoacetamide is somehow restricted, the low concentration of dithiothreitol might antagonize the action of iodoacetamide added after sampling, leaving free cysteines unmodified, which will then be oxidized after cell pelleting and solubilization in SDS.

ACKNOWLEDGEMENTS

We thank Dr. John Joly for generously providing the dsbC deletion strain, Dr. Guy Condemine for pDS30, and Kiyoko Mochizuki for laboratory supplies.


REFERENCES

  1. Anfinsen, C. B. (1973) Science 181, 223-230 [Medline] [Order article via Infotrieve]
  2. Bardwell, J. C. A. (1994) Mol. Microbiol. 14, 199-205 [Medline] [Order article via Infotrieve]
  3. Bardwell, J. C. A., McGovern, K., and Beckwith, J. (1991) Cell 67, 581-589 [Medline] [Order article via Infotrieve]
  4. Kamitani, S., Akiyama, Y., and Ito, K. (1992) EMBO J. 11, 57-62 [Abstract]
  5. Zapun, A., and Creighton, T. E. (1994) Biochemistry 33, 5202-5211 [Medline] [Order article via Infotrieve]
  6. Akiyama, Y., Kamitani, S., Kusukawa, N., and Ito, K. (1992) J. Biol. Chem. 267, 22440-22445 [Abstract/Free Full Text]
  7. Holmgren, A. (1989) J. Biol. Chem. 264, 13963-13966 [Free Full Text]
  8. Bardwell, J. C. A., Lee, J.-O., Jander, G., Martin, N., Belin, D., and Beckwith, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1038-1042 [Abstract]
  9. Kishigami, S., Kanaya, E., Kikuchi, M., and Ito, K. (1995) J. Biol. Chem. 270, 17072-17074 [Abstract/Free Full Text]
  10. Guilhot, C., Jander, G., Martin, L. N., and Beckwith, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9895-9899 [Abstract]
  11. Kishigami, S., and Ito, K. (1996) Genes Cells 1, 201-208 [Abstract/Free Full Text]
  12. Missiakas, D., Georgopoulos, C., and Raina, S. (1994) EMBO J. 13, 2013-2020 [Abstract]
  13. Missiakas, D., Schwager, F., and Raina, S. (1995) EMBO J. 14, 3415-3424 [Abstract]
  14. Shevchik, V. E., Condemine, G., and Robert-Baudouy, J. (1994) EMBO J. 13, 2007-2012 [Abstract]
  15. Crooke, H., and Cole, J. (1995) Mol. Microbiol. 15, 1139-1150 [Medline] [Order article via Infotrieve]
  16. Zapun, A., Missiakas, D., Raina, S., and Creighton, T. E. (1995) Biochemistry 34, 5075-5089 [Medline] [Order article via Infotrieve]
  17. Bradshaw, R. A., Cancedda, F., Ericsson, L. H., Neumann, P. E., Piccoli, S. P., Schlesinger, M. J., Shriefer, K., and Walsh, K. A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3473-3477 [Abstract]
  18. Derman, A. I., and Beckwith, J. (1991) J. Bacteriol. 173, 7719-7722 [Medline] [Order article via Infotrieve]
  19. Akiyama, Y., and Ito, K. (1993) J. Biol. Chem. 286, 8146-8150
  20. Sone, M., Kishigami, S., Yoshihisa, T., and Ito, K. (1997) J. Biol. Chem. 272, 6174-6178 [Abstract/Free Full Text]
  21. Strauch, K. L., and Beckwith, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1576-1580 [Abstract]
  22. Shimoike, T., Taura, T., Kihara, A., Yoshihisa, T., Akiyama, Y., Cannon, K., and Ito, K. (1995) J. Biol. Chem. 270, 5519-5526 [Abstract/Free Full Text]
  23. Pollitt, S., and Zalkin, H. (1983) J. Bacteriol. 153, 27-32 [Medline] [Order article via Infotrieve]
  24. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  25. Akiyama, Y., Ogura, T., and Ito, K. (1994) J. Biol. Chem. 269, 5218-5224 [Abstract/Free Full Text]
  26. Miller, J. H. (1972) Experiments in Molecular Genetics, p. 431, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  27. Kishigami, S., Akiyama, Y., and Ito, K. (1995) FEBS Lett. 364, 55-58 [CrossRef][Medline] [Order article via Infotrieve]
  28. Frech, C., Wunderlich, M., Glockshuber, R., and Schmid, F. X. (1996) Biochemistry 35, 11386-11395 [CrossRef][Medline] [Order article via Infotrieve]
  29. Nelson, J. W., and Creighton, T. E. (1994) Biochemistry 33, 5974-5983 [Medline] [Order article via Infotrieve]
  30. Zapun, A., Cooper, L., and Creighton, T. E. (1994) Biochemistry 33, 1907-1914 [Medline] [Order article via Infotrieve]
  31. Grauschopf, U., Winther, J. R., Korber, P., Zander, T., Dallinger, P., and Bardwell, J. C. A. (1995) Cell 83, 947-955 [Medline] [Order article via Infotrieve]
  32. Alksne, L. E., Keenney, D., and Rasmussen, B. A. (1995) J. Bacteriol. 177, 462-464 [Abstract]
  33. Alksne, L. E., and Rasmussen, B. A. (1996) J. Bacteriol. 178, 4306-4309 [Abstract]
  34. Rietsch, A., Belin, D., Martin, N., and Beckwith, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13048-13053 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.