©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
- Subunit Interaction Is Required for Catalysis by H-ATPase (ATP Synthase)
beta SUBUNIT AMINO ACID REPLACEMENTS SUPPRESS A SUBUNIT MUTATION HAVING A LONG UNRELATED CARBOXYL TERMINUS (*)

(Received for publication, May 31, 1995; and in revised form, July 10, 1995)

Catherine Jeanteur-De Beukelaer Hiroshi Omote (§) Atsuko Iwamoto-Kihara (¶) Masatomo Maeda (**) Masamitsu Futai (§§)

From the Division of Biological Science, the Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The mechanisms of energy coupling and catalytic cooperativity are not yet understood for H-ATPase (ATP synthase). An Escherichia coli subunit frameshift mutant (downstream of Thr-277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M.(1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta52 Cys or Gly-beta150 Asp replacement could suppress the deleterious effects of the frameshift. The membranes of the two mutants ( frameshift/Cys-beta52 with or without a third mutation, Val-beta77 Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the frameshift/Asp-beta150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.


INTRODUCTION

H-ATPase (ATP synthase, F(0)F(1)) synthesizes ATP coupled with an electrochemical gradient of protons (for reviews see (1, 2, 3) ). The catalytic site of the enzyme is located in the beta subunit of the F(1) sector (alpha(3)beta(3)). The membrane-intrinsic F(0) sector (ab(2)c) functions as a proton pathway. Studies on a negatively stained specimen labeled with maleimidogold established that the central mass of F(1) contains the amino-terminal part of the subunit(4) . The x-ray structures of the mitochondrial F(1) sector of bovine heart and rat liver have been studied by two groups independently(5, 6, 7) . In the crystal structure of bovine F(1), the alpha and beta subunits are arranged alternatively around the amino and carboxyl-terminal alpha helices of the subunit(7) . Consistent with its central location in the F(1) sector, the active role of the subunit in catalysis and its energy coupling with proton transport have been supported by biochemical and molecular biological studies(8, 9, 10, 11, 12) .

Mutational studies on the Escherichia coli enzyme suggested the importance of the amino- and carboxyl-terminal regions of the subunit (286 total residues) in catalysis and energy coupling. Amino acid substitutions in the region between Gln-269 and Thr-277 decreased the membrane ATPase activity and growth by oxidative phosphorylation(8, 10) . The amino-terminal mutants, Met-23 Lys and Met-23 Arg, exhibited substantial ATPase activities (65 and 100%, respectively, of the wild type level), although they grew only very slowly by oxidative phosphorylation(9) . The uncoupled phenotype of the Met-23 Lys mutant was suppressed by eight different amino acid replacements mapped between residues 269 and 280 in the carboxyl-terminal domain(10, 13) . These results suggest that the two terminal regions functionally interact to mediate efficient energy coupling. The subunit with the Ser-8 Cys mutation cross-linked with a different beta subunit region in the presence of Mg-ADP or Mg-ATP, suggesting that conformational changes related to the catalytic site event occur around Ser-8(11) . Furthermore, a fluorescence probe introduced into the subunit changed its spectrum on the addition of ATP or AMPPNP(^1)(12) . However, it is unknown which region(s) of the beta subunit interacts functionally with the subunit.

The interaction(s) between different subunits within the F(1) sector can be clearly shown by mutation studies(14) ; suppression of the defective energy coupling of the Ser-beta174 Phe mutation by an Arg-alpha296 Cys replacement indicated the importance of the functional alpha-beta interaction in energy coupling. In this study, a similar approach was adopted for the beta subunit- subunit interaction. We previously isolated an interesting frameshift mutant that had 16 unrelated residues due to a nucleotide deletion from the Gln-278 codon(8) ; the subunit frameshift has 7 additional residues at its carboxyl terminus together with 9 altered residues downstream of Thr-277. The mutant cells exhibited low membrane ATPase activity and could not grow by oxidative phosphorylation. Thus, the long altered carboxyl-terminal domain may interact deleteriously with the beta subunit leading to lower catalytic cooperativity and defective energy coupling. The defect of the frameshift mutation was suppressed by betaR52C (Arg-beta52 Cys) with or without a betaV77A (Val-beta77 Ala) or betaG150D (Gly-beta150 Asp) replacement in the beta subunit, suggesting that the deleterious beta- interaction in the frameshift was suppressed by the second beta subunit mutation. It is surprising that growth of the three mutants can be supported by oxidative phosphorylation.


EXPERIMENTAL PROCEDURES

E. coli and Growth Conditions

Strains DK8 (DeltauncB-C, ilv::Tn10, thi) (15) lacking the entire unc (ATP synthase) operon and KF10rA (thi, thy, recA1, uncG10 (Gln-14 end)) carrying pBMG293fs (frameshift in the uncG gene for the subunit) (8) were also used. The rich medium (L broth) supplemented with 50 µg/ml thymine with or without ampicillin and the minimal medium (16) containing 2 µg/ml thiamine, 50 µg/ml thymine, 50 µg/ml isoleucine, 50 µg/ml valine, and a carbon source (10 mM glucose, 15 mM succinate, or 0.5% glycerol) were described previously.

Construction of pBMUG15fs Carrying the Entire unc Operon with the Subunit Frameshift Mutation

The frameshift mutation was introduced into a recombinant plasmid, pBWU1(17) , carrying the entire wild type unc operon (see Fig. 1). A unique SpeI site was introduced into the noncoding region (9 base pairs downstream of the last letter of the uncG end codon) by mutagenesis of pBWU1 using polymerase chain reaction (PCR) (Perkin-Elmer); the combinations of primers I and III and primers II and IV were used for the first PCR, and then primers I and II were used for the second PCR (see Fig. 1a). The primers used were: I, 5`-TGCCCAGGTCACCGGCATGG-3`; II, 5`-TAACCGGAGTGCTCGATCGC-3`; III and IV, as shown in Fig. 1. After removing the NdeI segment from the resulting plasmid, pBWU15 carrying the entire unc operon was constructed. pBMG293fs (8) carrying uncG with the frameshift mutation was subjected to PCR using primers I and FS (see Fig. 1b). The RsrII-SpeI fragment was isolated from the amplified product and ligated with pBWU15 digested with the same endonucleases. The resulting recombinant plasmid, pBMUG15fs, was used for further studies.


Figure 1: Construction of a recombinant plasmid (pBMUG15fs) carrying the unc (H-ATPase) operon with the subunit frameshift mutation and introduction of random mutations into the beta subunit. a, an SpeI site was introduced by PCR between the and beta subunit genes carried by pBWU1; primers I/III and II/IV were used. The product was subjected to a second PCR (primer I/II). The resulting amplified fragment was digested with RsrII and SacI and then inserted into the corresponding site of pBWU1. The NdeI fragment was deleted from the resulting plasmid, and the final construct was named pBWU15. b, an SpeI site was also introduced downstream of the subunit gene of pBMG293fs using primers I and FS. The Rsr II-SpeI fragment was obtained and introduced into the corresponding site of the derivative of pBWU15 constructed in a. The derivative was named pBMUG15fs. It should be noted that five restriction sites were introduced previously into the carboxyl-terminal region without changes in the amino acid residues coded(8) . c, the SpeI-SacI fragment of pBMUG15fs was transferred to pBluescript II SK+ and then subjected to PCR mutagenesis with sequence primers. The amplified products were digested with SpeI and SacI and then ligated with the large SpeI-SacI fragment of pBMUG15fs. Three plasmids conferred the ability of oxidative phosphorylation-dependent growth on strain DK8 lacking the unc operon.



Random Mutagenesis of the beta Subunit Gene (uncD)

The SpeI-SacI fragment corresponding to the amino-terminal half of the uncD gene was isolated and introduced into pBluescript II SK+ (see Fig. 1c). A modified PCR (18) was employed to generate random mutations using universal primers (vector segments), and the SpeI-SacI fragment was isolated and ligated with pBMUG15fs treated with the same enzymes. The resulting plasmids were transformed into strain DK8, and cells showing positive growth on succinate by oxidative phosphorylation were isolated. These cells harbored recombinant plasmids carrying the frameshift mutation and beta subunit mutation(s). A plasmid, pBMUD13-G150D, having a single mutation, Gly-beta150 Asp (GGT GAT) was constructed as described previously(17) .

Other Procedures

Membrane vesicles were prepared from logarithmic phase cells (19) and suspended in a TKDG buffer (10 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mM dithiothreitol, and 10% glycerol). The formation of an electrochemical H gradient(8) , ATPase activity(19) , the amount of protein(20) , and the rate of ATP synthesis in the presence of 10 mM NADH (14) were assayed by the published procedures. DNA manipulation and sequencing were performed as described previously(21) . Immunoblotting was carried out using an Amersham ECL Western blotting detection kit.

Materials

Oligonucleotides were synthesized with a Pharmacia LKB (Uppsala) Gene Assembler Plus. The restriction and modifying enzymes for DNA were from Takara Shuzo Co. (Kyoto, Japan), Nippon Gene Co. (Toyama, Japan), or New England Biolabs (Beverly, MA). Taq polymerase and deoxynucleotides were from Perkin-Elmer.


RESULTS

Construction of a Recombinant Plasmid, pBMUG15fs, Carrying the unc Operon with the Subunit Frameshift Mutation

A plasmid, pBMG293fs, carrying the frameshift subunit gene was derived from a mutant recombinant plasmid, pBMG278Q (Glu-278 Gln) by one nucleotide deletion from the Gln-278 codon (CAG AG) (8) . The frameshift subunit was 7 residues longer than the wild type (286 total residues) and had 16 different residues. Plasmid pBMG293fs could not support the growth of the mutant, KF10rA (Gln-14 end), by oxidative phosphorylation, although membranes of KF10rA/pBMG293fs exhibited substantial ATPase activity(8) .

For further studies, the frameshift subunit gene was transferred from pBMG293fs into pBWU15 (Fig. 1, a and b). The resulting plasmid, pBMUG15fs, carried all cistrons of the unc operon for HATPase with the frameshift mutation. Similar to KF10rA/pBMG293fs, DK8 harboring pBMUG15fs could not grow by oxidative phosphorylation and exhibited about 23% of the membrane ATPase activity of the same strain with wild type plasmid pBWU15 (Table 1). As shown previously(22) , DK8 harboring pBWU15 had 10-fold more enzyme than the regular E. coli strain. Thus, mutant cells even with enzyme having 10% of the specific activity of the wild type can grow by oxidative phosphorylation, if they can couple H transport and ATP synthesis properly. The Ser-beta174 Leu mutant is an example of such cells; this mutant exhibited 10% membrane ATPase activity and a 50% growth yield on oxidative phosphorylation(14) . Therefore, it is clear that DK8/pBMUG15fs (with 23% membrane ATPase activity) is defective in energy coupling because no growth by oxidative phosphorylation could be observed. Consistent with no growth, the rate of ATP synthesis by DK8/pBMUG15fs membrane vesicles was less than 5% of the wild type level (Table 1).



beta Subunit Amino Acid Substitutions Suppressed the Frameshift Mutation

A DNA fragment corresponding to the amino-terminal half of the beta subunit (Ala-beta1 to Leu-beta162) was randomly mutagenized by PCR and ligated into pBMUG15fs (Fig. 1c). Surprisingly, three plasmids could confer the ability of oxidative phosphorylation-dependent growth on strain DK8 (Table 1). DNA sequencing of the two plasmids (pBMUG15fsR1 and pBMUG15fsR2) revealed different beta subunit mutations, betaR52C and betaG150D, respectively (Table 1). The third plasmid (pBMUG15fsR3) had two mutations, betaR52C and betaV77A. The amounts of enzyme (F(1) sector) found in membranes of the frameshift with or without the beta subunit mutation(s) were similar to those in the wild type (Fig. 2a). Consistent with the 7 additional residues attached to the carboxyl terminus, the mobilities of the frameshift subunits on electrophoresis were significantly lower than that of the wild type. This was clear with partially purified F(1) (Fig. 2b). Essentially the same amounts of the mutant and wild type subunits were also identified on Western blotting (data not shown).


Figure 2: Polyacrylamide gel electrophoresis of F(1)-ATPase fractions from DK8 harboring various mutant plasmids. Cells were grown in a synthetic medium with glycerol as the sole carbon source. Their membranes (40 µg of protein) were subjected to polyacrylamide gel electrophoresis (12%) in the presence of sodium dodecyl sulfate and then stained with Coomassie Brilliant Blue (a). The solubilized F(1) sectors (EDTA extract, 15 µg of protein) were also subjected to electrophoresis, and portions of the gel are shown to indicate mobility differences between the wild type and frameshift subunits (b). The positions of F(1) subunits are indicated by arrows, together with the frameshift (fs). Lane 1, purified F(1) (15 µg); lane 2, frameshift; lane 3, frameshift/R52C; lane 4, frameshift/R52C/V77A; lane 5, frameshift/G150D; lane 6, wild type; lane 7, DK8/pBR322 (no unc operon); lane 8, G150D.



Second Site Mutations Restored Oxidative Phosphorylation and Energy Coupling

Growth by oxidative phosphorylation was substantially recovered in DK8 harboring recombinant plasmids with the frameshift and the beta subunit mutation, whereas no growth was observed for the frameshift (Table 1). DK8 harboring pBMUG15fsR1 (betaR52C) and pBMUG15fs R2 (betaG150D) gave about 70% of the growth yield of the wild type. The unc operon carried by pBMUG15fsR3 had two amino acid substitutions (betaR52C and betaV77A) in the beta subunit together with the frameshift. We think that both the betaR52C and betaV77A mutations suppressed the frameshift mutation because DK8/pBMUG15fsR3 always gave a higher growth yield and rate than DK8/pBMUG15fsR1 having only a betaR52C replacement in the beta subunit. The growth rate of DK8/pBMUG15fsR1 was about 40% of the wild type level, whereas those of other mutants were essentially the same as that of the wild type (data not shown).

Membrane vesicles from the frameshift/betaR52C mutant and that with betaV77A exhibited high membrane ATPase activities (70 and 100% of the wild type level, respectively) and could synthesize ATP at about 15 ( frameshift/betaR52C) and 30% ( frameshift/betaR52C/betaV77A) of the wild type rate (Table 1). These results suggest that the beta subunit mutations suppressed the defective catalytic cooperativity of the frameshift and increased the membrane ATPase activity. Consistent with higher growth by oxidative phosphorylation, the frameshift/betaR52C/betaV77A mutant exhibited higher ATPase activity and ATP synthesis than the frameshift/betaR52C. Membrane vesicles from the frameshift with beta subunit mutations exhibited lower ATP synthesis than the wild type, suggesting that they are still defective in energy coupling.

The membrane ATPase activity of frameshift/betaG150D was found to be low when assayed by the standard procedure. The frameshift/betaG150D enzyme was unstable under these conditions. As shown previously(14) , the Arg-alpha296 Cys/Ser-beta174 Phe enzyme was quickly inactivated but stabilized with high concentration of KCl(14) . Similar to this mutant enzyme, the frameshift/betaG150D enzyme was stabilized by KCl, and ATPase activity was found to be highest (2.6 units/mg protein, about 10% of the wild type level) when assayed in the presence of 70 mM KCl (Fig. 3a). The optimal pH values of the mutant enzyme with and without KCl were 7.0-8.0 and 6.5, respectively, whereas that of the wild type was 8.5 regardless of the presence of KCl (Fig. 3, a and b); the specific activity of the mutant ATPase was found to be about 40% of the wild type level when assayed at pH 6.5. Thus the high growth yield of the mutant by oxidative phosphorylation may be due to the restored energy coupling because the mutant exhibited lower ATPase activity than the wild type even assayed under different conditions. Repeated trials to obtain membrane vesicles with ATP synthesis from the double mutant were not successful; we tried buffers with different pH values and ionic strengths for the preparation of membranes and assaying ATP synthesis, but the mutant membranes were always leaky to protons. The altered properties of the mutant were due to the combination of the frameshift and betaG150D; as separate mutants, the frameshift and betaG150D exhibited 23 and 17% of the membrane ATPase activity of the wild type, respectively, when assayed at pH 8.0, and membranes of the betaG150D single mutant were not leaky to protons, showing 13% of the ATP synthesis level of the wild type (Table 1).


Figure 3: Effects of pH and KCl on the membrane ATPase activity of the frameshift/betaG150D mutant. pH activity profiles of the frameshift/betaG150D (a) and wild type (b) membrane enzymes are shown. Membranes were diluted with TKDG buffer and then ATPase activity was measured with (closed symbols) and without (open symbols) 70 mM KCl. The standard assay conditions were used except that different buffers were used: pH 6 and 6.5, 20 mM MES-NaOH; pH 7 and 7.5, MOPS-NaOH; pH 8 and 8.5, Tris-HCl.




DISCUSSION

Protons transported through F(0) may cause a series of conformational changes in different subunits and finally drive ATP synthesis in the beta subunit. Similarly in the reverse reaction, ATP hydrolysis causes the beta subunit conformational change(s), which is transmitted through other subunits and finally to the H transport pathway in the F(0) sector. The frameshift mutant had two defects: a defect in catalytic cooperativity, giving low ATPase activity, and one in energy coupling, resulting in low efficiency in ATP synthesis. The frameshift subunit had a longer altered sequence downstream of Thr-277. With two methods(23, 24) , it was predicted that the mutant carboxyl-terminal forms a beta-strand, whereas with the same methods a wild type alpha helix similar to the x-ray structure was predicted(7) . The beta-strand may possibly extend about 60 Å from Thr-277 and interact with the upper beta-barrel and/or a part of the catalytic domain of the beta subunit (Fig. 4). Thus, the mutant enzyme became defective in energy coupling and catalytic cooperativity, possibly because the long carboxyl-terminal beta-strand inhibited the proper conformational transmission between the beta and subunit. It may also be possible that the transmission is carried out through the rotation of the subunit (7) and such mechanical movement was inhibited in the frameshift by the interaction of its carboxyl beta-strand with the beta subunit.


Figure 4: A model of F(1) with the frameshift mutation and suppression by the beta subunit mutations. A model was drawn based on the x-ray structure of bovine F(1)(7) . The frameshift has an unrelated sequence of 16 residues (additional 7 residues at the carboxyl terminus together with 9 altered residues downstream of Thr-277). With two methods(23, 24) , it was predicted that the carboxyl-terminal region of the frameshift forms a beta-strand of about 60 Å (shaded arrow). The betaR52C (with or without betaV77A) and betaG150D mutations restored membrane ATPase activity and ATP synthesis. Mutant residues (C, A, and D) are located following the bovine F(1) structure. The amino (N) and carboxyl (C) termini of the wild type and the frameshift (Cfs) subunit are shown. The glycine-rich phosphate loop (25, 26) containing the catalytic residues is also shown (P loop).



The defective energy coupling and catalytic cooperativity of the frameshift mutant were suppressed by amino acid replacements in the beta subunit, indicating that the altered beta- interaction in the frameshift was restored by the beta subunit mutations. Two different replacements, betaR52C with or without betaV77A and betaG150D, suppressed the frameshift, possibly through different mechanisms. The Arg-beta52 residue is conserved in all the beta subunit so far sequenced (57 different species; SWISS PROT rel. 30), and the corresponding bovine residue is located in the beta-barrel domain(7) . Val-beta77 corresponds to bovine Ile-84, which is located in the beta-sheet connecting the beta-barrel and nucleotide binding domains. Therefore, the deleterious interaction between the putative beta-strand of the carboxyl-terminal domain of the frameshift and the beta-barrel was restored by the second mutation (betaR52C) in the same domain. This is further supported by an additional suppressing effect of the betaV77A mutation.

Gly-beta150 is located in the phosphate loop containing the catalytic residues(25, 26) . The corresponding bovine loop may show a large conformational change during catalysis, because structures of this region in the nucleotide-bound and empty beta subunit are strikingly different(7) . Thus, the betaG150D mutation may affect the orientation of another loop located above the glycine-rich sequence or that of the carboxyl-terminal alpha helical domain. These structural considerations suggest that the defective energy coupling of the frameshift was suppressed by the Asp-beta150 mutation, possibly because the beta subunit mutation changed the mode of conformational transmission between the beta catalytic site and the subunit. Further studies with the present approach will be helpful for clarifying the conformational transmission among subunits during ATP synthesis.


FOOTNOTES

*
This work was supported by grants from the Japanese Ministry of Education, Science, and Culture and the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Postdoctoral Fellowship from the Japan Society for the Promotion of Science.

Present address: Dept. of Biology, College of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan.

**
Present address: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565, Japan.

§§
To whom correspondence should be addressed. Tel.: 81-6-879-8480; Fax: 81-6-875-5724; m-futai{at}sanken.osaka-u.ac.jp.

(^1)
The abbreviations used are: AMPPNP, 5`-adenylyl-beta,-imidodiphosphate; PCR, polymerase chain reaction; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid.


REFERENCES

  1. Futai, M., Noumi, T., and Maeda, M. (1989) Annu. Rev. Biochem. 58,111-136 [CrossRef][Medline] [Order article via Infotrieve]
  2. Fillingame, R. H. (1990) in The Bacteria (Krulwich, T. A., ed) pp. 345-391, Academic Press, New York
  3. Senior, A. E. (1990) Annu. Rev. Biophys. Biophys. Chem. 19,7-41 [CrossRef][Medline] [Order article via Infotrieve]
  4. Wilkens, S., and Capaldi, R. A. (1992) Arch. Biochem. Biophys. 299,105-109 [Medline] [Order article via Infotrieve]
  5. Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1991) J. Biol. Chem. 266,21197-21201 [Abstract/Free Full Text]
  6. Abrahams, J. P., Lutter, R., Todd, R. J., van Raaij, M. J., Leslie, A. G. W., and Walker, J. E. (1993) EMBO J. 12,1775-1780 [Abstract]
  7. Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370,621-628 [CrossRef][Medline] [Order article via Infotrieve]
  8. Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265,5043-5048 [Abstract/Free Full Text]
  9. Shin, K., Nakamoto, R. K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267,20835-20839 [Abstract/Free Full Text]
  10. Nakamoto, R. K., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268,867-872 [Abstract/Free Full Text]
  11. Aggeler, R., Cai, S. X., Keana, J. F. W., Koike, T., and Capaldi, R. A. (1993) J. Biol. Chem. 268,20831-20837 [Abstract/Free Full Text]
  12. Turina, P., and Capaldi, R. A. (1994) J. Biol. Chem. 269,13465-13471 [Abstract/Free Full Text]
  13. Nakamoto, R. K., Al-Shawi, M. K., and Futai, M. (1995) J. Biol. Chem. 270,14042-14046 [Abstract/Free Full Text]
  14. Omote, H., Park, M.-Y., Maeda, M., and Futai, M. (1994) J. Biol. Chem. 269,10265-10269 [Abstract/Free Full Text]
  15. Klionsky, D. J., Brusilow, W. S. A., and Simoni, R. D. (1984) J. Bacteriol. 160,1055-1060 [Medline] [Order article via Infotrieve]
  16. Tanaka, S., Lerner, S. A., and Lin, E. C. C. (1967) J. Bacteriol. 93,642-648 [Medline] [Order article via Infotrieve]
  17. Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265,21279-21284 [Abstract/Free Full Text]
  18. Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268,3156-3160 [Abstract/Free Full Text]
  19. Futai, M., Sternweis, P. C., and Heppel, L. A. (1974) Proc. Natl. Acad. Sci. U. S. A. 71,2725-2729 [Abstract]
  20. Lowry, O. H., Rosebrough, N. J., Farr, A. C., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 [Free Full Text]
  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Moriyama, Y., Iwamoto, A., Hanada, H., Maeda, M., and Futai, M. (1991) J. Biol. Chem. 266,22141-22146 [Abstract/Free Full Text]
  23. Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13,212-245
  24. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120,97-120 [Medline] [Order article via Infotrieve]
  25. Omote, H., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267,20571-20576 [Abstract/Free Full Text]
  26. Senior, A. E., and Al-Shawi, M. K. (1992) J. Biol. Chem. 267,21471-21478 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.