©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The ATP Synthase Subunit
SUPPRESSOR MUTAGENESIS REVEALS THREE HELICAL REGIONS INVOLVED IN ENERGY COUPLING (*)

Robert K. Nakamoto (§) , Marwan K. Al-Shawi , Masamitsu Futai

From the (1) Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908 (2) Institute for Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A role in coupling proton transport to catalysis of ATP synthesis has been demonstrated for the Escherichia coli FF ATP synthase subunit. Previously, functional interactions between the terminal regions that were important for coupling were shown by finding several mutations in the carboxyl-terminal region of the subunit (involving residues at positions 242 and 269-280) that restored efficient coupling to the mutation, Met-23 Lys (Nakamoto, R. K., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 867-872). In this study, we used suppressor mutagenesis to establish that the terminal regions can be separated into three interacting segments. Second-site mutations that cause pseudo reversion of the primary mutations, Gln-269 Glu or Thr-273 Val, map to an amino-terminal segment with changes at residues 18, 34, and 35, and to a segment near the carboxyl terminus with changes at residues 236, 238, 242, and 246. Each second-site mutation suppressed the effects of both Gln-269 Glu and Thr-273 Val, and restored efficient coupling to enzyme complexes containing either of the primary mutations. Mapping of these residues in the recently reported x-ray crystallographic structure of the F complex (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628), reveals that the second-site mutations do not directly interact with Gln-269 and Thr-273 and that the effect of suppression occurs at a distance. We propose that the three subunit segments defined by suppressor mutagenesis, residues 18-35, 236-246, and 269-280, constitute a domain that is critical for both catalytic function and energy coupling.


INTRODUCTION

In the FF ATP synthase, energy coupling between proton transport and catalysis of ATP synthesis occurs via conformational changes transmitted through a complex made up of at least eight different subunits (for reviews see Refs. 1-7). A key subunit in the coupling mechanism is the subunit, which appears as a single copy in the complex. Changes in chemical cross-linking patterns (8, 9, 10, 11, 12, 13) , protease sensitivity (14) , intensities of fluorescence probes (15) , and immunoelectron micrographic images (16) have demonstrated that the subunit undergoes conformation changes in response to catalysis or proton motive force. A structural model of bovine F based on x-ray diffraction verifies that the subunit has specific interactions with the and subunits that contain the catalytic sites and suggests that these interactions may be critical for function (17) .

Through the use of mutagenesis, we established that the conserved terminal regions of the subunit are involved in coupling. By changing conserved residue Met-23 of the Escherichia coli subunit (286 amino acids in length) to lysine, energy coupling was rendered extremely inefficient (18) . Functional interactions between terminal regions were realized by identification of several second-site mutations near the carboxyl terminus that restored efficient coupling to the Met-23 Lys mutant (19) . Two such second-site mutations were the replacements, Gln-269 Arg and Thr-273 Ser. Other replacements of these two conserved residues invariably caused reduced turnover and coupling efficiency (20) ; the most severe mutations were Gln-269 Glu and Thr-273 Val. In this paper, we describe the identification of several intragenic second-site mutations that suppress Gln-269 Glu and Thr-273 Val. Taken together, the suppressor mutations reveal three subunit regions that functionally interact to mediate energy coupling.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides were synthesized with a Pharmacia LKB Gene Assembler Plus. [-P]dCTP (3000 Ci/mmol) and [P]P were from Amersham Corp. Restriction endonucleases and other DNA modifying enzymes were from Takara Shuzo Co., Nippon Gene Co., Toyobo Co., or New England Biolabs. Taq polymerase and deoxynucleotides were from Perkin-Elmer. For all other chemicals and enzymes, the highest grades commercially available were used.

Bacterial Strains, Plasmids, and Growth Conditions

The subunit-deficient E. coli strain, KF10rA (thi, thy, recA1, uncG10 (Gln-14 end)), was described previously (21) and grown as before (20) . Unless otherwise indicated, all strains were grown at 37 °C. To assure that mutations in chromosomal or plasmid-borne copies of uncG did not revert during an experiment, the phenotype of all strains were checked after growths and plasmids were isolated and sequenced. Expression and mutagenesis of uncG were performed in derivatives of plasmid pBMG15 (18) .

Random Mutagenesis, Selection of Pseudo Revertants, and Manipulation of pBMG15

The Gln-269 Glu and Thr-273 Val mutations first described by Iwamoto et al.(20) were moved to plasmid pBMG15 to facilitate replacement of the entire uncG coding sequence with the randomly mutagenized gene as was done by Nakamoto et al.(19) . uncG (Gln-269 Glu or Thr-273 Val) was randomly mutagenized using a modified polymerase chain reaction (19, 22) . Transformation of KF10rA with mutagenized plasmids, genetic selection and analysis of isolates able to grow on succinate minimal medium (suc) were done as before (19) .

To assure that no extraneous mutations accompanied the identified suppressor mutations, each suppressor mutation was isolated on a restriction fragment and ligated into the original pBMG15 (Gln-269 Glu) and pBMG15 (Thr-273 Val). Second-site mutations near the amino terminus were isolated on the NcoI to XbaI fragment, mutations between codons 233 and 246 were isolated on the RsrII to PstI fragment, and mutations of codon 269 were isolated on the PstI to BglII fragment. Reconstructed plasmids were sequenced to assure both mutations were present.

Molecular biological manipulations (23) and DNA sequencing (24) were done by standard protocols.

Biochemical Procedures

Membrane vesicles were prepared from strains grown at 37 °C in minimal medium containing 0.2% glucose. Logarithmic phase cells were passed through a French press at 16,000 p.s.i. and membranes isolated by differential centrifugation as described previously (25) . Protein (26) , ATPase activity (25, 27) , and ATP synthesis (28, 32) were assayed as described previously.

For immunoblotting, membrane proteins were prepared as described by Nakamoto et al.(29) and separated on a 12.5% SDS-polyacrylamide gel (30) . Proteins were then transferred to nitrocellulose (31) and reacted with polyclonal antibodies raised against E. coli F and subunits (obtained from Dr. Alan Senior, University of Rochester) or the subunit. Immunoreactive bands were detected using the TMB membrane peroxidase system (Kirkegaard & Perry Laboratories, Inc.).


RESULTS

Suppression of Met-23 Lys by Various Amino Acids at Positions 269 and 273 of the Subunit

Previously, residues Gln-269 and Thr-273 were implicated in energy coupling because mutations Gln-269 Arg and Thr-273 Ser were able to suppress the effects of the primary mutation, Met-23 Lys, and restored efficient energy coupling (19) . Interestingly, a number of other mutations at these same positions also suppressed Met-23 Lys and resulted in oxidative phosphorylation-dependent growth when succinate was used as the sole carbon source (suc). In addition to the original suppressor mutations, replacement of Gln-269 with Leu and Glu, and Thr-273 with Gly and Val conferred intragenic suppression of Met-23 Lys (). Of these, Gln-269 Glu and Thr-273 Val were the most deleterious and, as single mutations, did not allow growth on solid succinate medium at 37 °C. Clearly, the suppression between either of these mutations and Met-23 Lys was mutual. Similar to previously described mutations at these positions (19), Gln-269 Glu and Thr-273 Val mutant strains were temperature-sensitive and were able to grow on succinate at 30 °C.

Subunit Mutations That Suppress Gln-269 Glu and Thr-273 Val

In turn, we searched for second-site mutations that would suppress the effects of Gln-269 Glu and Thr-273 Val. Random mutations were generated in uncG (Gln-269 Glu or Thr-273 Val) and screened for the ability to grow by oxidative phosphorylation. Twelve stable suc colonies arose, and plasmids were isolated and sequenced. As listed in , six different mutations that resulted in amino acid changes were found as single second-site mutations. Two other second-site mutations, Glu-233 Gly and Ala-240 Val, were accompanied by changes of Glu-269. Finally, two second-site mutations, Asp-36 Gly and Met-246 Leu, were found on the same plasmid with Thr-273 Val. To assure that the clustering of suppressor mutations near the ends of the gene was not due to a bias of the mutagenesis system, several randomly selected plasmids were sequenced. Mutations were found distributed throughout the mutagenized segment which included the entire uncG coding sequence (data not shown). These observations indicated that mutations were randomly introduced throughout the gene.

The suppression behavior of each second-site mutation was tested by isolating it on a restriction fragment and ligating into the original plasmid with either Gln-269 Glu or Thr-273 Val. Based on increased growth yields in liquid succinate medium, we confirmed that Ser-34 Leu, Gln-35 Arg, Ala-236 Thr, Glu-238 Gly, Arg-242 His, and Met-246 Leu partially suppressed the effects of the original primary mutation. In addition, we found that each of these mutations suppressed both Gln-269 Glu and Thr-273 Val. Although the growth yield was lower than the others, Lys-18 Met was able to suppress Gln-269 Glu, but only imparted a slight increase in growth to Thr-273 Val. The remaining mutations listed in were unable to suppress; however, strains carrying Glu-269 Lys or Gly as single mutations grew on succinate (data not shown).

In summary, the suppressor mutations fell into two groups: three were found in the conserved amino-terminal region between positions 18 and 35, and four in the conserved carboxyl-terminal region between positions 236 and 246. The mutations near the amino terminus changed residues that are conservatively replaced in the known subunit sequences and are adjacent to residues that are completely conserved, while those near the carboxyl terminus changed residues that are conserved or nearly so. In general, the second-site mutations near the amino terminus were not as effective as ones near the carboxyl terminus in suppressing effects of the primary mutations.

Suppressor Mutations Restore Efficient Coupling

The ATPase activities of Gln-269 Glu or Thr-273 Val mutant enzymes were greatly reduced compared to wild-type enzyme (I), as were ATP-dependent proton pumping (Fig. 1A) and NADH-dependent ATP synthesis rates (I). The Val-273 enzyme generated a smaller electrochemical gradient of protons and had a lower rate of ATP synthesis than the Glu-269 enzyme despite having similar ATPase activities. Both properties suggested that the Val-273 enzyme was less efficient at coupling proton transport to catalysis. As an indicator of coupling efficiency, ATP synthesis and ATP hydrolysis rates were compared. Assuming that hydrolysis rates represent the catalytic competence of the enzyme, we can use synthesis:hydrolysis ratios to indicate the ability of the FF complex to couple energy between proton transport and catalysis. The synthesis:hydrolysis ratios listed in I suggest that the coupling efficiency of the Glu-269 mutant enzyme was similar to wild type, whereas the Val-273 mutant enzyme was significantly lower.


Figure 1: Effect of subunit mutations on formation of ATP- or NADH-dependent electrochemical gradients of protons in membrane vesicles. 100 µg of membrane vesicle protein from strain KF10rA harboring the indicated mutant subunits were suspended in 1.0 ml of the buffer described in Table III. Fluorescence intensity at 530 nm (excitation at 460 nm) was monitored at 37 °C. A, ATP-driven quenching. At the indicated times (arrows), 5 µl of 0.2 M ATP (1 mM final concentration) or 1 µl of 1 mM carbonylcyanide-m-chlorophenylhydrazone (CCCP) (1 µM final) were added. B, NADH-driven quenching. At the indicated times (arrows), 20 µl of 0.1 M NADH (2 mM final), 10 µl of 0.3 M KCN (3 mM final), or 1 µl of 1 mM carbonylcyanide-m-chlorophenylhydrazone (CCCP) were added.



When the Glu-269 and Val-273 mutations were combined with each of the second-site mutations, the ATPase hydrolysis rates were essentially unchanged; however, proton pumping and ATP synthesis rates were increased (Fig. 1A and I). Significantly, the ATP synthesis:hydrolysis ratios for the double mutant enzymes (Glu-269 or Val-273 plus a suppressor mutation) were 2-4-fold higher than for the single mutants. These data indicate that coupling between proton transport and catalysis became more efficient and even exceeded that of wild-type enzyme.

Interestingly, the differences in activity between the Glu-269 and Val-273 mutant enzymes and wild type were not as striking as the differences in oxidative phosphorylation-dependent growth (). A possible reason was that activities were measured in conditions optimal for the Glu-269 and Val-273 enzymes (pH 7.5 and 200-300 mM KCl),() and that in vivo conditions, especially during oxidative phosphorylation-dependent growth, may have caused the Glu-269 and Val-273 mutations to perturb enzyme function to a greater extent.

The behavior of the Glu-269 and Val-273 mutant complexes could be explained by loosely associated or unstable F, which readily dissociates from the membrane leaving F to passively conduct protons. Mutant membranes were tested for the ability to generate a proton motive force from NADH via electron transport (Fig. 1B). Similar electrochemical gradients of protons were generated regardless of the mutation present; therefore, the mutant F complexes appear to remain bound to the membranes and no free F exposed under our experimental conditions. In fact, immunoblot analysis of membranes using polyclonal antibodies against , , and subunits demonstrated that the F subunits were membrane-associated in all mutant complexes except when was not synthesized (Fig. 2). Interestingly, a small but significant amount of and subunits were membrane-associated even in the case of strain KF10rA harboring plasmid pBR322, which lacks uncG and expresses no subunit.


Figure 2: Immunoblot detection of , , and subunits in membrane preparations from strain KF10rA harboring pBMG(Glu-269 or Val-273) with selected suppressor mutations (Ser-34 Leu, Glu-238 Gly, and Arg-242 His). 25 µg of membrane protein from strains grown at 37 °C were separated on a 12.5% SDS-polyacrylamide gel and , , and subunit polypeptides detected by immunoblotting (see ``Experimental Procedures''). Lane1, wild type (pBWG15); lane2, no subunit (no uncG on plasmid pBR322); lane3, Glu-269; lane4, Glu-269/Ser-34 Leu; lane5, Glu-269/Glu-238 Gly; lane6, Glu-269/Arg-242 His; lane7, Val-273; lane8, Val-273/Ser-34 Leu; lane9, Val-273/Glu-238 Gly; lane10, Val-273/Arg-242 His.




DISCUSSION

The multiplicity of second-site mutations that suppress Gln-269 Glu and Thr-273 Val, in addition to those that suppress Met-23 Lys (19) , reveals three regions of the subunit that are involved in coupling (Fig. 3). One region is between residues 269-280 near the carboxyl terminus and was defined by a series of second-site mutations that suppressed Met-23 Lys (19) . The other two regions encompassing residues 18-35 and 236-246 were described in this study. We further note that changes at position Arg-242 suppressed Met-23 Lys as well as Gln-269 Glu and Thr-273 Val. We conclude that the three regions functionally interact because each shares primary mutation/suppressor mutation combinations with the other two regions. Moreover, because the primary mutations, Met-23 Lys, Gln-269 Glu, and Thr-273 Val, affect coupling and the second-site mutations restore coupling to varying degrees, we propose that the three regions are directly involved in coupling transport to catalysis. Interestingly, the three regions coincide with the conserved portions of the subunit (see Ref. 5).


Figure 3: Three interacting regions of the subunit involved in energy coupling. The -helical termini of the E. coli subunit (residues 1-45 and 223-286) are indicated by the stripedverticalbars. The position of the helices relative to one another was approximated from the structural model of Abrahams et al. (17) based on x-ray crystallographic analysis. In the model, the two helices are in a coiled-coil conformation. The three interacting regions involved in coupling and defined by suppressor mutagenesis are indicated by the brackets (see ``Discussion''). The position of the three primary mutations, Met-23 Lys, Gln-269 Glu, and Thr-273 Val, are indicated by the smallnumbers.



We are fortunate that the three subunit regions are found in the partial x-ray crystallographic structure of bovine F that was recently presented by Abrahams et al.(17) . Two important features of the structural model apply to our mutagenesis results. First, the structure shows that Gln-269 (E. coli numbering; equivalent of bovine residue Gln-255) forms a hydrogen bond with one of the subunits near its nucleotide binding site (17). The effect of replacing this residue confirms its significance. Although formation of the hydrogen bond in not essential (Arg, Gly, Leu, and Lys replacements retain function), turnover of the enzyme is greatly reduced as evidenced by low membrane ATPase and ATP synthesis rates (I and Refs. 19-20). Because the coupling efficiency of the Gln-269 Glu mutant enzyme was the same as wild type, this mutation appears to disrupt catalytic turnover in both hydrolysis and synthesis directions. One turn of the helix away, replacement of the conserved Thr-273 with valine caused the same decrease in ATP hydrolysis rate and, in addition, a proportionally larger decrease in ATP synthesis rate. These results suggest that changes of Thr-273 can influence catalysis and coupling, possibly by perturbing the conformational changes involved in linking proton transport to catalysis.

The second important feature derived from the crystal structure is the relative position of the three subunit regions identified by suppressor mutagenesis. Even though the coordinates of the structure are not yet available (17) , the model clearly shows that the amino acid side chains of residues 269-280 do not directly contact those of residues 236-246 or 18-35 (see Fig. 3). Instead, the 18-35 segment appears to be adjacent to residues 236-246 in the coiled coil and interaction with the 269-280 segment is through the intervening -helix. Considering the structure and the functional interactions, we propose that the three regions of the subunit defined by the suppressor mutations constitute a domain responsible for transmitting energy between proton transport and catalytic sites.

Our results also suggest that the structural integrity of the domain is extremely important for efficient energy coupling. We base this notion on two observations. First, several different amino acid changes were able to suppress the same primary mutations. These results indicate that suppression is not the repair of a single, specific interaction between two residues, but the restabilization of interactions between segments of the subunit and possibly the subunit as well. Second, the temperature sensitivity caused by the three primary mutations suggests that the structural stability of the complex was perturbed. Further structural and mutagenesis studies are required to decipher the mechanism by which the mutations affect coupling and catalysis. This information should provide an understanding of the mechanism by which proton transport is coupled to catalysis.

  
Table: Oxidative phosphorylation-dependent growth of Gln-269 and Thr-273 mutations with or without Met-23 Lys


  
Table: Second-site mutations found in plasmid-borne uncG (Gln-269 Glu or Thr-273 Val)


  
Table: Activities of mutant FF in membrane vesicles from strains grown at 37 °C



FOOTNOTES

*
This research was supported by Grant GM50957 from the National Institutes of Health (to R. K. N.) and grants from the Japan Society for the Promotion of Science (to R. K. N.), the Ministry of Education, Science and Culture of Japan (to M. F.), and the Human Frontiers in Science Program (to M. F.). 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.

§
To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, University of Virginia, Jordan Hall, Box 449, Charlottesville, VA 22908. Tel.: 804-982-0279; Fax: 804-982-1616; E-mail: rkn3c@virginia.edu.

M. K. Al-Shawi and R. K. Nakamoto, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Alan Senior of the University of Rochester for the gift of anti-/ antiserum and Alistair Erskine for technical assistance.


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