INTRODUCTION

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The F₁F₀ ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (for recent reviews, see Refs. 1-4). It comprises an F₁ complex, which contains the nucleotide-binding subunits involved in catalysis, and an F₀ complex, which conducts protons across the membrane. The enzyme from E. coli appears to be a minimal form of the ATP synthase, with eight essential subunits. Five different subunits are found in F₁: , , , , and , in a stoichiometry of 3:3:1:1:1. Three different subunits, a, b, and c, form F₀ with a likely stoichiometry of 1:2:9-12 (5). The movement of protons through F₀, from the periplasm to the cytoplasm, drives the net synthesis of ATP by F₁.

The pathway of protons through F₀ is thought to involve subunits a and c. Subunit b is essential for assembly of a functional F₀ (6, 7) but is not likely to participate directly in proton translocation. The subunits b are embedded in the membrane through a span of about 30 hydrophobic amino acids at the NH₂ terminus. NMR studies of c (8, 9) have confirmed the -helical hairpin structure of the two predicted transmembrane spans and have provided details about the environment of the essential residue Asp⁶¹ (10, 11). Questions remain about the oligomeric structure of c subunits and how they interact with subunits a and b. Atomic force microscopy (12, 13) and electron spectroscopic imaging (14) have provided some evidence for a ring of 9-12 c subunits adjacent to the subunits a and b.

Subunit a is the largest of the F₀ subunits (271 residues). Mutagenesis has revealed that in addition to Asp⁶¹ of subunit c, three residues in subunit a seem to be important in proton translocation: Arg²¹⁰, Glu²¹⁹, and His²⁴⁵ (15-19). Of these three, only Arg²¹⁰ is strictly conserved among all known sequences, which suggests a unique role for this residue. No substitutions at this position permit ATP synthesis, but an alanine substitution allows limited passive proton permeability (20). The positions of the Glu and His residues are found to be reversed in some organisms, such as human and bovine mitochondria. Other sequences lack an ionizable residue at one of these positions, such as Bacillus subtilis and spinach chloroplast. Correspondingly, some amino acid substitutions at positions 219 and 245 in E. coli have been found to be partially functional (16, 21). Therefore, these two residues can be considered to be of secondary importance.

In a previous study (22), we probed subunit a by a series of single amino acid insertions at seven distinct locations between residues 187 and 222. Alanine insertions after residues 212, 217, and 222 were highly disruptive of function, consistent with the importance of this region in proton translocation. Studies of other membrane proteins by single-amino acid insertions have been described, including bacteriorhodopsin (23) and lac permease (24). In the present study, we have extended the analysis of subunit a from residue 229 to 245. The results indicate a single region of disruption, between residues 238 and 245, but insertions after 245 have little effect.

	EXPERIMENTAL PROCEDURES

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Materials-- Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs, Inc. Materials for silver sequencing and plasmid minipreps were obtained from Promega Corp. Synthetic oligonucleotides were obtained from Operon Technologies, Inc. or National Biosciences. Urea was from International Biotechnologies, Inc. ACMA¹ was obtained from Molecular Probes. Dicyclohexylcarbodiimide (DCCD) and octyl glucoside were obtained from Sigma. Immunoblotting reagents and detergent-compatible protein assay materials were obtained from Bio-Rad. Nickel-nitrilotriacetic acid-agarose was obtained from Qiagen. ATP and anti-HA antibody were obtained from Boehringer Mannheim.

Bacterial Strains-- Strain XL1-Blue (recA1, endA1, gyrA96, thi, hsdR17(r_k,mk⁺), supE44, relA1, ,(lac), F', proAB, lacI^q ZM15, Tn10(Tet^R)) was obtained from Stratagene and was used for subclonings and mutagenesis. Strain RH305 (uncB205, recA56, srl::Tn10, bglR, thi-1, rel-1, Hfr PO1) (25) was used to characterize mutations in uncB. It produces an a subunit that is truncated near Pro²⁴⁰ (26) and is complemented by plasmids containing a wild type uncB gene. Cultures were grown at 37 °C, and cell density was monitored by optical density at 600 nm using a Milton Roy 1001 spectrophotometer. Rich medium was LB supplemented with 0.2% glucose, and minimal medium was A salts supplemented with succinate (0.2%) or with glucose, as indicated (27). Media were supplemented with chloramphenicol (34 mg/liter) or tetracycline (12.5 mg/liter) as appropriate. Growth yields were determined in minimal A supplemented with 7 mM glucose.

Construction of Plasmids and Insertions-- Plasmids used in this study are shown in Fig. 1. The uncB mutations analyzed in this study were constructed using the cassette mutagenesis technique, as described previously (28). To construct pSBV18, plasmid pSBV16 (21) was digested with BsaHI and BamHI to excise a 686-bp fragment. It was replaced in a two-step procedure. First, a synthetic 69-bp fragment (Fig. 2A) was ligated to the remaining large fragment of pSBV16. This construct retained the BsaHI site, but the BamHI site was lost. However, a BstYI site was created at the same position, which can generate compatible cohesive ends. Second, the new plasmid was digested with BstYI, producing a single fragment, and was ligated to the 617-bp BamHI fragment from pSBV11 (29). This produced pSBV18, shown in Fig. 1. Plasmid pBJA1018 was constructed from pSBV10 and pSBV18 by digesting each with BsaHI and BamHI. The 686-bp fragment from pSBV18, containing most of uncB, was ligated to the 3222-bp fragment of pSBV10 to produce pBJA1018. Insertion mutations (Fig. 3A) and missense mutations (Fig. 3B) at position 245 were constructed in pSBV18 and moved to pBJA1018 in the same way. Insertion mutations at positions 225, 229, and 233 (Fig. 3A) were constructed in pBJA1018.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Plasmids used to analyze insertion mutants in this study. The gene for subunit a, uncB, is shown at the left. Regions that have been replaced with synthetic DNA to introduce new restriction sites by silent mutations are shown in black. The boundaries of these regions are identified by restriction sites. Other identified restriction sites were used in mutagenesis. The gene coding for chloramphenicol resistance is indicated by Cm.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Synthetic DNA introduced into uncB to generate new unique restriction sites. A, the region between Pro8 and Val29 was constructed from two oligonucleotides of 68 and 70 nucleotides. B, the region between Val141 and Leu180 was constructed from four oligonucleotides. The top strand was derived from two oligonucleotides with lengths of 69 and 57 nucleotides, while the bottom strand was derived from oligonucleotides of 65 and 60 nucleotides (5'-end indicated first).

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Oligonucleotides used to construct mutations. A, insertion mutations. The site of the insertion follows the residue number shown at the left. The restriction sites used in the cassette mutagenesis are also indicated. The nucleotides inserted are GMC or GMT (M represents A plus C), resulting in an equal mixture of alanine and aspartic acid. B, missense mutations. The residue number of the missense mutation is indicated at the left, and the restriction sites used are also shown. At position 245, nucleotides KST (K represents G plus T; S represents G plus C) code for glycine, alanine, cysteine, and serine. At position 241, nucleotides TGT code for cysteine. At position 162, nucleotides CWC (W represents A plus T) code for leucine (wild type) and histidine.

Preparation of Cell Fractions and Assays-- Fractionation of cells and isolation of membranes and stripped membranes were carried out as described previously (15). Protein concentrations were determined by a detergent-compatible protein assay (Bio-Rad) using bovine serum albumin as standard. ATP hydrolysis assays and fluorescence quenching assays were performed essentially as described previously (28). ATP hydrolysis was measured in 50 mM Tris-HCl (pH 9.1), 1 mM MgCl₂, 3 mM ATP at 37 °C, and fluorescence assays were measured using 400 mg/liter of membrane protein in a solution of 50 mM MOPS (pH 7.3), 10 mM MgCl_2, 1 µM ACMA, and either 0.5 mM NADH or 0.1 mM ATP, as appropriate. The excitation wavelength was 410 nm, and the emission wavelength was 490 nm. Inhibition of ATP hydrolysis by DCCD was measured as described previously (28).

Immunoblotting-- Membranes were prepared as above from 250-ml cultures. The membrane pellets were suspended in 1.5 ml of a detergent solution containing 0.1% deoxycholate, 0.5% cholate, 1.5% octyl glucoside, and 50 mM Tris-HCl, pH 7.5, and incubated for 1 h at 25 °C. The solubilized membranes were centrifuged for 10 min at 16,000 × g in a microcentrifuge, and the supernatant fraction was mixed with 0.4 ml of nickel-nitrilotriacetic acid-agarose and incubated overnight at 4 °C. The agarose was washed according to the manufacturer's (Qiagen) batch method, using the detergent solution above, and eluted by suspending the agarose in 80 µl of detergent solution plus 0.5 M imidazole. After a 10-min incubation at 25 °C and 10 min of microcentrifugation at 16,000 × g, the supernatant fraction was collected as partially purified subunit a. The samples were electrophoresed and transferred to nitrocellulose as described previously (21). The a subunits were detected by probing first with mouse anti-HA antibody (5 mg/liter) followed by goat anti-mouse IgG-alkaline phosphatase conjugate, according to the manufacturer's instructions.

	RESULTS

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A series of single amino acid insertions, both alanine and aspartic acid, were constructed after residues 225, 229, 233, 238, 243, and 245 in the a subunit of the F₁F₀ ATP synthase, as described under "Experimental Procedures." These sites span the two residues of secondary importance in proton translocation, Glu²¹⁹ and His²⁴⁵. Growth yields of E. coli strains carrying these mutations were determined and are shown in Tables I and II. The four mutants with the lowest growth yields, those at positions 238 and 243, were all unable to grow on succinate minimal medium, indicating a deficiency in oxidative phosphorylation.

Membrane vesicles were prepared from cells carrying each of the 12 insertion mutations. The membrane and supernatant fractions were tested for ATPase activity to determine the relative binding of the F₁ sector to the membranes. The results are reported in Tables I and II. Alanine insertions (Table I) had little effect on F₁ binding after residues 225, 243, and 245, but after residue 238 significant loss of F₁ binding occurred. The ATPase activities of the membrane fractions were also tested for sensitivity to DCCD, a reagent that reacts specifically with the subunit c of the F₀ complex (31). These results are also presented in Tables I and II. Alanine insertions (Table I) after residues 225 and 245 had no detectable effect, while those after residues 238 and 243 caused a loss of sensitivity.

The membrane vesicles from all 12 insertion mutants were also tested for ATP-dependent proton translocation activity, using the fluorescent dye ACMA, and the results are shown in Fig. 4. Nearly normal ATP-driven proton translocation was seen with the mutants 225A, 225D, and 245A. Little or no proton translocation was seen with mutants 238A, 238D, 243A, and 243D. The other insertion mutants showed intermediate levels of proton translocation that were at least 50% of the wild type level. The membranes were also stripped of F₁ and assayed for passive proton permeability, using NADH to generate a proton gradient. These results are presented in Fig. 5. In general, the same trends were observed, but the aspartate insertions had a noticeably more significant effect on passive proton permeability than did the alanine insertions. Membranes from the four mutants unable to grow on succinate minimal medium (238A,D, and 243A,D) were tested for the level of subunit a by immunoblotting. The results, presented in Fig. 6, indicate that subunit a is present at somewhat reduced levels in the 238A,D mutants and at even lower levels in the 243A,D mutants.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   ATP-driven proton translocation assays of alanine insertions (A) and aspartate insertions (B). Proton gradient formation is indicated by quenching of the fluorescence of ACMA, as described under "Experimental Procedures."

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Passive proton permeability assays of stripped membranes prepared from alanine insertions (A) and aspartate insertions (B). Proton permeability through F0 stripped of F1 is indicated by diminished ability to quench fluorescence of ACMA in the presence of NADH, which rapidly builds a proton gradient. Preparation of stripped membranes and assay conditions are described under "Experimental Procedures."

View larger version (69K): [in this window] [in a new window]   Fig. 6.   Immunoblot analysis of insertion mutants that fail to grow on succinate minimal medium. Prestained protein standards in the first and last lanes are 143, 97, 50, 35, 30, and 22 kDa. Lane 2, wild type subunit a with HA and oligohistidine carboxyl terminus from pTW1HisHA. Lanes 3-6, insertion mutants as indicated.

The residue His²⁴⁵ was replaced by Gly, Ser, and Cys to see if small or polar residues at that position would retain any function. None of the three mutants was able to grow on succinate minimal medium after 2 days of growth at 37 °C. Some growth was seen when the H245S strain was supplemented with 10-100 mM sodium acetate. This had no effect on the inability of the uncB strain RH305 to grow on succinate minimal medium. Attempts to demonstrate enhanced ATP-dependent proton translocation, by preparing membranes or performing assays in the presence of 10-100 mM acetate, all failed to show significant differences. In contrast, membranes prepared from W241C showed normal rates of proton translocation, although passive proton permeability was somewhat reduced from the wild type level (results not shown).

In a previous study (21), residue Leu¹⁶² of subunit a in E. coli was identified as corresponding to a histidine that is conserved among all known Bacillus species. Three double mutants were constructed to see if relocating His²⁴⁵ to position 162 would retain function: L162H/H245G, L162H/H245S, and L162H/H245C. It was determined that none could grow on succinate minimal medium.

	DISCUSSION

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All subunits of the E. coli F₁F₀ ATP synthase are essential for ATP synthesis. Subunit a has been implicated in proton translocation through F₀, but its precise role remains largely unknown. Single amino acid insertion-scanning mutagenesis has been applied to a region of subunit a between the important residues Glu²¹⁹ and His²⁴⁵. The goal was to detect amino acid residues in subunit a that are important in proton translocation and regions of subunit a that are in close contact with other subunits. This study is a continuation of a previous study (22) in which insertions of alanine and aspartate were made after residues 187, 193, 198, 202, 212, 217, and 222 of subunit a. In that study, it was found that insertions of both alanine and aspartate after residues 212, 217, and 222 reduce or abolish ATP-driven proton translocation. In particular, the 222 insertions were the most severe and showed disruption of F₁ binding to membranes. Interestingly, this region of subunit a had also been found to be the location of several second-site suppressors of mutations in subunit c (32). Most of the rest of the insertions had little or no effect on function; therefore, no other residues were implicated in function.

In this study, a similar pattern of results was obtained. Insertions of alanine or aspartate after residues 238 or 243 disrupted oxidative phosphorylation such that the strains could not grow on succinate minimal medium. Assays of the membrane fractions indicated that F₁ binding was decreased significantly in the case of 238A, while some ATP-driven proton translocation remained. In contrast, membranes from the 243A mutant had normal F₁ binding but no ATP-driven proton translocation. In both cases, the DCCD sensitivity of ATPase activity was very low. These results indicate two different types of disruption by the insertions of alanine at 238 and at 243.

The phenotype of 238A is consistent with a disruption of a-b or a-c interactions. In a previous study, Kumamoto and Simoni (33) discovered that Pro²⁴⁰ was the site of two suppressor mutations of subunit b mutation G9D. This subunit b mutation had been isolated and characterized by two groups (34, 35) and was further analyzed by immunoprecipitation (36). Its properties include inability to grow on succinate minimal medium, reduced binding of F₁ to membranes, and greatly diminished ATP-driven proton translocation and sensitivity of ATPase activity to DCCD. Two virtually identical partial suppressors were identified as P240A and P240L in subunit a (33). The suppressors permitted growth on succinate minimal medium and significant ATP-driven proton translocation but had little effect on F₁ binding or DCCD sensitivity. Detergent solubilization and immunoprecipitation studies of the original mutant, G9D, indicated very low levels of subunit a in F₁F₀ complexes, suggesting that the mutation affects a-b interactions. In the presence of the suppressor mutation, a normal complex of subunits a, b, and c was detected (36). The results presented here, including loss of F₁ binding and DCCD sensitivity, are also consistent with an important interaction between subunit a in the region of Pro²⁴⁰ and either the membrane-spanning NH₂ terminus of subunit b or subunit c.

The phenotype of 243A was quite different, in that F₁ remained bound to the membranes, but ATP-driven proton translocation and sensitivity to DCCD were completely missing. The immunoblot using anti-HA showed at least a low level of subunit a, which presumably is necessary for normal F₁ binding. Therefore, the loss of function is not consistent with a large scale disruption of subunit interactions but rather suggests a more specific effect. Further insight into the 243A,D mutants may be gained by considering the insertions after residue 245 and the conservation of residues 241-245. Although His²⁴⁵ seems to be a functionally important residue from the results of mutagenesis (15, 19), the insertion of alanine following it has very little effect on function. One interpretation is that His²⁴⁵ does not interact with other residues toward the carboxyl terminus of subunit a but that the insertion at 243 disrupts an important interaction between the side chain of His²⁴⁵ and a nearby residue toward the amino terminus. The best candidate would be Trp²⁴¹, for the following two reasons. First, a tryptophan is found at this position in all subunit a sequences that have a histidine at position 245 (Fig. 7). Second, a recent study (37) of model peptides has revealed a pH-dependent stabilization of an -helical conformation mediated by Trpⁿ and His^n + 4. This stabilization was not seen for peptides with Trpⁿ/His^n + 3 or for Hisⁿ/Trp^n + 4. An analysis of proteins of known three-dimensional structure has revealed a significant number of instances of interactions in -helices between Trpⁿ and His^n + 4. The side chains were found to be oriented for an interaction between the protonated imidazolium of His and the planar indole ring of tryptophan. Such an interaction at the periplasmic surface of F₀ could function as a proton gate, regulated by the pK_a of His²⁴⁵. In this way, at sufficiently low pH, the imidazole of His²⁴⁵ would be protonated, allowing an interaction with Trp²⁴¹, and thereby stabilizing the -helix. This interaction might, through conformational or pK_a shifts, permit access of protons to Asp⁶¹ of subunit c. In such a scheme, neither His²⁴⁵ nor any other residues of subunit a, would be on the pathway of protons from the periplasm to Asp⁶¹ of subunit c. Rather, the pathway would be a pH-gated, water-filled "half-channel." It is interesting to note that another second-site suppressor of the subunit b G9D mutation was isolated in subunit c, A62S, near Asp⁶¹ (38).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Amino acid sequence comparison. The region of the E. coli subunit a from Asn238 to Ala267 is shown (41), along with the homologous regions from Haemophilus influenzae (42), Vibrio alginolyticus (43), and V. cholerae (the Institute for Genomic Research, personal communication). The regions spanning Trp241 and His245 are absolutely conserved and are shown in boldface type. The residue Gln252, which is strictly conserved among all species, is also shown in boldface type.

Analysis of the other insertions from residue 225 to 233 revealed, at most, intermediate effects on function. Both insertions after residue 225 were nearly identical to the wild type, suggesting that this position is not near a catalytic residue or an important subunit interaction. Insertions after residues 229 and 233 showed some loss of function, in particular the 233D mutant. These results seem to reflect the proximity of these positions to the 238 region and may be affecting subunit interactions or, indirectly, the conformation of His²⁴⁵.

The replacement of His²⁴⁵ with the small side chain amino acids glycine, serine, and cysteine led to total loss of function, as indicated by an inability to grow on succinate minimal medium. This suggested that water or fortuitous, neighboring side chains could not substitute for His²⁴⁵. Chemical rescue has been achieved for certain Asp mutants of bacteriorhodopsin using acetate and azide (39) and with carbonic anhydrase His mutants using imidazole (40), but our success was limited. Only when acetate, but not imidazole, was added to the growth medium of H245S was growth detectable on succinate minimal medium. These results are consistent with His²⁴⁵ being involved with gating rather than being on the proton pathway, because it is unlikely that an imidazole-Trp²⁴¹ interaction would induce a conformational change, but the smaller acetate might facilitate proton entry to a suboptimal channel.

The recent results of labeling studies (30) essentially eliminated the possibility that histidine at position Leu¹⁶² could substitute for the mutated His²⁴⁵, since the findings reported here were that no suppression occurred. In light of the findings here and of the membrane topology indicated by labeling studies, a revised model for a-c interactions and proton translocation is presented in Fig. 8. In this model, the half-channel from the periplasm to Asp⁶¹ of subunit c is gated by both His²⁴⁵ and Glu²¹⁹. Alanine insertions at residues 238, 241, and 222 are more disruptive than those at 245 and 217, indicating important conformations on the periplasmic side of these residues. Arg²¹⁰ resides at the other end of this half-channel, interacting with Asp⁶¹ of a subunit c. The half-channel from the second Asp⁶¹ to the cytoplasmic surface is drawn accessible to solvent, and this would keep the aspartate unprotonated. Glu¹⁹⁶, a conserved residue, is located near the cytoplasmic surface of this half-channel as a facilitator of proton transport. Mutagenesis of this residue (28) showed a charge- and polarity-dependent effect on rates of proton translocation. High resolution structural information about F₀ subunits will be necessary for further insight into the mechanism of proton translocation.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Model for the proton translocation pathway through F0. Arg210 of subunit a has a key electrostatic interaction with an ionized Asp61 of one subunit c. Glu219 and His245 of subunit a are located near the periplasmic surface and control access of protons to the ionized Asp61. His245 may be interacting directly with Trp241 as part of its gating function. Glu196 of subunit a is located near the cytoplasmic surface and may facilitate proton release there. For other details, see "Discussion."