THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 31, Issue of November 5, pp. 16106-16112,1988 Printed in U. S. A.

Fo Portion of Escherichia coli H’-ATPase CARBOXYL-TERMINAL REGION OF THE b SUBUNIT IS ESSENTIAL FOR ASSEMBLY OF FUNCTIONAL Fo*

(Received for publication, May 24, 1988)

Michiyasu Takeyama, Takato Noumi, Masatomo Maeda, and Masamitsu Futai From the Department of Organic Chemistry and Biochemistry, The Institute of Scientific and industrial Research, Osaka Uniuersity, Ibaraki, Osaka 567, Japan

Six chromosomal uncF mutants of Escherichia coli defective in the b subunit of H+-ATPase (156 amino acid residues) were identified (KF92, Met-1 + Val; KF164, Gln-64 + end; KF61 and KF144, Gln-104 + end; KF138, Gln-106 + end; and KF79, Gln-123 + end). The membranes of all these mutants had low ATPase activities (less than 5% of that of the wild type), and no functional H+ pathway, although the truncated b subunits were integrated into these mem- branes. These findings suggest that about 30 carboxyl- terminal amino acid residues of the b subunit are es- sential for formation of the F1-binding site and H+ pathway. For examination of the role(s) of the car- boxyl-terminal region(s) or residue(s) of the b subunit, recombinant plasmids carrying truncated uncF genes of various lengths were constructed by in vitro muta- genesis and introduced into a recAl derivative of strain KF92 (Met- 1 +Val). Analyses of the membranes from the resulting strains demonstrated that almost the entire carboxyl-terminal region of the b subunit is necessary for formation of functional Fo, since loss of the carboxyl-terminal residue resulted in significant reduction of both F1 binding and H+ translocation, and loss of two or more residues abolished both activities completely.

The H+-ATPase (FoF,) of Escherichia coli catalyzes the synthesis or hydrolysis of ATP coupled with an electrochem- ical gradient of H+ (for reviews, see Refs. 1-4). The extrinsic membrane portion of the enzyme, F1, has a catalytic function and is formed from a, 0, y, 6, and t subunits. The intrinsic membrane portion of the enzyme, Fo, acts as a H+ pathway and is formed from a, b, and c subunits. The Fo becomes a passive H+ pathway after removal of F1 and the movement of H+ through Fo is inhibited by the binding of dicyclohexylcar- bodiimide (DCCD)’ to the Asp-61 residue of the c subunit (5) or the replacement of this residue by Gly or Asn (6, 7).

Subunit b, coded by the uncF gene, has a unique primary structure consisting of the following two clearly defined do- mains: an amino-terminal hydrophobic domain (26-amino acid residues), presumably embedded in the membrane, and a hydrophilic domain (130-amino-acid residues between po- sition 27 and the carboxyl terminus) probably exposed to the

* This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, the Science and Technology Agency of the Japanese Government, and Mitsubishi Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviation used is DCCD, dicyclohexylcarbodiimide.

cytoplasm (2, 3, 8). Thus, the amino-terminal domain could be essential for the H+ pathway, and the carboxyl-terminal domain for the F1-binding site. This model is supported by studies using proteolytic enzymes (9, 10) and hydrophobic photoreactive reagents (11, 12). However, mutant analyses demonstrated that “Gly-131 -+ Asp” substitution impaired formation of the H+ pathway and the F1-binding site, indicat- ing that the two functions of FO could not be strictly separated by a single mutation (13,14). The “Gly-9 + Asp” substitution also affected the assembly of this subunit (13,15), while Trp- 26 could be replaced by an acidic or basic amino acid residue (16).

In this study, we identified five nonsense mutants (strains KF61, KF79, KF138, KF144, and KF164) and one missense mutant (strain KF92) of the b subunit. In strain KF92, the codon for Met-1 was replaced by Val, and no translation of the b subunit was observed. None of the six mutants has functional Fo, indicating the importance of the carboxyl- terminal region of the b subunit for Fo assembly. We also constructed recombinant plasmids carrying wild-type and mu- tant uncF genes and introduced them into the recAl derivative of strain KF92. Analyses of the membranes of the strains carrying various mutant b subunits demonstrated that loss of the carboxyl-terminal residue resulted in significant reduction of Fo functions and that loss of 2 or more residues abolished these functions completely.

EXPERIMENTAL PROCEDURES

Bacteria and Growth Conditions-E. coli mutants defective in the b subunit, KF61, KF79, KF92, KF138, KF144, and KF164, were

asparagine after transduction of KY7230 ( a n , thi, thy) with P1 phage isolated from strains unable to grow on succinate and not requiring

mutagenized with hydroxylamine (17). Strain KF96 (uncH96) defec- tive in the 6 subunit was isolated by a similar method. Strain KF92rA (uncF92, thi, recAl), a recAl derivative of KF92, was constructed as described previously (18). A deletion strain of the unc operon DK8 (AuncB-C) (19) and a UV-sensitive strain N1790 (trp, recA99, uurA54, rspL) (20) were kindly provided by Dr. R. D. Simoni and Dr. H. Ogawa, respectively. Minimal medium supplemented with thy- mine, thiamine, and a carbon source (either glucose or succinate) and a rich medium (L-broth) with or without 20 pg/ml of ampicillin or tetracycline were used for genetic studies (21). The same minimal

transducing phage, XhpSU+2, was kindly provided by Dr. H. Ozeki medium with glycerol was used for preparation of membranes. A

and used after propagation in E. coli strain W3110 (22). Genetic Analysis-Hybrid plasmids pRPG45 (carrying the entire

uncB, uncE, uncF, and uncH genes), pRPG56 (carrying the entire uncB and uncE genes and the amino-terminal part of the uncF gene between amino acid residues 1 and 63) and pRPG57 (carrying part of the uncF gene between residues 64 and 156 and the entire uncH gene) (23) were kindly provided by Dr. R. D. Simoni, and pKY159-16 (carrying the entire uncB and uncE genes and part of the uncF gene between residues 1 and 130) was described previously (24) (Fig. 1). Plasmid DNA was prepared and mixed with competent cells by the published procedure (25). Mutants with a defective uncF gene were

16106

H+-ATPase b Subunit 16107

identified in a group of strains that became Uric' (wild type) after complementation with pRPG45, and their alleles were localized on defined DNA segments by testing for Unc+ or Unc- (unable to grow on succinate by oxidative phosphorylation) cells after recombination with other hybrid plasmids (21).

The total chromosomal DNA of each mutant was prepared as described previously (26), digested with BamHI and EcoRI and sub- jected to electrophoresis on polyacrylamide gel (5.0% acrylamide, 0.17% bisacrylamide). The fraction of DNA fragments corresponding to a size of 1500 base pairs (Fig. L4) was ligated with pBR322 as described previously (26), and the recombinant plasmids were intro- duced into KF96 (uncH96). Plasmids carrying the mutant alleles converted strain KF96 to Unc+, and the inserts were subcloned into pUC18 (27) with appropriate restriction endonucleases for DNA sequencing (28). An oligonucleotide corresponding to the 463rd to 483rd bases of the antisense strand (numbered from the first letter of the initiation codon of the uncF cistron) was synthesized and used as a primer for DNA sequencing with other oligonucleotides available commercially.

Construction of Recombinant Plasmids Carrying the uncF Gene Mutagenized in Vitro-The HpaI-BssHII fragment carrying a part of the unc operon from the intracistronic region (between the uncE and uncF genes) to the amino-terminal half of the uncH gene was obtained from pRPG45, treated with the Klenow fragment of DNA polymerase I and ligated into the HincII site of pUC18. The resulting plasmid, pUWF02, was used for oligonucleotide-directed mutagenesis (Fig. 2 A ) . For replacing Glu-142 by an end codon (TGA), the two DNA fragments (41 base sense and 32 base antisense strand DNA) were synthesized and annealed (Fig. 2 8 , IZ). The double-stranded DNA formed has HaeII and BstEII sites at the promoter proximal and distal end, respectively, and was ligated into pUWFO2 digested with HaeII and BstEII. The resulting plasmid pUMF142e had the trun- cated gene with a termination codon at position 142 and newly introduced AccI site. The plasmid pUMF153e had the truncated gene with a termination codon at position 153. The double-stranded DNA was synthesized with AccI and BstEII sites at the promoter proximal and distal end (Fig. 2B, IIZ), respectively, and artificially introduced EcoRV and HindIII sites, and ligated with pUMF142e digested with AccI and BstEII endonucleases. Similarly, the following recombinant plasmids were constructed by ligating synthetic DNA (Fig. 2B, ZV- X ) into HindIII and BstEII sites of pUMF153e (Fig. 2B, IZI): pUMF155e, Glu-155 + end; pUMF156e, Leu-156 + end pUMF155Q, Glu-155 -P Gln; pUMF155D, Glu-155 + Asp; pUMF155A, Glu-155 -+ Ala; pUMF155K, Glu-155 - Lys; pUWFS3, wild type. pUMF79 (Gln-123 + end) was constructed by ligating the PuuII-BstEII frag- ment of cloned DNA from strain KF79 (Gln-123 “-f end) with the corresponding sites of pUWFO2. pUMF92 (Met-1 + Val) was con- structed by ligating the HpaI-BstEII fragment of cloned DNA from KF92 with the corresponding sites of pUWFO2. These recombinant plasmids were used for studying the synthesis of the membrane- bound b subunit. However, the growth yields of strain KF92rA har- boring these plasmids were extremely low in synthetic medium. Thus, the BamHI-SphI fragment (Fig. 2 A ) was cut out from these plasmids and ligated with the corresponding sites of PBR322. The resulting plasmids were named pBWFO2 (derived from pUWFOP), pBMF79 (pUMF79), pBMF142e (pUMF142e), pBMF153e (pUMF153e), pBMF155e (pUMF155e), pBMFl56e (pUMF156e), pBMF155Q (pUMF155Q), pBMF155D (pUMF155D), pBMF155A (pUMF155A), pBMF155K, (pUMF155K), and pBWFS3 (pUWFS3). Membranes of strain KF92rA carrying these plasmids were used for further studies.

Protein Synthesis Dependent on Recombinant Plasmids-The re- combinant plasmids carrying genes for the wild-type and mutant b subunits (Fig. 2B) were introduced into strain N1790. The recombi- nant plasmid pMCR533 (18) carrying genes for the a, c, b, 6, and CY

subunits was also introduced into N1790 and used as a control. The procedures for protein synthesis in UV-irradiated N1790 (maxicell system) were as described previously (29). Briefly, about 1 X lo9 cells/ ml in synthetic medium supplemented with casamino acids (0.5%) (Difco) and tryptophan (40 pg/ml) were irradiated with UV-light, and cycloserine (100 pg/ml) was added to the culture. Then [35S]methio- nine (40 pCi) was added, and the cultures were incubated for 60 min. Membrane and cytoplasmic fractions of the labeled cells were sepa- rated and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (30).

Other Procedures-Membranes for biochemical analysis were pre- pared from cells (in the late logarithmic phase, grown on glycerol) that had been passed through a French press (17). Membrane vesicles depleted of F1 (washed membranes) were obtained by washing mem-

branes with dilute buffer containing EDTA (17). ATPase activity (31), formation of an electrochemical gradient of protons (17), and the amount of protein (32) were determined by published procedures. Subunit c was identified as described previously (29). Briefly, subunit c labeled with [“CIDCCD was extracted from the membranes with a mixture of chloroform and methanol, and precipitated with ether. The preparation was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radiolabeled subunit was located by autoradiography. The radioactivity associated with the subunit was measured after solubilizing the gel with HZ02.

Amounts of the p subunit in lysates (13,000 X g, 15 min supernatant of cells disrupted in a French press) were estimated immunochemi- cally after separation of proteins by polyacrylamide gel electropho- resis (in the presence of sodium dodecyl sulfate) and their electro- phoretic transfer to nitrocellulose paper (22). The band corresponding to the p subunit was tagged with fluorescein isothiocyanate-labeled anti-F1 antibodies. The amount of p subunit was estimated by scan- ning the nitrocellulose paper with a Shimadzu dual wave length thin layer chromatography scanner (3930.

Materials-Anti-F1 antibodies (22) and F, (17) were prepared as described previously. Restriction endonucleases BssHII and BstEII were from New England BioLabs, Inc. Other restriction endonucle- ases, Klenow fragment, and T4 DNA ligase were purchased from Takara Shuzo Co., Kyoto, Japan. [cY-~’P]~CTP (400 Ci/mmol), [36S] methionine (1250 Ci/mmol), and [“CIDCCD (57 mCi/mmol) were obtained from Amersham Corp. Other reagents used were of the highest grade available commercially.

RESULTS

Determination of Altered Bases in Mutants Defective in the b Subunit-The DNA fragments carrying mutant alleles were cloned and both DNA strands of each allele were sequenced (Fig. 1, Table I). The sequences indicated one mutation in the initiation codon (Met-1 + Val) (strain KF92) and four non- sense mutations introducing termination codons at amino

1 436 650 851 1055 1094 1500 BP 0 HD P HI Bt Es E l

I I pRPG45 I I I

I I I 1 4 I 1 I

B pRPG56

pRPG57

pKY159-16

c / a l r - q IJ)I 6 J a D >-;-< ::$A

KF92 KF164 KFl44 KF138

FIG. 1. Mapping of the mutant alleles defective in the b subunit. A, sites of E. coli chromosomal DNA cleaved by restriction endonucleases: B, BarnHI; Bs, BssHII; Bt, BstEII; Hp, HpaI; P, PuuII; Ha, HaeII; EI, EcoRI. Numbers indicate base pairs (BP), taking the BamHI site as 1. B, DNA segments carried by recombinant plasmids used for mapping the mutants. C, reading frames of the a, c, b, 6, and CY subunits. D, locations of mutant alleles for strains KF61, KF79, KF92, KF138, KF144, and KF164.

TABLE I Determination of mutation sites in strains defective in the b subunit

Mutant alleles of strains isolated in this study were cloned and sequenced as described in the text.

Strain Codon change”

KF92 GTG(1) -+ GTA KF164 CAG(64) * TAG

Met-1 -+ Val Gln-64 -P end

KF61, KF144 CAG(104) -TAG Gln-104 + end KF138 CAG(106) -P TAG Gln-106 + end KF79 CAA(123) + TAA Gln-123 - end

Amino acid residue replaced

a Altered codons in the antisense strands are shown.

H+-ATPase b Subunit 16108

(6)

CTC TTC TAG TAG CTT CCA AGG CAC CTA CTT CGA CGA TTG TCG CTG TAG CAC CTA TTT GAA CAG CGA CTT GAC A T T GGC GCC GAG M G ATC ATC GAA CGT TCC GTG GAT G M GCT GCT M C AGC GAC ATC GTG GAT A M CTT GTC GCT GM CTG T M

H.

*I pW142e (G1u-142-end) CTC TTC TAG TAG CTT CCA AGG SAG C T G H C GAG AAC ATC ATC GM CGT K C GTC GAC G

Ha Ac

111 pUY153e IVaI-153-end) C GAC G A A GCT CCT AAC AGC GAT ATC GTG GAT M G CTT - TG CTT CGA CGA TTG TCG C m CAC CTA TTC GAA $A TTG Ac E V Hd 01

FIG. 2. Construction of recombinant plasmids carrying the uncF gene mutagenized in vitro. A, a plasmid carrying the wild-type uncF gene (pUWFO2) was constructed from pRPG45 (20) and pUC18 (25). Reading frames for the a, c , b, and 6 subunits, amino-terminal region (6') of the 6 subunit, promoter operator region (PO) of the lac operon, the genes for tetracycline resistance (Tet') a d &lactamme (Amp') are shown. See "Experimental Procedures" for details. B, amino acid sequence of the carboxyl-terminal region of the b subunit and corresponding nucleotide sequences of the two strands are shown together with reading frames for the a (umB cistron), c (uncE), b (uncF), 6 (uncH), and a (uncA) subunits (I). Plasmid pUMF142e (ZI) carrying a mutant gene (Glu-142 + end) was constructed by replacement of the corresponding region of pUWF02 by a synthetic double-stranded DNA. Plasmid pUMF153e carrying a "Val-153 + end" mutation was constructed from pUMF142e and a synthetic double-stranded DNA (ZZI) in a similar manner. Other plasmids were constructed from pUMF153e (Val-153 + end) and the DNA fragments indicated in the figure: pUMF155e (Glu-155 + end), IV; pUMF156e (Leu-156 + end), V; pUMF155Q (Glu-155 + Gln), VI; pUMF155D (Glu-155 +Asp), VIZ; pUMF155A (Glu-155 + Ala), VIII; pUMF155K (Glu-155 + Lys), IX, pUWFS3 (wild type), X . Codons for termination and new amino acid residues

Ha, H a d ; Bs, BssHII; Bt, BstEII; Ac, AccI; EV, EcoRV; Hd, HindIII; Af, AfZII; EI, EcoRI; P,. PuuII; Bg, BglII; D, in the synthetic DNA are indicated by bones. Abbreviations of restriction sites are as follows: Hp, HpaI; Hc, HincII;

DraI; Ba, BalI; S, Sack Sp, SphI; B, BamHI. BamHI-SphI fragments were cut out from all these recombinant plasmids and ligated with the corresponding sites of pBR322 to construct the series of recombinant plasmids used in further studies. (Both BamHI and SphI sites are in the multicloning site of pUC18.) For further details, see "Experimental Procedures."

acid residues 64 (KF164), 104 (KF61 and KF144), 106 (KF138), and 123 (KF79) of the b subunit. Consistent with these results, all nonsense mutants (CAG + TAG) except KF79 (CAA + TAA) became Unc+ (able to grow by oxidative phosphorylation) after lysogenization with a transducing phage carrying SU+2 suppressor, which reads the termination codon (TAG) as a Gln codon. These nonsense mutants are similar to those isolated by Simoni and co-workers (131, who mutagenized the uncF gene carried by a recombinant plasmid. Strain KF92 may not form a fragment of the b subunit, because initiation from the Val codon seems to be minor, and the Shine-Dalgarno sequence (33) cannot be found upstream of the rest of the Met codons (Met-22 and Met-30) and potential initiation codons (ATA, TTG, GTG) which may serve for minor initiation. Consistent with this prediction, strain N1790 with plasmid pUMF92 carrying the mutant uncF gene did not synthesize any detectable amount of the b subunit in either the membrane or cytoplasmic fraction (Fig. 3, lane 2).

Absence of R-ATPase in Mutant Membranes-The specific activities of membrane ATPase of the five mutants were less than 5% of that of the wild type (Table 11). The ATPase activities (azide-sensitive activities, see Ref. 34) in the cyto-

plasmic fractions of these mutants were slightly higher than that of the wild type.

The washed membranes of these mutants could bind only a small amount of F1 even when incubated with an excess amount of purified F1, whereas wild-type-washed membranes could bind enough F1 to give the same specific activity as that of unwashed membranes (Table 11). These results indicate that membranes of mutants lack most of the binding sites for F1.

Loss of H+ Translocation through Mutant Membrane- The formation of a H' gradient in wild-type or mutant mem- brane vesicles was determined semiquantitatively by meas- uring the quenching of quinacrine fluorescence (Fig. 4). Wild- type membranes showed ATP or respiration (with 5 mM lactate)-dependent quenching (Fig. 4A), whereas its washed membranes (without F1) did not show the quenching (Fig. 4E). The respiratory quenching was restored in the presence of DCCD, which seals the H+ pathway of Fo. However, as expected from the absence of ATPase activity, neither un- washed nor washed membranes from the mutants listed in Table I showed ATP-dependent quenching of fluorescence, but they showed respiration-dependent quenching. The traces were essentially the same as those for KF92rA (Fig. 4, B and

H+-ATPase b Subunit 16109 1 2 3 4 5 6 7 8

M C M C M C M M M M M - “ ”

Amp‘ - a -

6 -

b l

C

FIG. 3. Syntheses of wild-type and truncated b subunits directed by cloned mutant DNA segments. About lo8 cells car- rying various plasmids were irradiated with UV light and [35S]methi- onine was added. Membrane (M) and cytoplasmic (C) fractions were prepared and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent autoradiography as described under “Experimental Procedures.” 1 , pMCR533 (carry- ing genes for a, c , b, 6, and a subunits); 2, pUMF92 (Met-1 .--, Val); 3, pUMF79 (Gln-123 + end); 4 , pUMF142e (Glu-142 .--, end), 5, pUMF153e (Val-153 + end); 6, pUMF155e (Glu-155 .--, end); 7, pUMFl56e (Leu-156 .--, end); 8, pUWFO2 (wild type). The bands identified as 8-lactamase (Amp’) and wild-type (closed triangles) and mutant (open triangles) b subunits are indicated. The positions of a, c, and 6 subunits are also shown. Although not shown in the figure, no radioactive bands corresponding to truncated or wild-type b sub- units were found in the cytoplasmic fractions of cells harboring pUMF142e, pUMF153e, pUMF155e, pUMF156e, and pUWFO2.

TABLE I1 ATPase activities of membranes and cytoplasm of mutants

Membrane vesicles (Membranes) and cytoplasmic fractions (Cy- toplasm) were prepared (17), and ATPase activities (31) were assayed as described previously. Strains are shown together with the positions of mutations (in parentheses). Membrane vesicles from each strain were washed with dilute buffer containing EDTA, and suspended in 10 mM Tris-HCI, pH 8.0, containing 140 mM KCI, 2 mM 8-mercap- toethanol and 10% glycerol (Washed membranes). The washed mem- branes (0.5 mg of protein) were incubated with saturation amounts of purified F, (80 pg) in 1.0 ml of the same buffer containing 5 mM MgCl, for 20 min at 25 “C. The mixtures were centrifuged at 100,000 X g for 60 min. The pellet was washed with 1.0 ml of the above buffer and finally suspended at about 1 mg/ml. The ATPase activities of the washed membranes incubated with (+F1) or without (-FJ puri- fied F, were assayed. Averages of duplicate measurements are shown. Independent preparations gave similar results (Deviations were less than 10%).

ATPase activity

Strain Membranes Cytoplasm

KY7230 (wild) KF92 (Met-1 + Val) KF164 (Gln-64 .--, end) KF61 (Gln-104 .--,end) KF138 (Gln-106 .--, end) KF79 (Gln-123 + end)

units/mg 1.80 0.18 0.06 0.27 0.07 0.27 0.08 0.31 0.08 0.29 0.08 0.26

Washed membranes

-F1 +FI

0.11 1.25 0.01 0.05 0.01 0.06 0.02 0.04 0.02 0.06 0.01 0.04

F) . Quenching was also observed with mutant membrane on zdding a low concentration of lactate (100 pM) (35), whereas wild-type-washed membranes showed respiratory quenching with 100 p~ lactate only in the presence of DCCD. These results suggest that membranes from the four nonsense mu- tants as well as strain KF92 were unable to translocate H+.

The a and c subunits were synthesized and inserted into the membranes of KF92, as this strain was complemented by introduction of a recombinant plasmid pBWFO2 carrying only the wild-type uncF cistron for the b subunit. The low ATPase activity (Table 11) in membranes of KF92 may be due to F1- binding to FO without the b subunit. It must be noted that the c subunit was inserted into membranes of KF92 judging from the labeling with [I4C]DCCD. 18 and 14 pmol of DCCD/ mg of membrane protein were incorporated into the c subunits of KF92 and the wild type, respectively, whereas essentially no radioactive DCCD was found in DK8 (AuncB-C) mem- branes (less than 0.5 pmol of DCCD/mg of membrane pro- tein). These results suggest that the defects of Fo in KF92 membranes were due to mutational loss of the b subunit. Furthermore, observations of the nonsense mutants, including strain KF79 (Gln-123 + end), indicated that the 34-amino acid residues between Gln-123 and Leu-156 (carboxyl termi- nus) may be important for formation of functional Fo.

Complementation of Strain KF92rA by Truncated b Sub- units Coded by Recombinant Plasm&-To determine how many of the 34 residues in the carboxyl-terminal region are essential, we constructed plasmids carrying mutant uncF genes having termination codons corresponding to amino acid residues 142 (Glu-142 + end), 153 (Val-153 + end), 155 (Glu- 155 + end), and 156 (Leu-156 + end), and introduced them into strain KF92rA (reCAI derivative of KF92) (Fig. 2). Strain KF92rA had essentially the same properties as those of KF92 and became able to grow on succinate (Unc’ phenotype) by oxidative phosphorylation after introduction of pBWF02 (car- rying the wild-type uncF gene cloned from the chromosome) or pBWFS3 (carrying the wild-type uncF gene synthesized in uitro) (Table 111). Thus, the wild-type b subunit was synthe- sized from the uncF gene carried by plasmids and assembled into functional Fo in these transformants. In contrast, strain KF92rA with all mutant plasmids except pBMF156e did not show the Unc+ phenotype. Strain KF92rA with the wild-type plasmid could form visible colonies on succinate plates in about 12 h, whereas the same strain with pBMF156e (lacking the carboxyl terminus of the b subunit) took 24 h to form visible colonies. Furthermore, pBMF155e lacking 2 residues from the carboxyl terminus could not support growth of strain KF92rA on succinate.

Fl binding and H + Translocation of Membranes with Mutant b Subunits Coded by Recombinant Plasmids-Next we exam- ined whether the Unc- phenotype in cells carrying recombi- nant plasmid was correlated with the absence of F1-binding sites and H+ pathways in the membranes of these cells. Truncated b subunits (Gln-123 + end, Glu-142 + end, Val- 153 + end, Glu-155 + end, and Leu-156 + end) coded by mutant uncF genes were associated with membranes and could not be found in the cytoplasmic fraction (Fig. 3, lanes 3-7), suggesting that the carboxyl-terminal region is not required for integration of the b subunit into membranes. Visual inspection suggested that the amounts of truncated subunits in mutant membranes were similar to that of the normal b subunit in wild-type membranes (Fig. 3, lanes 3-8), regardless of whether the mutant genes were constructed in uitro or cloned from unc strains. However, F1 could not bind to the membranes with any mutant b subunit except that with “Leu-156 + end” mutation (Table 111).

The F1-ATPase activity on the membranes of strain KF92rA carrying a recombinant plasmid with the wild-type uncF gene (Table 111) was about 1h of that of membranes of wild-type cells where all the genes for FoFl are in the chro- mosome (Table 11). Expression of the wild-type b subunit in KF92rA cells restored ATP-driven H+ translocation in their

H+-ATPase b Subunit 16110

KY7230 (wild

E

L lactate A\

DCCD \

KF92rA

0 v ATP c

F w ATP

D V

H V ATP

L v - lactate

FIG. 4. Formation of a proton gradient in membrane vesicles of mutants measured by fluorescence quenching of quinacrine. Membrane vesicles (100 pg of protein) from the wild type ( A and E ) , KF92rA ( B and F ) , KF92rA/pBMF156e ( b subunit with Leu-156 --* end) (C and G), and KF92rA/pBWF02 (wild-type b subunit) (D and H ) in 1.0 ml of 10 mM Tricine-choline buffer (pH 8.0), containing 140 mM choline chloride and 1 p~ quinacrine were mixed with 20 pl of 1 M MgCl2, and the fluorescence (emission, 500 nm; excitation, 420 nm) was monitored. Washed membranes were prepared as described in the legend of Table 11. Fluorescence quenching was monitored after addition (open triangles) of 10 pl of 1.0 M D-lactate or 10 p1 of 0.20 M ATP. At the indicated time (closed triangles) 2 111 of 20 mM DCCD (ethanol solution) was added. Membranes prepared from all chromosomal mutants (Table I) and KF92rA with pBMF79 (Gln-123 -+ end), pBMF142e (Glu-142 -+ end), pBMF153e (Val-153 + end), or pBMF155e (Glu-155 + end) showed the same behavior as those of strain KF92rA. Membranes from KF92rA with pBMF155Q (Glu-155 + Gln), pBMF155D (Glu-155 + Asp), pBMF155K (Glu-155 + Lys), pBMF155A (Glu-155 + Ala), or pBWFS3 (synthetic wild-type b subunit) gave similar results to those for KF92rA with pBWFO2 (cloned wild-type b subunit).

membranes (Fig. 4D). Their washed membranes could not establish respiration-dependent H+ translocation, but this increased on addition of DCCD (Fig. 4 H ) , suggesting that normal FO was formed. The reason for the low membrane ATPase activity is unknown but may be due to the genetic polar effect (36) and reduced assembly of Fo subunits, since the level of F1 binding was similar even after long incubation of the washed membranes with an excess amount of purified F1 (Table 111). Consistent with this notion, the amount of the fi subunit in a KF92rA lysate, estimated immunochemically, was about 50% of that of the wild type (data not shown). Furthermore, Aris et al. (37) showed that the relative amounts of the a and b subunits in membranes were lower when the subunits were coded by recombinant plasmids than when they were coded by chromosome.

Membranes with the mutant Leu-156 + end b subunit had significantly higher ATPase activity than those of KF92rA (with or without other truncated b subunits), and washed membranes could bind F1 resulting in similar higher specific activity to that of membranes before washing (Table 111). The F1-ATPase bound to membranes from the mutant Leu-156 + end was slightly less sensitive to DCCD than wild-type mem- brane ATPase. The sensitivities of mutant and wild-type membranes were 46 and 78%, respectively. In accordance with the Fl-ATPase activity, membranes with the mutant b subunit (Leu-156 + end) could translocate H' dependent on ATP (at about 20% of the initial velocity of fluorescence quenching of the wild type) (Fig. 4, C and D). On the other hand, the membranes of KF92rA with a mutant b subunit of 154-amino acid residues or less did not show ATP-dependent quenching

like membranes of KF92rA (Fig. 4B). However, no passive H+ pathway could be observed in any washed membrane preparations obtained from strain KF92rA with mutant b subunits including the Leu-156 + end mutation (Fig. 4G; other mutants gave similar traces). These results indicate that the entire carboxyl-terminal region of the b subunit is neces- sary for formation of a normal Fo. Partially functional Fo could be formed, or rate of assembly became extremely low with a synthetic b subunit of 155 amino acid residues.

Effect of Glu-155 Substitution on the Function of Fo-As described above, the mutant b subunit (Leu-156 --., end) could form partially functional Fo, but the subunit lacking one more residue (Glu-155 + end) could not, suggesting the importance of the residue at position 155. Thus, we constructed mutant uncF genes in which Glu-155 was replaced by Asp, Gln, Lys, or Ala and introduced them into strain KF92rA. All these mutant b subunits were essentially as functional as the wild type, since strain KF92rA with any one of the mutant uncF genes could grow on succinate as the sole carbon source, and the F1-binding and H' translocation of the membranes of these mutants were all indistinguishable from those with wild- type plasmids (Table I11 and legend to Fig. 4). These results suggest that a specific amino acid residue is not required at position 155.

DISCUSSION

Both F1 binding and €3' translocation are prerequisite functions of Fo in H'-ATPase. Genetic analyses of mutant strains lacking one of the three Fo subunits a, b, and c (38) and reconstitution experiments with isolated subunits (39)

H+-ATPase b Subunit 16111

TABLE I11 Properties of strain KF92rA harboring plasmids with

mutant uncF genes The recA derivative of strain KF92, KF92rA, (uncF92 (Met-1 -+

Val), thi, recAl) was unable to grow on succinate. Recombinant plasmids carrying wild-type or mutant uncF genes were introduced into this strain.

ATPase activityb

Plasmid uncF Growth on" Washed mutation succinate Mem- mem-

branes branes -F, +F1

unitslmgprotein

pBWFO2 none (wild) + 0.47 0.02 0.43

pBWFS3 none (wild) + 0.45 0.03 0.37

pBMF79 Gln-123 -+ end - 0.06 0.01 0.03 pBMF142e Gln-142 -+ end - 0.07 0.01 0.03 pBMF153e Val-153 -P end - 0.06 0.01 0.03 pBMF155e Glu-155 -+ end - 0.06 0.01 0.03 pBMF156e Leu-156 -+ end + 0.12 0.01 0.10

pBMF155Q Glu-155 -+ Gln + 0.41 0.02 0.35

pBMF155A Glu-155 -+ Ala + 0.36 0.02 0.30 pBMF155K Glu-155 -+ Lys + 0.37 0.02 0.29

pBMF155D Glu-155 -+ ASP + 0.39 0.02 0.33

Strain KF92rA carrying different plasmids was plated on agar medium containing succinate as the sole carbon source: +, growth after incubation for 48 h; -, no growth.

* Membrane vesicles (Membranes) were obtained (17) and ATPase activities (31) were assayed. Washed membranes were prepared and incubated with (+F1) or without (-FI) purified F1 as described in the legend to Table 11. Strain KF92rA with or without pBR322 gave the same results as strain KF92 (see Table 11). Averages of duplicate measurements are shown. Independent preparations gave similar results (Deviations were less than 10%).

have indicated that all three proteins are necessary for a functional FO complex. Consistent with these results, we found that in the absence of synthesis of the 6 subunit (Met- 1 + Val) a functional Fo was not formed. Furthermore, membranes with truncated 6 subunits, including the subunit lacking 2-amino acid residues from the carboxyl terminus, were similar to those without a 6 subunit. Cells with mutant 6 subunits could not grow on succinate and had no functional Fo. These results suggest the importance of the carboxyl- terminal region of (including carboxyl terminus Leu-156) the 6 subunit for the assembly of Fo. In contrast to the 6 subunit, an a subunit lacking the carboxyl half of the polypeptide chain could form FO capable of binding about 50% as much F1 as the wild type (29), although no H' pathway was formed. All the b subunit mutants so far isolated are nonsense mutants except for two missense ones (Gly-9 + Asp and Gly-131 + Asp) (13-15). The primary sequences of 6 subunits and related polypeptide (6') from several species have been determined (40-44). The homologies between the sequences of the sub- units of different species are low, but their hydropathy profiles are similar. These results suggest that only a few amino acid residues in the 6 subunit are essential.

About 10% of the F1 binding of the wild-type strain (KY7230) was detected in the membranes of strain KF92rA (pBMF156e) with the mutant 6 subunit (Leu-156 + end). As KF92rA (pBMF156e) could grow slowly on succinate by oxi- dative phosphorylation, about 10% of the wild-type activity of H+-ATPase could support slow growth. We showed previ- ously that about 15% of the wild-type H+-ATPase activity was sufficient to support growth by oxidative phosphoryla-

tion, although the growth rate was about half of the wild type (45).

It is of interest to compare the present results with those of proteolysis experiments. Previous studies showed that pro- teolysis of washed membranes abolished their abilities to rebind F1 due to loss of the carboxyl-terminal region of the b subunit (9, 10). However, the passive H' pathway was still functional even without 20-25 residues from the carboxyl terminus of the 6 subunit (9). Thus, once functional FO is formed from the a, 6, and c subunits, the carboxyl-terminal region of b subunit is not required for maintaining the H+ pathway. However, the present studies demonstrated that almost the entire carboxyl-terminal region of the 6 subunit is required for formation of the H' pathway and F1-binding site. Consistent with our observations, a truncated 6 subunit (12 or 8.3 kDa) was reported to be unable to reconstitute a functional Fo with the a and c subunits (46). Thus the car- boxyl-terminal region is required for the assembly of the amino-terminal region of the b subunit with the a and c subunits. In this regard, it will be of interest to identify the proteolytic product(s) corresponding to the carboxyl-terminal region of 6 subunit, since the carboxyl-terminal fragment(s) may remain attached to the entire Fo complex and so maintain a functional H+ pathway.

1. 2.

3. 4. 5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20. 21.

22.

23.

24.

25.

26.

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