ARCHIVES
Vol.
OF BIOCHEMISTRY
284, No. 1, January,
AND
BIOPHYSICS
pp. 71-77,
1991
Role of the Carboxyl Terminal Region of H+-ATPase (F,F,) a Subunit from Escherichia co/i Seiji Eya, Masatomo
Maeda,
and Masamitsu
Department of Organic Chemistry and Biochemistry, Osaka University, Osaka 567, Japan
Received
June
11, 1990, and in revised
form
August
Futai The Institute
Academic
Press,
Inc.
Research,
located only in this region asjudged by comparison of the known amino acid sequences of the corresponding subunits from various sources (7, 10). Site-directed mutagenesis in this region suggestedthat Arg-210 is an essential component for proton translocation, and that the Glu219 and His-245 residues may also be directly involved in the proton translocation mechanism (7, 11-16). Furthermore, from analysis of nonsense mutants, we proposed that essential residues may be located in the region between Gln-252 and Ser-268 (17). In the present study, mutations were introduced into the Glu-269, Ser-265, Tyr263, Gln-252, His-245, Pro-230, Glu-219, and Arg-210 residues of the a subunit. Analysis of these mutant subunits suggestedthat the seven carboxyl-terminal residues are not required for the functional proton pathway, but that other residues or their vicinities have critical roles in maintaining the correct conformation of the proton pathway. We confirmed that Arg-210 is an essential residue for proton translocation. EXPERIMENTAL
H+-ATPase (F,F,) synthesizes ATP when coupled with an electrochemical gradient of protons and is located in membranes of bacteria, mitochondria, and chloroplasts [for reviews, see Refs. (l-5)]. The intrinsic membrane sector (F,) of Escherichia coli consists of three different subunits, a, b, and c coded by the genes uncB, uncF, and uncE, respectively, and acts as a proton pathway (l-5). Genetic analyses and in vitro reconstitution experiments have suggested that all three F0 subunits are required for formation of a functional proton pathway (6-9). The carboxyl-terminal third of the a subunit (271 amino acid residues) has been the focus of mutagenesis studies to identify the amino acid residues involved in proton translocation, since conserved hydrophilic residues are 0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
and Industrial
10, 1990
The effects of amino acid substitutions in the carboxyl terminal region of the H+-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263 + end, Trp-231 + end, Glu-219 + Gln, and Arg-210 + Lys (or Gln) were fully defective in ATPdependent proton translocation, and those with Gln-252 --t Glu (or Leu), His-245 + Glu, Pro-230 + Leu, and Glu-219 + His were partially defective. On the other hand, the phenotypes of the Glu-269 --, end, Ser-265 + Ala (or end), and Tyr-263 + Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation. 0 1991
of Scientific
PROCEDURES
E. coli strains. Strains KF116 [Pro-230 (CCG) -* Leu (CTG)] and KF135 [Tip-231 (TGG) + end (TGA)] defective in the u&3 cistron for the a subunit of H+-ATPase were isolated in this study after transduction of KY7230 (asn, thi, thy) with Pl phage mutagenized with hydroxylamine (18). Strains KF9 (uncB, Gln-20 + end) (lo), KF24A (uncB, Trplll + end; recAl) (17), and MV1184 (19) were described previously. Strain KY7485 (a lysogen of a X phage carrying the entire uric operon) (20) was used for the preparation of the F,F-ATPase complex. Construction of mutant uncB genes. pBBW (17) was digested with Hind111 and AuaI, and the 1106-bp fragment carrying the entire uncB gene was ligated into pUC19. The ZfindIII-EcoRI fragment (1122 bp) was recovered from the resulting plasmid (pUBW-l), and inserted into the HindIII-EcoRI site of pUC119. The recombinant plasmid was designated as pUBW-2. The plasmid pUB269e-2, carrying the mutant uncB gene (Glu-269 + end), was constructed from pMYB269e (17) as described above. The antisense strand DNA of pUBW-2 or pUB269e-2 was prepared from strain MV1184, harboring each plasmid and a helper phage M13K07. Oligonucleotides (Table I) were synthesized using a 71
Inc. reserved.
72
EYA,
MAEDA,
AND
TABLE
Oligonucleotides
Note. Oligonucleotides (boldface) were introduced
-+ + + + + + + + + + +
I
used for Construction
Mutation Arg-210 Arg-210 Glu-219 Glu-219 His-245 Gln-252 Gln-252 Tyr-263 Tyr-263 Ser.265 Ser-265
FUTAI
Synthetic Gln Lys Gln His Glu Glu Leu Phe end Ala end
of uncB Mutants oligonucleotide
3’.TGAGCCAAACGTCGACAAGCCAT-5’ 3’-GTGAGCCAAACTTTGACAAGCCAT-5 J-ACATACGGCCAGTCGACTAAAAGT-5’ Y-CATACGGCCAGTGGACTAAAAGT-5’ 3-CCGGTAAAAGCTTTAGGACTAGT-5 3’-TAATGCGACCTTCGGAAGTAG-5’ ZTAATGCGACGACCGGAAGTAG-5 b-CCAAGACTGGTAGCAGAAGGACAGCTAC-5’ 3’-ACCAAGACTGGTAGCAGATTGACAGCTA-5’ 3’-ACCAAGACTGGTAGCAGATAGACCGATACCGCAGAC-5’ 3’.TAGCAGATGAATACTTACCGATCGATTG-5
for sense strands were synthesized with codon changes (italics) to introduce to remove PuuI sites in the oligonucleotides for Tyr-263 + Phe (or end),
Model 381A DNA synthesizer (Applied Biosystems). Synonymous base changes were introduced into the oligonucleotides for the Tyr-263 + Phe (or end) and Ser-265 + Ala mutations to remove PuuI sites from the uncE genes (Table I, boldface). For in vitro mutagenesis (21), about 10 pmol of each mutagenic oligonucleotide was annealed with the antisense strand of pUB269e-2 (4 pmol, for Ser-265 + end mutation) or pUBW-2 (4 pmol, for other mutations), and closed circular doublestranded DNA was synthesized. Mutant recombinant plasmids were identified by hybridization with corresponding oligonucleotides as probes, or by the absence of specific restriction sites, and the mutations were confirmed by the dideoxy termination method (22). The HindIII-AuaI fragment carrying each mutant gene (962 bp for Ser-265 + end and Glu-269 + end; 1106 bp for other mutations) was inserted into the HindIII-AuaI site of pBR322, and the recombinant plasmids were designated as pBB21OQ (Arg-210 + Gln), pBB21OK (Arg-210 + Lys), pBB219Q (Glu-219 + Gin), pBB219H (Glu219 --, His), pBB245E (His-245 + Glu), pBB252E (Gln-252 + Glu), pBB252L (Gin-252 + Leu), pBB263F (Tyr-263 + Phe), pBB263e (Tyr263 + end), pBB265A (Ser.265 --, Ala), pBB265e (Ser-265 + end), and pBB269e (Glu-269 4 end), respectively. The pKF116 was obtained by ligating the HindIII-AuaI fragment (1106 bp) carrying the mutant uncB gene from strain KF116 (Pro-230 + Leu) into pBR322. pKF2 (uncB, Trp231 -* end), and pKF24 (uncB, Tip-111 * end) were described previously (10). The absence of mutations other than at the manipulated site was confirmed genetically by replacing the mutant restriction fragment by that of the wild-type and introducing the resulting plasmid into strain KF24A. Genetic complementation and bacterial growth. In the complementation test, strain KF24A was transformed with each recombinant plasmid and ampicillin (50 pg/ml)-resistant colonies on rich medium (10) were transferred to plates of minimal medium (23) containing thymine (50 pg/ml), thiamine (2 pg/ml), and a carbon source (11 mM glucose or 15 mM succinate), and incubated for 24 to 48 h at 37°C. At least 10 colonies for each mutant showing negative growth with succinate were isolated. For determining growth yield, KF24A harboring each plasmid was grown at 37’C in the same medium containing ampicillin and a carbon source (15 mM succinate or 5 mM glucose) and the optical density at 650 nm of the culture was monitored. Preparations and assays. Strain KF24A harboring each plasmid was grown at 37’C in minimal medium supplemented with 0.5% glycerol and ampicillin (50 pg/ml) and harvested in the logarithmic phase. Cells were suspended (at about 10 OD,,,/ml) in 10 mM Tris-HCI (pH 8.0), containing 140 mM KCl, 2 mM p-mercaptoethanol and 10% glycerol. Inverted membrane vesicles were prepared after disrupting cells in a French press and were washed once with dilute buffer to obtain F,depleted membranes (20). The ATPase activity (24), formation of a
mutations. and Ser-265
The synonymous + Ala mutations.
base changes
proton gradient and the amount
(20), DCCD’ sensitivity of the membrane ATPase (lo), of protein (25) were assayed by published methods. Mutant subunits Identification of mutant a subunits in membranes. in membranes were identified by Western blotting (26). Membranes (100 /Lg protein) from strain KF9 or KF24A harboring different recombinant plasmids were subjected to electrophoresis in polyacrylamide gel (12.5-17.5% or 12.5-22.5%) in the presence of sodium dodecyl sulfate. Gradient gels were used to improve identification of truncated subunits. Electroblotting of protein bands onto a nitrocellulose filter (0.45 bm, Toyo Roshi Co. Ltd. Japan) was performed at 10 V/cm for 5 h in a semidry blotting apparatus (ATT0 Model AE6670). Rabbit anti-u subunit antiserum (recognizing the amino terminal region between residues 2 and 11 of the a subunit) pretreated with membranes of the strain lacking the uric operon was generously supplied by Drs. J. Hermolin, and R. H. Fillingame. The antiserum was diluted (1.5 X 1O3-fold) with 10 mM Tris-HCl (pH 7.4) containing 5% bovine serum albumin and 0.9% NaCl. The binding of the anti-a subunit antibodies to the a subunit on the nitrocellulose filter was detected with peroxidase-conjugated goatanti-rabbit antibodies (Jackson Immune Research Laboratories). Wild-type F,F,-ATPase was obtained from the membranes of KY7485 after solubilization with 10 mM Tris-HCl (pH 8.0), containing 1.5% (w/v) octylglucoside and 1.0% (w/v) sodium cholate, and further purified by density gradient (lo-30% glycerol) centrifugation. Materials. The reagents used were obtained as follows: restriction endonucleases, Klenow fragment, and T4 DNA ligase from Nippon Gene Co. (Toyama. Japan); DNA polymerase I and exonuclease III from Takara Shuzo. (Kyoto); and (a-32P)dCTP (400 Ci/mmol) and (y-32P)ATP (4000 Ci/mmol) from Amersham Corp. All other reagents used were of the highest grade available commercially.
RESULTS
Construction of the a subunit mutants. From analyses of a group of nonsense mutants, we suggested that important residues for proton translocation were located between residues 252 and 271 (carboxyl terminus) of the a subunit (10). In contrast, we found that the three carboxy1 terminal residues (Glu-269, Glu-270, and His-271) were not required for a functional F0 (17). Therefore, we focused on the three hydrophilic residues between Gln1 Abbreviations used: DCCD, carboxylcyanide-m-chlorophenylhydrazone.
iV,N’-dicyclohexylcarbodiimide;
CCCP,
H+-ATPase
a SUBUNIT
252 and Ser-268, and introduced, Gln-252 + Gh (or Leu), Tyr-263 + Phe (or end), and Ser-265 + Ala (or end) mutations by oligonucleotide-directed mutagenesis (Table I). It is noteworthy that Gln-252 and Tyr-263 are conserved in the a subunits of other organisms (13). In addition, to confirm the role(s) of Arg-210, Glu-219, and His-245 in proton translocation (7,11-16), we constructed Arg-210 + Lys (or Gln), Glu-219 + His (or Gln), and His-245 + Glu mutants and compared their phenotypes with those previously observed. We also isolated a new un& mutant strain KF116 (Pro-230 + Leu) by random mutagenesis. Each mutant gene was cloned into pBR322 and introduced into strain KF24A (Tip-111 --* end), which is defective in ATP synthesis. Our system is suitable for examining the effects of mutant a subunits expressed from recombinant plasmids, since the short a subunit fragment (Trp-1113 end) synthesized from chromosomal DNA of KF24A did not interfere with the assembly of the longer a subunit from recombinant plasmids. Detailed results are shown below. Presence of mutant subunits in membranes. Membranes from KF24A harboring recombinant plasmids were subjected to immunoblot analysis using two gel electrophoresis systems and mutant subunits were detected immunochemically (Figs. 1A and 1B). Subunits with missense mutations showed essentially the same mobilities as the wild-type a subunit. Truncated subunits (Glu269 + end, Ser-265 + end, Tyr-263 + end, Trp-231 + end, and Trp-111 + end) showed significantly increased mobilities on electrophoresis, consistent with their lower molecular weights due to the loss of carboxyl terminal residues (Fig. lA, lanes 14-1’7; Fig. lB, lanes 7 and 9). The amounts of Tyr-263 -+ end (Fig. lA, lane 16), and Arg-210 * Lys (Fig. lA, lane 12) subunits were slightly less than those of the wild-type. It is noteworthy that the amount of the truncated subunit (Trp-111 --* end) coded by the host (KF24A) chromosome was very low (essentially not detectable) and the longer subunits coded by recombinant plasmids became predominant. Thus the properties of the longer subunits could be studied using this experimental system. Effectsof the a subunit mutations ongrowth by oxidative phosphorylation. The abilities of the mutant a subunits to support growth by oxidative phosphorylation were tested. AS shown in Table II, cells carrying the o subunits with Tyr-263 + end, Glu-219 + Gln, Arg-210 --f Lys, and Arg-210 + Gln mutations did not grow significantly on succinate, and showed greatly reduced growth on glucose, indicating that the H+-ATPases with these mutant subunits were defective in oxidative phosphorylation. The a subunits with Gln-252 --t Leu, Gln-252 --* Glu, His245 + Glu, Pro-230 + Leu, and Glu-219 + His mutations allowed growth by oxidative phosphorylation, but the growth yields were lower than that with the wild-type subunit. On the other hand, the growth and oxidative
FROM
Escherichia
A
1
2
3
73
coli
4
!i
6
7
6
9 10 11 12 13 14 15 16
17
a*
B 1
2
3
4
5
6
7
8
9
FIG. 1. Presence of mutant a subunits in membranes. Membranes (100 pg protein) from strain KF24A haboring different plasmids were subjected to 12.5-17.5% (A) or 12.5-22.5% (B) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and then proteins were transferred onto nitrocellulose filters. Mutant a subunits were detected immunochemically with anti-a subunit antibodies. The amount --, end) coded by the chromosome of of the truncated subunit (Trplll strain KF24A was reduced to an undetectable level when longer subunits were supplied from recombinant plasmids (horizontal arrowhead, (3). (A) Lane 1, FoF,; lane 2, pBBW (wild-type a subunit); lane 3, pBB265A (Ser-265 + Ala); lane 4, pBB263F (Tyr-263 + Phe); lane 5, pBB252E (Gln-252 + Glu); lane 6, pBB252L (Gin-252 + Leu); lane 7, pBB245E (His-245 + Glu); lane 8, pKF116 (Pro-230 + Leu); lane 9, pBB219Q (Glu-219 + Gln); lane 10, pBB219H (Glu-219 + His); lane 11, pBB2lOQ (Arg-210 --, Gln); lane 12, pBB21OK (Arg-210 + Lys); lane 13, pBBW; lane 14, pBB269e (Glu-269 + end); lane 15, pBB265e (Ser.265 + end); lane 16, pBB263e (Tyr-263 + end); lane 17, pKF2 (Trp-231 + end). (B) Lane 1, pBBW; lane 2, pBB252E; lane 3, pBB252L; lane 4, pBB219Q; lane 5, pBB219H; lane 6, pBB21OQ; lane 7, pBR322; lane 8, KF9 (Gln20 -, end); lane 9, pKF24 (Trp-111 * end).
phosphorylation activities of the Glu-269 + end, Ser265 --* Ala, Ser-265 + end, and Tyr-263 --+ Phe mutants were essentially similar to those with the wild-type subunit. Mutant subunits similar to the wild-type. The a subunits with Glu-269 + end, Ser-265 + Ala, and Tyr263 --* Phe mutations gave FoF, similar to that of the wild-type, consistent with the positive growths of these mutants by oxidative phosphorylation: the membrane ATPase activities of these mutants were similar to that of the wild-type, and were sensitive to DCCD (Table II). The mutant membranes showed the wild-type level of ATP-dependent proton translocation (quinacrine fluorescence quenching) (Fig. 2A). Wild-type membranes depleted of Fi were permeable to protons and showed only low level quenching of quinacrine fluorescence dependent on respiration (D-lactate), confirming previous results (17). However, DCCD sealed the proton pathway of the wild-type F0 in F,-depleted
74
EYA,
MAEDA, TABLE
AND
FUTAI
II
Growth Yields and Membrane ATPase Activities of Mutants Growth
Plasmid pBBW pBB269e pBB265A pBB265e pBB263F pBB263e pBB252E pBB252L pBB245E pKF116 pBB219H pBB219Q pBB210K pBB2lOQ pBR322
Mutation in a subunit
Membrane ATPase (units/mg)
15mM
5mM
glucose
(Wild-type) E269 + end S265 --, A S265 -+ end Y263 + F Y263 -+ end 6252 + E Q252 + L H245 + E P230 + L E219 + H E219 -+ Q R210 + K R210 + Q (No a subunit)
yield
100 106 102 84 98 52 93 78 62 50 33 48 45 26 30
succinate 100 110 95 73 92
Vol.
OF BIOCHEMISTRY
284, No. 1, January,
AND
BIOPHYSICS
pp. 71-77,
1991
Role of the Carboxyl Terminal Region of H+-ATPase (F,F,) a Subunit from Escherichia co/i Seiji Eya, Masatomo
Maeda,
and Masamitsu
Department of Organic Chemistry and Biochemistry, Osaka University, Osaka 567, Japan
Received
June
11, 1990, and in revised
form
August
Futai The Institute
Academic
Press,
Inc.
Research,
located only in this region asjudged by comparison of the known amino acid sequences of the corresponding subunits from various sources (7, 10). Site-directed mutagenesis in this region suggestedthat Arg-210 is an essential component for proton translocation, and that the Glu219 and His-245 residues may also be directly involved in the proton translocation mechanism (7, 11-16). Furthermore, from analysis of nonsense mutants, we proposed that essential residues may be located in the region between Gln-252 and Ser-268 (17). In the present study, mutations were introduced into the Glu-269, Ser-265, Tyr263, Gln-252, His-245, Pro-230, Glu-219, and Arg-210 residues of the a subunit. Analysis of these mutant subunits suggestedthat the seven carboxyl-terminal residues are not required for the functional proton pathway, but that other residues or their vicinities have critical roles in maintaining the correct conformation of the proton pathway. We confirmed that Arg-210 is an essential residue for proton translocation. EXPERIMENTAL
H+-ATPase (F,F,) synthesizes ATP when coupled with an electrochemical gradient of protons and is located in membranes of bacteria, mitochondria, and chloroplasts [for reviews, see Refs. (l-5)]. The intrinsic membrane sector (F,) of Escherichia coli consists of three different subunits, a, b, and c coded by the genes uncB, uncF, and uncE, respectively, and acts as a proton pathway (l-5). Genetic analyses and in vitro reconstitution experiments have suggested that all three F0 subunits are required for formation of a functional proton pathway (6-9). The carboxyl-terminal third of the a subunit (271 amino acid residues) has been the focus of mutagenesis studies to identify the amino acid residues involved in proton translocation, since conserved hydrophilic residues are 0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
and Industrial
10, 1990
The effects of amino acid substitutions in the carboxyl terminal region of the H+-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263 + end, Trp-231 + end, Glu-219 + Gln, and Arg-210 + Lys (or Gln) were fully defective in ATPdependent proton translocation, and those with Gln-252 --t Glu (or Leu), His-245 + Glu, Pro-230 + Leu, and Glu-219 + His were partially defective. On the other hand, the phenotypes of the Glu-269 --, end, Ser-265 + Ala (or end), and Tyr-263 + Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation. 0 1991
of Scientific
PROCEDURES
E. coli strains. Strains KF116 [Pro-230 (CCG) -* Leu (CTG)] and KF135 [Tip-231 (TGG) + end (TGA)] defective in the u&3 cistron for the a subunit of H+-ATPase were isolated in this study after transduction of KY7230 (asn, thi, thy) with Pl phage mutagenized with hydroxylamine (18). Strains KF9 (uncB, Gln-20 + end) (lo), KF24A (uncB, Trplll + end; recAl) (17), and MV1184 (19) were described previously. Strain KY7485 (a lysogen of a X phage carrying the entire uric operon) (20) was used for the preparation of the F,F-ATPase complex. Construction of mutant uncB genes. pBBW (17) was digested with Hind111 and AuaI, and the 1106-bp fragment carrying the entire uncB gene was ligated into pUC19. The ZfindIII-EcoRI fragment (1122 bp) was recovered from the resulting plasmid (pUBW-l), and inserted into the HindIII-EcoRI site of pUC119. The recombinant plasmid was designated as pUBW-2. The plasmid pUB269e-2, carrying the mutant uncB gene (Glu-269 + end), was constructed from pMYB269e (17) as described above. The antisense strand DNA of pUBW-2 or pUB269e-2 was prepared from strain MV1184, harboring each plasmid and a helper phage M13K07. Oligonucleotides (Table I) were synthesized using a 71
Inc. reserved.
72
EYA,
MAEDA,
AND
TABLE
Oligonucleotides
Note. Oligonucleotides (boldface) were introduced
-+ + + + + + + + + + +
I
used for Construction
Mutation Arg-210 Arg-210 Glu-219 Glu-219 His-245 Gln-252 Gln-252 Tyr-263 Tyr-263 Ser.265 Ser-265
FUTAI
Synthetic Gln Lys Gln His Glu Glu Leu Phe end Ala end
of uncB Mutants oligonucleotide
3’.TGAGCCAAACGTCGACAAGCCAT-5’ 3’-GTGAGCCAAACTTTGACAAGCCAT-5 J-ACATACGGCCAGTCGACTAAAAGT-5’ Y-CATACGGCCAGTGGACTAAAAGT-5’ 3-CCGGTAAAAGCTTTAGGACTAGT-5 3’-TAATGCGACCTTCGGAAGTAG-5’ ZTAATGCGACGACCGGAAGTAG-5 b-CCAAGACTGGTAGCAGAAGGACAGCTAC-5’ 3’-ACCAAGACTGGTAGCAGATTGACAGCTA-5’ 3’-ACCAAGACTGGTAGCAGATAGACCGATACCGCAGAC-5’ 3’.TAGCAGATGAATACTTACCGATCGATTG-5
for sense strands were synthesized with codon changes (italics) to introduce to remove PuuI sites in the oligonucleotides for Tyr-263 + Phe (or end),
Model 381A DNA synthesizer (Applied Biosystems). Synonymous base changes were introduced into the oligonucleotides for the Tyr-263 + Phe (or end) and Ser-265 + Ala mutations to remove PuuI sites from the uncE genes (Table I, boldface). For in vitro mutagenesis (21), about 10 pmol of each mutagenic oligonucleotide was annealed with the antisense strand of pUB269e-2 (4 pmol, for Ser-265 + end mutation) or pUBW-2 (4 pmol, for other mutations), and closed circular doublestranded DNA was synthesized. Mutant recombinant plasmids were identified by hybridization with corresponding oligonucleotides as probes, or by the absence of specific restriction sites, and the mutations were confirmed by the dideoxy termination method (22). The HindIII-AuaI fragment carrying each mutant gene (962 bp for Ser-265 + end and Glu-269 + end; 1106 bp for other mutations) was inserted into the HindIII-AuaI site of pBR322, and the recombinant plasmids were designated as pBB21OQ (Arg-210 + Gln), pBB21OK (Arg-210 + Lys), pBB219Q (Glu-219 + Gin), pBB219H (Glu219 --, His), pBB245E (His-245 + Glu), pBB252E (Gln-252 + Glu), pBB252L (Gin-252 + Leu), pBB263F (Tyr-263 + Phe), pBB263e (Tyr263 + end), pBB265A (Ser.265 --, Ala), pBB265e (Ser-265 + end), and pBB269e (Glu-269 4 end), respectively. The pKF116 was obtained by ligating the HindIII-AuaI fragment (1106 bp) carrying the mutant uncB gene from strain KF116 (Pro-230 + Leu) into pBR322. pKF2 (uncB, Trp231 -* end), and pKF24 (uncB, Tip-111 * end) were described previously (10). The absence of mutations other than at the manipulated site was confirmed genetically by replacing the mutant restriction fragment by that of the wild-type and introducing the resulting plasmid into strain KF24A. Genetic complementation and bacterial growth. In the complementation test, strain KF24A was transformed with each recombinant plasmid and ampicillin (50 pg/ml)-resistant colonies on rich medium (10) were transferred to plates of minimal medium (23) containing thymine (50 pg/ml), thiamine (2 pg/ml), and a carbon source (11 mM glucose or 15 mM succinate), and incubated for 24 to 48 h at 37°C. At least 10 colonies for each mutant showing negative growth with succinate were isolated. For determining growth yield, KF24A harboring each plasmid was grown at 37’C in the same medium containing ampicillin and a carbon source (15 mM succinate or 5 mM glucose) and the optical density at 650 nm of the culture was monitored. Preparations and assays. Strain KF24A harboring each plasmid was grown at 37’C in minimal medium supplemented with 0.5% glycerol and ampicillin (50 pg/ml) and harvested in the logarithmic phase. Cells were suspended (at about 10 OD,,,/ml) in 10 mM Tris-HCI (pH 8.0), containing 140 mM KCl, 2 mM p-mercaptoethanol and 10% glycerol. Inverted membrane vesicles were prepared after disrupting cells in a French press and were washed once with dilute buffer to obtain F,depleted membranes (20). The ATPase activity (24), formation of a
mutations. and Ser-265
The synonymous + Ala mutations.
base changes
proton gradient and the amount
(20), DCCD’ sensitivity of the membrane ATPase (lo), of protein (25) were assayed by published methods. Mutant subunits Identification of mutant a subunits in membranes. in membranes were identified by Western blotting (26). Membranes (100 /Lg protein) from strain KF9 or KF24A harboring different recombinant plasmids were subjected to electrophoresis in polyacrylamide gel (12.5-17.5% or 12.5-22.5%) in the presence of sodium dodecyl sulfate. Gradient gels were used to improve identification of truncated subunits. Electroblotting of protein bands onto a nitrocellulose filter (0.45 bm, Toyo Roshi Co. Ltd. Japan) was performed at 10 V/cm for 5 h in a semidry blotting apparatus (ATT0 Model AE6670). Rabbit anti-u subunit antiserum (recognizing the amino terminal region between residues 2 and 11 of the a subunit) pretreated with membranes of the strain lacking the uric operon was generously supplied by Drs. J. Hermolin, and R. H. Fillingame. The antiserum was diluted (1.5 X 1O3-fold) with 10 mM Tris-HCl (pH 7.4) containing 5% bovine serum albumin and 0.9% NaCl. The binding of the anti-a subunit antibodies to the a subunit on the nitrocellulose filter was detected with peroxidase-conjugated goatanti-rabbit antibodies (Jackson Immune Research Laboratories). Wild-type F,F,-ATPase was obtained from the membranes of KY7485 after solubilization with 10 mM Tris-HCl (pH 8.0), containing 1.5% (w/v) octylglucoside and 1.0% (w/v) sodium cholate, and further purified by density gradient (lo-30% glycerol) centrifugation. Materials. The reagents used were obtained as follows: restriction endonucleases, Klenow fragment, and T4 DNA ligase from Nippon Gene Co. (Toyama. Japan); DNA polymerase I and exonuclease III from Takara Shuzo. (Kyoto); and (a-32P)dCTP (400 Ci/mmol) and (y-32P)ATP (4000 Ci/mmol) from Amersham Corp. All other reagents used were of the highest grade available commercially.
RESULTS
Construction of the a subunit mutants. From analyses of a group of nonsense mutants, we suggested that important residues for proton translocation were located between residues 252 and 271 (carboxyl terminus) of the a subunit (10). In contrast, we found that the three carboxy1 terminal residues (Glu-269, Glu-270, and His-271) were not required for a functional F0 (17). Therefore, we focused on the three hydrophilic residues between Gln1 Abbreviations used: DCCD, carboxylcyanide-m-chlorophenylhydrazone.
iV,N’-dicyclohexylcarbodiimide;
CCCP,
H+-ATPase
a SUBUNIT
252 and Ser-268, and introduced, Gln-252 + Gh (or Leu), Tyr-263 + Phe (or end), and Ser-265 + Ala (or end) mutations by oligonucleotide-directed mutagenesis (Table I). It is noteworthy that Gln-252 and Tyr-263 are conserved in the a subunits of other organisms (13). In addition, to confirm the role(s) of Arg-210, Glu-219, and His-245 in proton translocation (7,11-16), we constructed Arg-210 + Lys (or Gln), Glu-219 + His (or Gln), and His-245 + Glu mutants and compared their phenotypes with those previously observed. We also isolated a new un& mutant strain KF116 (Pro-230 + Leu) by random mutagenesis. Each mutant gene was cloned into pBR322 and introduced into strain KF24A (Tip-111 --* end), which is defective in ATP synthesis. Our system is suitable for examining the effects of mutant a subunits expressed from recombinant plasmids, since the short a subunit fragment (Trp-1113 end) synthesized from chromosomal DNA of KF24A did not interfere with the assembly of the longer a subunit from recombinant plasmids. Detailed results are shown below. Presence of mutant subunits in membranes. Membranes from KF24A harboring recombinant plasmids were subjected to immunoblot analysis using two gel electrophoresis systems and mutant subunits were detected immunochemically (Figs. 1A and 1B). Subunits with missense mutations showed essentially the same mobilities as the wild-type a subunit. Truncated subunits (Glu269 + end, Ser-265 + end, Tyr-263 + end, Trp-231 + end, and Trp-111 + end) showed significantly increased mobilities on electrophoresis, consistent with their lower molecular weights due to the loss of carboxyl terminal residues (Fig. lA, lanes 14-1’7; Fig. lB, lanes 7 and 9). The amounts of Tyr-263 -+ end (Fig. lA, lane 16), and Arg-210 * Lys (Fig. lA, lane 12) subunits were slightly less than those of the wild-type. It is noteworthy that the amount of the truncated subunit (Trp-111 --* end) coded by the host (KF24A) chromosome was very low (essentially not detectable) and the longer subunits coded by recombinant plasmids became predominant. Thus the properties of the longer subunits could be studied using this experimental system. Effectsof the a subunit mutations ongrowth by oxidative phosphorylation. The abilities of the mutant a subunits to support growth by oxidative phosphorylation were tested. AS shown in Table II, cells carrying the o subunits with Tyr-263 + end, Glu-219 + Gln, Arg-210 --f Lys, and Arg-210 + Gln mutations did not grow significantly on succinate, and showed greatly reduced growth on glucose, indicating that the H+-ATPases with these mutant subunits were defective in oxidative phosphorylation. The a subunits with Gln-252 --t Leu, Gln-252 --* Glu, His245 + Glu, Pro-230 + Leu, and Glu-219 + His mutations allowed growth by oxidative phosphorylation, but the growth yields were lower than that with the wild-type subunit. On the other hand, the growth and oxidative
FROM
Escherichia
A
1
2
3
73
coli
4
!i
6
7
6
9 10 11 12 13 14 15 16
17
a*
B 1
2
3
4
5
6
7
8
9
FIG. 1. Presence of mutant a subunits in membranes. Membranes (100 pg protein) from strain KF24A haboring different plasmids were subjected to 12.5-17.5% (A) or 12.5-22.5% (B) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and then proteins were transferred onto nitrocellulose filters. Mutant a subunits were detected immunochemically with anti-a subunit antibodies. The amount --, end) coded by the chromosome of of the truncated subunit (Trplll strain KF24A was reduced to an undetectable level when longer subunits were supplied from recombinant plasmids (horizontal arrowhead, (3). (A) Lane 1, FoF,; lane 2, pBBW (wild-type a subunit); lane 3, pBB265A (Ser-265 + Ala); lane 4, pBB263F (Tyr-263 + Phe); lane 5, pBB252E (Gln-252 + Glu); lane 6, pBB252L (Gin-252 + Leu); lane 7, pBB245E (His-245 + Glu); lane 8, pKF116 (Pro-230 + Leu); lane 9, pBB219Q (Glu-219 + Gln); lane 10, pBB219H (Glu-219 + His); lane 11, pBB2lOQ (Arg-210 --, Gln); lane 12, pBB21OK (Arg-210 + Lys); lane 13, pBBW; lane 14, pBB269e (Glu-269 + end); lane 15, pBB265e (Ser.265 + end); lane 16, pBB263e (Tyr-263 + end); lane 17, pKF2 (Trp-231 + end). (B) Lane 1, pBBW; lane 2, pBB252E; lane 3, pBB252L; lane 4, pBB219Q; lane 5, pBB219H; lane 6, pBB21OQ; lane 7, pBR322; lane 8, KF9 (Gln20 -, end); lane 9, pKF24 (Trp-111 * end).
phosphorylation activities of the Glu-269 + end, Ser265 --* Ala, Ser-265 + end, and Tyr-263 --+ Phe mutants were essentially similar to those with the wild-type subunit. Mutant subunits similar to the wild-type. The a subunits with Glu-269 + end, Ser-265 + Ala, and Tyr263 --* Phe mutations gave FoF, similar to that of the wild-type, consistent with the positive growths of these mutants by oxidative phosphorylation: the membrane ATPase activities of these mutants were similar to that of the wild-type, and were sensitive to DCCD (Table II). The mutant membranes showed the wild-type level of ATP-dependent proton translocation (quinacrine fluorescence quenching) (Fig. 2A). Wild-type membranes depleted of Fi were permeable to protons and showed only low level quenching of quinacrine fluorescence dependent on respiration (D-lactate), confirming previous results (17). However, DCCD sealed the proton pathway of the wild-type F0 in F,-depleted
74
EYA,
MAEDA, TABLE
AND
FUTAI
II
Growth Yields and Membrane ATPase Activities of Mutants Growth
Plasmid pBBW pBB269e pBB265A pBB265e pBB263F pBB263e pBB252E pBB252L pBB245E pKF116 pBB219H pBB219Q pBB210K pBB2lOQ pBR322
Mutation in a subunit
Membrane ATPase (units/mg)
15mM
5mM
glucose
(Wild-type) E269 + end S265 --, A S265 -+ end Y263 + F Y263 -+ end 6252 + E Q252 + L H245 + E P230 + L E219 + H E219 -+ Q R210 + K R210 + Q (No a subunit)
yield
100 106 102 84 98 52 93 78 62 50 33 48 45 26 30
succinate 100 110 95 73 92
