THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc

Vol. 261, No. Issue of August 5, pp. 10043-10050,1986 Printed in U.S.A.

Impaired Proton Conductivity Resulting from Mutations in the a Subunit of FIFO ATPase in Escherichia coli*

(Received for publication, December 16, 1985)

Brian D. Cain$ and Robert D. Simoni From the Department of Biological Sciences, Stanford Uniuersity, Stanford, California 94305

Mutations in the uncB gene which encodes the a subunit of FIFo-ATPase in Escherichia coli were iso- lated and characterized. Eight mutations caused pre- mature polypeptide chain termination. Two mutations were single amino acid substitutions resulting in the replacements of serine 206 with leucine (ser-2064eu) and histidine 245 with tyrosine (his-245+tyr). The ser-206+1eu mutation does not alter F1 binding and allows ATP driven membrane energization at a low level. Stripping of F, from membranes containing the ser-2064eu mutation does not render the membranes permeable to protons indicating impaired proton con- ductivity. The his-245+tyr mutation also blocks Fo- mediated proton conduction but has normal F1 binding properties. F1 bound to membranes with both ser-206+ leu and his-245+tyr mutant a subunits is sensitive to dicyclohexylcarbodiimide. Apparently, both missense mutations impair proton conduction without altering assembly of the FIFo-ATPase complex. The direct in- volvement of the a subunit in proton translocation is discussed.

The proton translocating adenosine triphosphate synthase (E.C. 3.6.1.3) of Escherichia coli is an amphipathic, multimeric enzyme complex (1, 2). All eight ATPase subunits plus a ninth polypeptide of unknown function are encoded by the unc operon; the unc1, B, E, F, H, A, G, D, C genes code for the i, a, c, b, 6 , a, y, @, e subunits, respectively (3, 4). The nucleotide sequence of the unc operon has been determined (4,5).

The hydrophilic F, portion of ATPase contains the a, p, 6 , y, t subunits. Biochemical studies have shown that a hexamer consisting of three a and three @ subunits arrayed about a single y subunit catalyzes ATP hydrolysis (1,6). The catalytic site is in the p subunit (7,8). The 6 and c subunits participate in the association of F, with the membrane bound subunits (6,9). Additionally, the t subunit also functions as an inhibitor of F, activity in vivo (9).

The hydrophobic Fo portion has the a, b, and c subunits in a stoichiometry of 1:2:6-10, respectively (10). The arrange- ment of the Fo subunits and the structure of the proton channel through the membrane are unknown. Primary se- quence data coupled with genetic and biochemical studies have revealed some characteristics of the individual subunits.

*This work was supported by Public Health Service Grant GM18539 (to R. D. S.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Recipient of Postdoctoral Fellowship GM09521 from the Na- tional Institutes of Health.

The c subunit, an extremely hydrophobic polypeptide of 8,288 daltons, is thought to have two transbilayer helices with the hairpin loop on the cytoplasmic side of the membrane (1). Characterization of missense mutations revealed that the c subunit participates in proton conduction and the coupling of F, activity to proton conduction (11-16). The c subunit con- tains the binding site for dicyclohexylcarbodiimide (DCCD’) which inhibits proton conduction. The b subunit (17,265 daltons) is thought to have a single transmembrane helix and an extensive hydrophilic domain exposed to the cytoplasm (1, 16, 17). The b subunit functions in the association of F1 with Fo (18-20). The transmembrane helix of the b subunit plays at least a structural role necessary for proton conduction (16- 20).

The largest and least understood Fo polypeptide is the a subunit (30,276 daltons). Others have suggested six or seven transmembrane helices (1, 4), but tangible topological data is lacking. Hydrophilic cross-linking reagents have revealed close association of the a and b subunits (17,21). Presumably this nearest neighbor relationship exists in the cytoplasm. Isolation of the second site mutation in the a subunit which partially suppresses the effects of a mutation in the b subunit hydrophobic domain supports a close association between these subunits in the bilayer.’ The c subunit also appears to associate strongly with the a subunit since an ac complex has been isolated (19). Early failures to isolate missense mutations led to the suggestion that the a subunit plays only a passive structural role and has no direct part in the function of FIFO ATPase (22).

The work presented here describes the isolation and partial characterization of the first two missense mutations in the uncB (a) gene. We present data suggesting that these muta- tions affect Fo-mediated proton conduction without altering FIFo complex assembly. The results are discussed in terms of the possible direct participation of the a subunit in proton translocation.

EXPERIMENTAL PROCEDURES

Materials-Lysozyme and deoxyribonuclease were the products of Sigma and Worthington. T-4 DNA ligase was prepared by standard procedures. Other enzymes were purchased from Bethesda Research Laboratories or New England Biolabs (Beverly, MA). [35S]Methio- nine was purchased from New England Nuclear (Boston, MA). Re- agents and radionucleotides for DNA sequencing were obtained from Amersham. Solvents and reagents were of the highest quality com- mercially available.

Organisms, Media, and Growth Conditions-Bacterial strains and plasmids used here are summarized in Table I. Luria broth supple- mented with 0.2% (w/v) glucose served as rich medium (23). Minimal media contained A salts and either glucose or succinate at 0.2% (w/v)

The abbreviations used are: DCCD, dicyclohexylcarbodiimide;

* Kumamoto, C. A., and Simoni, R. D. (1986) J. Biol. Chem., in MOPS, 3-[N-morpholino]propanesulfonic acid.

press.

10043

10044 ATPase a Subunit Mutants

TABLE I List of strains and plasmids

Strain/plasmid Genotype/description Source/Ref.

1100 bglR thi-1 rel-I HfrPOl 1100 i1u::TnlO BC2000 BC2001 BC2003 BC2019 RH305 DK8 c2110 cK1aaa cK1aa9 CK1891 pDJK5 pAP55 pBDC4 pRPG56 pUNCB1.O1, etc. pUNCB1l.O1, etc. pBDCl pBDC6 pUNCB13.01, etc. pUNCB23.01. etc.

llO0 i1u::TnlO 1100 carrying uncB2000 deletion from pBDC4 1100 carrying uncB2001 from pUNCB11.01 1100 carrying uncB2003 from pUNCB11.03 1100 carrying uncB2019 from pUNCB11.19 1100 uncB205 recA56 sr1::TnlO 1100 i1u::TnlO (uncB-uncC deletion) polAl his F- uncB2000 lacU169 araD139 thiA rpsL relA recA F- uncB2000 supE" lacU169 araD139 thiA rpsL relA F- uncB2000 supFb lacU169 araD139 thiA rpsL relA Cm' uncI+B-E+F+H+A+G+D+C+ Cm' uncI+B+E+F+H+A+G+D+C+ Cm' uncI+B-E+F+H+A+G+D+C+ Ap' uncB+E+ Ap' uncB-E+; mutated derivatives of pRPG56 Ap' uncB-E+; mutated derivatives of pRPG56 Ap' uncB+ Cm' uncB+ Cm' uncB-; mutated derivatives of pBDC6 Cm' uncB-; mutated derivatives of pBDC6

- pUNCB7 Ap' uncB2000, uncE+

O From strain XACSUDE. J. Beckwith. From strain XAcSLpF, J. Beckwith.

final concentration as a carbon source (23). Antibiotics were included as appropriate at concentrations of 100 pg/ml ampicillin and 18 fig/ ml chloramphenicol. Liquid cultures were mixed continuously on a rotary shaker or a roller drum. All incubations were at 37 "C. Growth was monitored turbidometrically using a Klett-Summerson colorim- eter.

Recombinant DNA Techniques-Large scale plasmid DNA was purified by the methods of Godson and Vapnek (24). Rapid prepara- tion of small amounts of plasmid DNA was according to Birnboim and Doly (25). Digestion with restriction endonucleases, ligation, transformation and agarose gel electrophoresis were standard proce- dures (26). DNA fragments were recovered from agarose gels as described by Porter et al. (27).

Plasmid Mutagenesis-Mutagenesis of plasmid pRPG56 (a,c) was essentially as described earlier (20). Plasmid DNA in buffer (0.1 mM KH2P04, 1 mM EDTA, pH 6.0) was incubated with hydroxylamine (32 mg/ml) for 24 h at 37 "C. The solution was dialyzed for 12 h at 4 "C against 500 volumes TE (10 mM Tris-HC1, 1 mM EDTA, pH 7.9). Sodium acetate (0.3 M, final concentration) and 2 volumes ethanol were added to precipitate the DNA. Mutagenized DNA was resuspended in TE buffer and then used to transform strain RH305 (uncB 205, recA-) to ampicillin resistance on minimal A glucose medium. Small colonies were picked and restreaked on rich medium

pUNCB1.O1 to pUNCB1.19. supplemented with antibiotic. Mutant plasmids were designated

Plasmid Constructions-Plasmids pBDCl (a) and pBDC6 (a) were made by the ligation of the 1.1-kilobase HindIIIIAuaI fragment of pRPG56 (a,c) to the 2.9-kilobase HindIIIIAuaI fragment of pBR322 and to the 2.8-kilobase HindIII/AuaI fragment of pACYC184, respec- tively (Table 1, Fig. 1). Mutant plasmids designated pUNCBll (a-,c) were constructed by the ligation of the 0.7-kb NcoIIAuaI fragment from a pUNCBl (a-,c) plasmid to the 3.1-kb NcoIIAuaI fragment of plasmid pRPG56 (a,c) (Fig. 1). Plasmids identified as pUNCB13 (a-) were made by the ligation of the 0.7-kb HindIIIIPstI fragment of a pUNCBl (a-,c) plasmid to the 3.2-kb HindIIIIPstI of plasmid pBDC6 (Fig. 1). Plasmids designated pUNCB23 (a-) were constructed by the ligation of the 1.1-kb HindIIIIAuaI fragment from a pUNCBl (a-,c) plasmid to the 2.9-kb HindIIIIAuaI fragment of plasmid pACYC184 (Fig. 1).

Plasmid pBDC4 (i, c,b, 6, a, 7, (3, e ) was constructed by digestion of pAP55 (i, a, c, b, 6, a, 7, (3, e) with BamHI and then ligation of the 12.1-kb fragment, resulting in the internal deletion of 0.6 kb of uncB (a) gene DNA. Similarly, plasmid pBDC7 (c) was constructed by digestion of pRPG56 (a,c) with BamHI and then ligation of the 3.2- kb fragment.

Nucleotide Sequence Determinution-For sequence determination

H

33 20 This study This study This study This study 33 9 34 Carol Kumamoto Carol Kumamoto Carol Kumamoto 35 27 This study 3 This study This study This study This study This study This study This study

H I

{ pUNCB pUNCB11 1 '<' pUf%?3 pUNCB 13 23 A

v u FIG. 1. Plasmids containing mutant uncB (a) genes. Plasmid

pRPG56 (a,c) (3) was mutagenized yielding plasmids denoted as pUNCB1. Plasmids designated pUNCBll contained a mutagenized fragment of DNA extending from the NcoI site to the AuaI site. Plasmids named pUNCB13 contained mutagenized DNA from the HindIII site to the PstI site. Plasmids designed pUNCB23 carried mutagenized DNA from the HindIII site to the AuaI site. Plasmids pUNCB23 served as the donors of PstIIEcoRI DNA fragments, for cloning into phage M13 for DNA sequence determination. Ap', am- picillin resistance; Cm', chloramphenicol resistance; A, AuaI; E, EcoRI; H, HindIII; N, NcoI; P, PstI.

of the middle one-third of the uncB (a) gene, the 0.7-kb HindIIIIPstI fragments of plasmids designated pUNCB23 were ligated with phage M13mp19 digested with HindIIIIPstI. For the rear one-third of the uncB (a) gene, the 1.5-kb PstIIEcoRI fragment of plasmids designated pUNCB23 were ligated with phage Ml3mplO digested with PstI/ EcoRI. Single-stranded phage DNA was prepared, and DNA se- quences were obtained using the Sanger dideoxynucleotide method

Construction of Chromosomal Mutant Strains-Mutations in plas- mids were transferred to the chromosome by the method of Porter et al. (20). The uncB (a) gene deletion plasmid pBDC4 (i, c, b, 6, a, 7,(3, e) and pUNCBll (a-,c) plasmids carrying point mutations in the uncB (a) gene served as donors for construction of mutant strains. Chromosomal mutations were subsequently moved into different strain backgrounds by phage Plvir-mediated generalized transduction using strain 1100 i1u::TnlO as the recipient (23). Presence of the deletion (uncB2000) was confirmed by Southern blot analysis (data not shown).

Preparation of Cell Fractions-Fractionation of cells was accom- plished essentially as described by Klionsky et al. (29). Approximately 1 g of fresh cells in TM buffer (50 mM Tris-HC1, 10 mM MgS04, pH 7.5) were disrupted in a French pressure cell (14,000 psi) in the

(28).

ATPase a Subunit Mutants 10045

presence of deoxyribonuclease (10 pglml). Unbroken cells and debris were removed by two consecutive centrifugations (2,000 X g, 10 min) to yield the cell lysate fraction. A subsequent high-speed centrifuga- tion (100,000 X g, 1.5 h) of the lysate generated soluble (cytoplasm) and particulate (membrane) fractions. Membranes used in proton conduction studies were washed with 7.5 ml of TM buffer prior to final resuspension (2 ml, final volume). Membranes used for F, stripping experiments were washed in 7.5 ml of SB buffer (1 mM Tris-HC1, 0.5 mM EDTA, 2.5 mM P-mercaptoethanol, 10% glycerol, pH 8.0). Membranes were resuspended in fresh SB buffer (7.5 ml) and incubated overnight a t 4" with continuous, gentle mixing. The stripped membranes were recovered by centrifugation (100,000 X g, 1 h) and then resuspended in TM (1 ml, final volume).

Assays-Protein concentrations were determined by the modified Lowry procedure of Markwell et al. (30). Assays of ATPase (F,) activity were as described earlier (31). Membrane energization was detected by the fluorescence quenching of 9-amino-6-chlor-2-meth- oxyacridine (32). Isolated membranes (0.5 mg of protein, in 100 pl of 50 mM MOPS, pH 7.3, 10 mM MgS04) were reacted with DCCD (50 pM, final concentration) for 10 min at 37 "c (32). In uitro transcrip- tion-translation of plasmid encoded genes and sodium dodecyl sulfate- polyacrylamide gel electrophoresis of products were as described by Gunsalus et al. (3).

RESULTS

Isolation of uncB Mutations-To isolate mutations in the uncB (a) gene and facilitate their manipulation, we elected to do in vitro mutagenesis on plasmids carrying an intact uncB (a) gene but a minimum of other urn operon DNA. The procedure was essentially the same as that used previously to alter the uncF (b ) gene (20). Plasmid pRPG56 (a,c), shown in Fig. 1, was treated with hydroxylamine and then trans- formed into E. coli strain RH305 (uncB205, recA-). Fourteen of the approximately 10,000 transformants tested displayed a clear uric- phenotype of reduced capacity to grow on succinate minimal medium. No growth was observed after incubating the succinate plates at 37 "C for 48 h. In a single instance, cells harboring plasmid pUNCB1.19, very small colonies ap- peared during the %day incubation. These pRPG56 (a,c)- derived plasmids were designated pUNCB1.O1 to pUNCB1.19; the plasmid decimal number and an allele number (uncB2001 to uncB2019) were assigned to a specific mutation and were used to refer to that mutation throughout this report.

Classification of uncB Mutations-Differentiation between chain terminating mutations and amino acid substitution mutations relied on two experimental approaches. First, in the presence of an appropriate nonsense suppressor mutation, plasmids encoding some chain terminating mutations would produce a functional a subunit and therefore could be expected to complement a strain with a chromosomal uncB (a) deletion, strain BC2000 (uncB2000). Six plasmids complemented the strains CK1889 (uncB2000, supE) or CK1891 (uncB2000, supF) and are listed in Table 11.

In order to further determine the nature of the mutation, a Zubay $30 in vitro transcription-translation system was used to study plasmid directed synthesis of mutant a subunit polypeptides. Nonsense mutations resulting in premature ter- mination of chain elongation were detected by the absence of a polypeptide product with mobility similar to authentic a subunit when analyzed by sodium dodecyl sulfate-polyacryl- amide gel electrophoresis. Conversely, amino acid substitution mutations resulted in apparently normal size a subunit bands. The autoradiogram shown in Fig. 2 depicts a typical experi- ment. An a subunit band of normal a subunit mobility was missing among the polypeptide products synthesized from p UNCB1.02 (uncB2002), pUNCB1.03 (uncB2003), and pUNCB1.ll (uncB2011) indicating chain terminating muta- tions. Plasmid pUNCB1.03 (uncB2003) directed synthesis of a polypeptide (apparent M, 19,000 daltons) which was prob- ably the truncated a subunit. Plasmids pUNCB1.O1 (unc-

TABLE I1 Clossijicution of mutations in the uncB (a) gene

SupE SupF a subunit 'Iasrnid suppression" suppressionb polypeptide'

pUNCB1.01 (un~B2001) - - pUNCB1.02 (uncB2002) + + pUNCB1.03 (uncB2003) + + - pUNCB1.05 (uncB2005) - -

pUNCB1.07 (uncB2007) + + pUNCB1.09 (uncB2009) + + pUNCB1.10 (un~B2010) - - pUNCB1.ll (uncB20II)

+ + pUNCB1.12 (uncB2012) + pUNCB1.17 (uncB2017) + + - pUNCB1.18 (uncB2018) - - pUNCB1.19 (umB2019) -

+ +

+ -

pUNCB1.04 ( u ~ B 2 0 0 4 ) - - + pUNCB1.06 ( ~ n ~ B 2 0 0 6 ) -

- - -

- -

- - - -

- a Complementation of strain CK1889 (uncB2000, supE). *Complementation of strain CK1891 (uncB2000, supF). e In uitro transcription-translation product of mobility similar to

authentic u subunit in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2).

a b c d e f q h - .. 7- -

-a "P

"E

" c FIG. 2. In vitro protein synthesis products of mutant uncB

(a) gene plasmids. In vitro transcription-translation of plasmids pUNCBl (Fig. 1) was followed by sodium dodecyl sulphate-polyac- rylamide gel electrophoresis and autoradiography. The locations of the ATPase subunits produced by the pAP55 (i, a, c, b, 6 , a, y, P, e) standard (27) are shown alongside. Lane a, pUNCB1.O1; lane b, pUNCB1.02; lane c, pUNCB1.03; lane d, pUNCBl.1O; inne e, pUNCB1.ll; lune j , pUNCB1.12; lane g, pUNCB1.1S; lane h, pAP55.

10046 ATPase a Subunit Mutants

B2001), pUNCB1.10 (uncB2010), pUNCB1.12 (uncB2012), and pUNCB1.19 (uncB2019) all generated polypeptides with mobilities identical to authentic a subunit. Normal size a subunit polypeptides were also observed in the products of plasmids pUNCB1.04 (uncB2004) and pUNCB1.18 (unc- B2018) (data not shown). The evidence suggesting that six mutations were missense mutations and eight mutations were nonsense mutations is summarized in Table 11.

Intragenic Mapping of uncB Mutations-We chose to clone the mutations from the mutagenized plasmids prior to char- acterization of their properties for two reasons. First, second- ary undetected mutations might complicate biochemical stud- ies; second, the cloning provided an approach for mapping mutations within the uncB ( a ) gene, thus reducing the amount of DNA sequencing necessary to identify mutations. Briefly, overlapping fragments of the mutagenized plasmids contain- ing the first (HindIIIIPstI, Fig. 1) and rear (NcoI/AuaI, Fig. 1) two-thirds of the altered uncB ( a ) genes were cloned into vectors carrying the complementary portion of the unmuta- genized gene (see "Experimental Procedures"). The resulting plasmids pUNCB13 and pUNCBll were analyzed by comple- mentation in strain RH 305 (uncB205, recA-). Failure of both plasmids, pUNCB13 and pUNCB11, to complement showed that the mutation was in the overlapping region of DNA contained in both plasmids, which corresponded to the middle one-third of the uncB ( a ) gene. However, if only pUNCB13 carried a defective uncB ( a ) gene, then the mutation was in the region of the cloned fragment which did not overlap with the fragment cloned into pUNCBll (i.e. the first one-third of the uncB ( a ) gene). Conversely, if only pUNCBll had a defective uncB ( a ) gene, then the mutation was in the rear one-third of the gene. The utility of the approach was tested by mapping the chain terminating mutations uncB2002 and uncB2003, which were judged from the in vitro transcription- translation experiments to occur early and late, respectively, in the uncB (a) gene (see Fig. 2). As predicted, plasmids pUNCB13.02 and pUNCB11.03 failed to complement RH305 (uncB205, recA-), while plasmids pUNCB11.02 and pUNCB13.03 both scored as wild-type for the uncB ( a ) gene. Each of the missense mutations were similarly mapped. Plas- mids pUNCB1l.O1, pUNCB11.18, and pUNCB11.19 all failed to complement strain RH305 (uncB205, recA-) and the cor- responding pUNCB13 plasmids were wild-type. Therefore, mutations uncB2001, uncB2018, and uncB2019 were localized to the rear one-third of the uncB (a) gene. No amino acid substitution mutations were found in the first one-third or middle one-third of the uncB ( a ) gene. Three mutations designated uncB2004, uncB2010, and uncB2012 did not confer the uncB- phenotype on either of the corresponding pUNCBll or pUNCB13 plasmids. Construction of the pUNCB23 plasmids by cloning the putative mutant uncB (a) genes intact (Fig. 1) confirmed that the apparent uncB- phenotype was not associated with the uncB ( a ) gene. Sub- sequent experiments showed that pUNCB1.10 encoded a mu- tation in the uncE ( c ) gene (hereafter referred to as uncE2010) (data not shown). Study of pUNCB1.04 and pUNCB1.12 was abandoned after we determined that neither carried a muta- tion in unc operon DNA (data not shown).

Sequence Determinations of uncB Mutations-DNA known to contain an uncB mutation was cloned into phage M13 for DNA sequence determination by the Sanger dideoxynucleo- tide method (28). Sequences of DNA from alleles in the rear one-third of the uncB (a) gene were determined from the PstI site through the termination codon (Fig. 1). Since we have confirmed the sequence of the uncB ( a ) gene reported by Walker et al. (4), mutant DNA was only sequenced from one

strand. However, the presence of each mutation was demon- strated in two separate phage isolates.

The locations of mutations in the uncB ( a ) gene were shown in Fig. 3. Mutations uncB2001 and uncB2018 were identical C to T transitions resulting in the substitution of histidine 245 with tyrosine. Allele uncB2019 was a C to T transition directing the replacement of serine 206 by leucine. Chain- terminating mutations uncB2003 and uncB2Ol7 were shown to be amber stop codons, resulting from G to A transitions, at tryptophan 235 and tryptophan 231, respectively. The former occurs in a BamHI endonuclease recognition sequence which enabled us to demonstrate that this was in fact the lesion in the original uncB2003 isolate; the other mutations do not affect an existing restriction site nor create a new one.

Construction of Chromosomal uncB Mutant Strains-Stud- ies of unc operon mutants have suggested that strains carrying mutations on multiple copy plasmids may differ in their properties when compared to strains encoding the same mu- tant allele in the chromosome (11,12). In order to detect gene copy number effects (if any) on the biochemical properties of the mutations considered here, it was necessary to construct strains carrying these mutations in the chromosome. The approach relied on recombination of mutant plasmids with the chromosome (20, see "Experimental Procedures"). Mu- tant plasmids pUNCB1l.O1 (ahis.245tyr,~) pUNCB11.03 (atrp. 23-&,c), pUNCB11.19 (aser-2-leu,c), and pBDC4 (i, c, b, 6, cy, y, p, t), which contains an internal 617 base pair deletion in the uncB ( a ) gene (uncB2000, see above), served as the donor plasmids for chromosomal mutant strains BC2001 (his-245-

ATC GCT TCA GAA AAT ATG ACG CCG CAG GAT TAC ATA GGA CAC CAC CTG AAT ARC CTT CAG Met Ala Ser G l u A s n Met T h r P r o G l n A s p T y r Ile G l y His His L e u Asn A m L e u G l n

10 20

CTG GAC CTG CGT ACA TTC TCG CTG GTG GAT CCA CAA AAC CCC CCA GCC ACC TTC TCG ACA L e u ASP Leu Rrg T h r P h e Sell Leu Val ASP P r o G l n As" P r c P r o A l a Thl' P h e T p p T h r

30 40

ATC AAT ATT CAC TCC ATG TTC TTC TCG GTG GTC CTG GGT CTG TTG TTC CTG GTT TTA TTC I l e A m Ile ASP Set- Met P h e P h e Ser Val Val L e u G l y L e u L e u P h e L e u Val L e u P h e

50 60

CGT AGC GTA GCC AAA AAG GCG ACC AGC GGT GTG CCA GGT AAG TTT CAG ACC GCG ATT GAG IO 80

Arg Ser Val A l a L y s L Y S la mr ser G l y V ~ I pro G l y LYS Phe GI" T h P ~ i a 11e r,lu

CTG GTG ATC GGC TTT GTT AAT GGT AGC GTG AAA GAC ATG TAC CAT GGC AAA AGC AAG CTG 90 100

L e u Val Ile G l y P h e Val A s n G l y Ser Val L y s A s p Met T y r His G l y L y s Ser L y s L e u

ATT GCT CCG CTG GCC CTG ACG ATC TTC GTC TGG GTA TTC CTG ATG AAC CTG ATG GAT TTA 110 1 2 0

Ile A l a P r o Leu A l a Leu T h r Ile P h e Val T r p Val P h e L e u Met As" L e u Met ASP L e u

CTG CCT ATC GAC CTC CTC CCG TAC ATT GCT GAA CAT GTA CTG GGT CTG CCT GCA CTG CGT 130 1 4 0

Leu P r o f l e A s p L e u Leu P r o T y r Ile A l a G l u HIS Val L e u G l y L e u P r o A l a L e u Arg

GTG GTT CCG TCT GCG GAC GTG ARC GTA ACG CTG TCT ATG GCA CTG GGC GTA TTT ATC CTG 1 5 0 160

Val Val P r o Ser A l a ASP va i AS" Val T h r ~ e u ser Met Ala L e u G l y Val P n e I l e L e u

ATT CTG TTC TAC AGC ATC AAA ATG AAA GGC ATC GGC GGC TTC ACG AAA GAG TTG ACG CTG 170 180

Ile Leu P 3 e T y r Ser I l e L y s Met L y s G l y Ile G l y G l y P h e T h r L y a Glu Leu T h r L e u

CAG CCG TTC ART CAC TGG GCG TCC ATT CCT GTC AAC TTA ATC CTT GAA GGG GTA AGC CTG 190 200

Gln P r o P h e A s n His T r p A l a P h e Ile P r o Val A s n Leu Ile L e u G 1 u G l y Val Ser Leu

CTG TCC AAA CCA GTT TCA CTC GGT TTC CGA CTG TTC GGT AAC ATG TAT GCC GGT GAG CTG 210 2 2 0

L e u Ser L y s P r o Val Ser L e u G l y Leu A r g L e u P h e G l y A s n Met T y r Ala C l y G l u Leu 1

D' 230

ATT TTC ATT CTG ATT GCT GGT CTG T X CCG TGG' TGG TCA CAG TGG ATC CTG AAT GTG CCG 240

Ile P h e I l e Leu Ile Ala G l y L e u Leu P r o T r p T r p Ser G l n T r p Ile L e u A m Val PPO L J D TGG GCC ATT TTC CAC ATC CTG ATC ATT ACG CTG CAA GCC TTC ATC TTC ATG GTT CTG ACG T r p A l a Ile P h e His I l e L e u Ile Ile T h r L e u Gln Ala P h e Ile P h e Met Val L e u T h r

2 5 0 260

ATC GTC TAT CTG TCG A X GCG TCT GAR GAA CAT TAA I l e Val T y r L e u Ser Met A l a Ser G l u G l u Hi3 End

270

FIG. 3. Sequence of the uncB (a) gene and mutants. Num- bering starts with the first amino acid of the a subunit. Boxes show the altered codon and the amino acid change.

ATPase a Subunit Mutants 1004 7

tyr), BC2003 (trp-235-wnd) BC2019 (ser-206+leu), and BC2000 (uncB deletion), respectively.

Growth of uncB Mutants-Mutations in FIFo complex ATPase typically result in a reduced capacity of cells to grow on glucose-limited medium (11). Each of the chromosomal mutant strains was grown in media containing 2 and 5 mM glucose. The deletion strain (uncB2000), the nonsense mutant t rp-2354nd (uncB2003) and the missense mutant his-245+ tyr (uncB2001) had identical, typical unc- growth character- istics (Table 111). A slightly higher growth yield was obtained from the strain harboring the ser-2064eu mutation (unc- B2019) (Table 111). Direct comparison of the growth rates and growth yields of strain CK1888 (uncB2000, recA-) har- boring plasmids pUNCB23.01 (ahb.24htrr) and pUNCB23.18 ( a h l s . B l ~ t y T ) revealed identical results. The uncB2028 isolate was not studied further.

Localization of F,-ATPase Activity in uncB Mutants-The distribution of F,-ATPase activity between the cytoplasm and the membrane in strains bearing chromosomal unc mutations is an indication of FI binding to the membrane. Therefore, cell free extracts of mutant strains were separated into soluble (cytoplasmic) and particulate (membrane) fraction by cen- trifugation and the ATPase activity determined.

The amount of F, bound to membranes was indistinguish- able within experimental error for the wild-type strain and strains carrying the his-245+tyr (uncB2001) and ser-206- leu (uncB2019) mutations (Table IV). In each case, approxi- mately 80% of the total F1 activity was localized to the membrane fraction. In contrast, the trp-235-nd (uncB2003) mutation displayed a severe reduction in the amount of F, associated with the membrane fraction. Only 40% of the total recovered activity was in the particulate fraction. Typically,

TABLE III Growth yield of uncB ( a ) gene mutants on glucose-limited media Growth was monitored turbidometrically and reported in Klett

units at culture saturation. Experiments used minimal medium A supplemented with glucose as the sole carbon source.

Glucose Strain (allele) Mutation concentration

2mM 5 m ~

1100 48 102 BC2000 (uncB2000) a subunit 31 59 BC2003 (uncB2003) trp-235 “-* end 29 61 BC2001 (uncB2001) his-245 + tyr 31 60 BC2019 (uncB2019) ser-206 + leu 36 71

cells with the trp-235-nd (uncB2003) mutation contained approximately 55% of the total F1 activity observed in the wild-type control cells for reasons which were unclear.

We also measured association of F, with the membrane in strains which had the uncB mutations on plasmids. This analysis was complicated, however, because the deletion of uncB from the chromosome, strain BC2000, decreased the amount of F1 in the cell free extracts by nearly 80% (Table IV). Apparently, some polar effect on the expression of down- stream genes in the unc operon occurs in the uncB deletion strain (uncB2000). Nevertheless, the F, activity in strain BC2000 (uncB2000) was measurable in comparison to strain DK8 which has a total deletion of the unc operon. Inclusion of plasmids carrying either normal or mutant a subunit did not affect the overall low level of F1 activity in the deletion strain (uncB2000), however, they did affect the intracellular distribution of the F, (Table IV). The inclusion of plasmids pRPG56 (a,c) , pUNCB1l.O1 (ahrs.245-+tyr,c) and pUNCB11.19 (aser.206rku,~) all resulted in the binding of approximately 80% of the F1 to the membrane. In cells harboring plasmid pUNCB11.03 ( a r r p . z 3 ~ ~ , c ) less than 50% of the F1 fraction- ated with the membrane. In general, the same pattern of F, binding emerged from cells with plasmid encoded mutant a subunits as cells carrying the mutations on the chromosome.

The data suggest that F1 binding was not affected by the his-245-tyr or ser-2064eu mutations. Moreover, DCCD in- hibition of the F1 activity in missense mutant membranes was 280% of that observed in wild-type membranes. We conclude that the his-245+tyr (uncB2001) and ser-2064eu (unc- B2029) mutations did not alter the subunit interactions nec- essary to assemble FIFo. Assembly of the complex was, how- ever, apparently disrupted by the truncation of the a subunit trp-235-nd (uncB2003).

Proton Conduction in Membranes from uneB Mutants- Since the his-245+tyr (uncB2001) and ser-206”tleu (unc- B2019) mutations did not seem to affect assembly of the ATPase complex, the next step was to determine if these mutations acted at the level of Fo-mediated proton conduc- tion. Therefore, membranes were prepared and examined using the quenching of the fluorescent dye 9-amino-6-chloro- 2-methoxyacridine as a probe of membrane energization.

NADH- and ATP-driven fluorescence quenching in mem- branes from mutant uncB (a ) strains are depicted in Fig. 4. The strong ATP-driven quenching observed in the mem- branes from wild-type strain 1100 was not observed with membranes derived from the his-245-tyr (uncB2001) and

TABLE IV Distribution of FI in cell fractions

Procedures.” ND, not determined. Cells were grown in rich medium to OD, = 1.0 and then fractionated as described under “Experimental

Specific activity Strain/plasmid Mutation

% F, membrane associated’ Soluble Particulate

1100 BC2003 trp-235 - end BC2001

0.35 & 0.01 0.30 k 0.13 35 his-245 - tyr 0.21 & 0.03 1.09 * 0.10 80

BC2019 DK8

ser-206 -+ leu 0.23 f 0.02 0.99 k 0.17 ATPase

77

BC2000 0.03 f 0.01 co.01 ND

BC2000/pRPG56 0.20 f 0.05

a/+ 0.05 k 0.02 16

BC2000/pUNCB11.03 0.08 f 0.01 0.29 k 0.01

a/trp-235 4 end 77

0.20 f 0.03 BC2000/pUNCB11.01

0.18 f 0.02 alhis-245 + tyr

48

BC2000/pUNCB11.19 0.06 & 0.01 0.27 k 0.02 83

a/ser-206 -+ leu 0.07 f 0.02 0.31 f 0.01 76

+ 0.22 f 0.03 1.16 f 0.20 81

a

‘ Specific activity = ATP hydrolyzed (Fmol Pi)/protein (mg)/min. Total activity = specific activity X total protein of fraction (mg). Percent F, membrane associated = total

activity (particulate)/total activity (particulate + soluble).

10048 ATPase a Subunit Mutants

A 1 1 1 NADHKCN ATP CCCP NADH KCN ATP CCCP

1 1 1 1 +DCCD

NADH KCN NADH KCN 1

E "1

lmin FIG. 4. Fluorescence quenching in membranes from uncB

(a) gene mutant strains. Membranes (0.5 mg of protein) were suspended in buffer (50 mM MOPS, 10 mM MgC12, pH 7.3) and 1 PM 9-amino-6-chloro-2-methoxyacridine was added. Arrows marked the subsequent additions of NADH (0.5 mM), KCN (0.5 mM), ATP (0.1 mM) and carbonyl cyanide rn-chlorophenylhydrazone (2.5 WM). Truces A and E also show the absence of ATP-driven quenching when membranes were treated with DCCD (50 PM) for 10 min at 37 "C. Truce A , 1100, wild-type; truce B, BC2000 (umB deletion); trace C, BC2003 ( t r p - 2 3 h n d ) ; trace D, BC2001 (his-245-+tyr); truce E, BC2019 ( s e r - 2 0 h k u ) .

trp-235-nd (uncB2003) mutant strains. A small quantity of ATP-driven quenching of 9-amino-6-chloro-2-methoxyacri- dine fluorescence was observed in membranes containing the ser-2064eu (uncB2019) mutation, which was abolished by treatment with DCCD (Fig. 4). Since the NADH-driven quenching was normal in membrane preparations from all strains, the loss of ATP-driven quenching in mutant mem- branes was not the result of increased proton permeability. Inclusion of a plasmid pDJK5 (c , b, 6, a, y, 8, 6 ) with plasmids carrying defective uncB (a) genes in the urn operon deletion strain DK8 resulted in membrane energization measurements identical to those shown for the corresponding chromosomal mutation strains (data not shown).

Stripping F, from the membranes eliminated ATP-driven quenching from all preparations (data not shown). NADH- driven quenching was diminished in the membranes from wild-type cells but not in the membrane preparations from mutant strains (Fig. 5) indicating that the mutant membranes were impermeable to protons (i.e. less than 2% of wild-type conduction). ATP-driven quenching was restored to the wild- type membranes upon addition of purified F, as was the small amount of ATP-driven quenching observed in membranes from the strain carrying the ser-2064eu (uncB2029) muta- tion (data not shown). However, membranes from cells with the his-245dtyr (uncB2001) and trp-235-nd (uncB2003) mutations lacked detectable ATP-driven quenching (data not shown).

Clearly, mutation ser-2064eu (uncB2019) allowed only limited Fl-linked membrane energization. Further, since Fo- mediated proton permeability was impaired in stripped mem-

FIG. 5. Proton permeability of stripped membranes from uncB (a) gene mutant strains. Assays were as described for Fig. 4. Truce A , 1100, wild-type; truce B, 1100 treated with DCCD; truce C , BC2003 (trp-235-nd); truce D, BC2001 (his-245+tyr); trace E , BC2019 ( ser -2064eu) .

brane preparations, we conclude that this mutation resulted in poor proton conduction. We also believe that the his-245- tyr (uncB2001) mutation results in altered Fo function since proton conduction was lost despite normal F, binding (see above).

DISCUSSION

This work describes the isolation and characterization of mutations in the uncB (a) gene. Eight mutations cause pre- mature termination of the a subunit polypeptide. Two lesions are single amino acid substitution mutations and are, to our knowledge, the first such mutations reported in the E. coli F,Fo-ATPase a subunit. Both of these missense mutations possess biochemically distinct properties. The missense mu- tations confer their unique characteristics regardless of whether the mutant gene is encoded on the chromosome or on a plasmid.

Truncation of the a subunit by introduction of the trp- 235-d mutation causes reduced F1 association with the membrane (Table IV). Bound F1 is insensitive to DCCD. The trp-235-wnd (uncB2003) mutation does not allow formation of a functional proton channel as evidenced by the absence of ATP-driven membrane energization in unstripped mem- branes and the proton impermeability of stripped membranes (Figs. 4 and 5). Similar results have been reported by Fillin- game et al. (36) for another chain-terminating mutation, uncB402. These data are consistent with a failure to assemble the Fo complex.

The ser-2064eu (uncB2019) mutation is unique in that the FIFo complex is partially functional. A small amount of DCCD-sensitive ATP-driven membrane energization is evi- dent (Fig. 4). Indeed, strains carrying the ser-206"tleu (uncB2019) mutation grow on succinate minimal medium although very slowly. Further, the distribution of F1 activity in cell fractions is identical to that for wild-type cells (Table IV). However, passive proton conductance in stripped mem- branes is impaired beyond the limits of detection (Fig. 5). We conclude that the a subunit with the ser-2064eu (uncB2019) mutation is capable of all intersubunit interactions necessary

ATPase a Subunit Mutants 10049

to the formation of the FIFO complex, but the resulting Fo has only very limited capacity to translocate protons. There- fore, the ser-20hleu (uncB2019) mutation would seem to directly influence the efficiency of the proton channel.

The his-245+tyr (uncB2001) mutation causes the abolition of proton conduction. Yet, F1 binding appears normal with respect to both the quantity bound and DCCD sensitivity. The logic discussed above for the ser-2064eu (uncB2019) mutation suggests that the his-245-tyr (uncB2001) mutation also directly affects the proton channel, but to a greater degree. A critical role for his-245 is supported by the fact that we isolated this mutation twice.

Cells carrying the trp-23l+end (uncB2017) or trp-235- end (uncB2003) mutations plus either the supE or supF amber suppressor mutations have unc+ phenotypes with respect to growth on succinate minimal medium. Apparently, substitu- tion of tyrosine or glutamine for tryptophan at either site results in no significant alteration of the a subunit. Although these data are of only a qualitative nature, they do suggest that the loop between the putative transmembrane helices (Fig. 6B) may not be critical to the function of the a subunit.

The possibility that the ser-2064eu (uncB2019) and his- 245-tyr (uncB2001) affect proton conduction without alter- ing intersubunit interactions necessary to FIFO complex as- sembly implies that the a subunit may constitute, at least in part, the proton channel of Fo. This proposal gains plausibility when the locations of a subunit mutations are considered relative to a region of strong evolutionary conservation in the primary structure (1,4). The carboxy-terminal82 amino acids of E. coli a subunit (4) with FIFO-ATPase 6 subunits from seven diverse mitochondrial sources (4, 37-42) are compared in Fig. 6. E. coli ser-206 is conserved among three ATPase-6 subunits and lies adjacent to a leucine which is conserved in all of the mitochondrial subunits. Ambiguity arises in the case of E. coli his-245, since this residue is not conserved in mitochondrial subunits, but occupies the position of a glu- tamic acid residue (oli-4) conserved in all of the mitochondrial

FIG. 6. Speculations on the struc- ture of the a subunit. Panel A , model of the a subunit containing five trans- membrane helices and showing the ap- proximate locations of mutations; panel B, structure of the two carboxy-terminal transmembrane helices of the a subunits indicated by boxes; panel C, evolutionary conservation in the carboxy-terminal re- gion of the E. coli a subunit (4), and the mitochondrial ATPase-6 subunits of As- pergillus nidulam (37), Neurospora crassa (38), Saccharomyces cerevisiue (39), Drosophila melanogaster (4), bovine (40), human (41), and mouse (42), re- spectively, labeled 1-8; boxes indicate identity among at least seven species.

A

n l l l l U J

ATPase-6 subunits. Interestingly, E. coli glu-219, is located at a position held by a histidine in each of the mitochondrial subunits. Possibly a critical charge pair exists between glu- 219 and his-245 in adjacent transmembrane helices (Fig. 6).

The number of transmembrane helices in the a subunit is open to conjecture. We show five helices in Fig. 6A based on hydropathy predictions (43), a bias toward placing the con- served residues from glu-196 to gly-218 in a single helix (see below), and to accommodate the possibility of a charge pair between glu-219 and his-245. Hydropathy analysis led Walker et al. (4) to suggest a total of six helices with two membrane- spanning regions between thr-179 and ile-236. This hypothe- sis is plausible, but then ser-206 would be outside the mem- brane bilayer making its role in proton conduction reported in this paper difficult to understand. Yet another transmem- brane helix can be envisioned from the hydropathy analysis of amino acids phe-75 to ual-90 (1). At present no convincing evidence exists to differentiate between these possibilities.

Assuming the structure shown in Fig. 6, the two carboxy- terminal helices constitute a plausible site for the formation of a transmembrane proton channel. Assuming an a helical structure, one finds that the conserved residues align verti- cally along one side of each helix. Hydrogen-bonding circuits have been suggested as a possible mechanism of transmem- brane proton channels (44-45). A circuit of this type has been proposed using the transmembrane helix containing amino acids glu-196 through gly-218 in conjunction with E. coli c subunit asp-61 (47). This proposal is based primarily on the realization of strong evolutionary conservation in this region of the a subunit. However, the presence of conserved amino acids in the helix containing amino acids trp-241 to leu-264 and the severe effects of altering his-245 lead us to suggest a central role for this helix as well. Indeed, the data presented here suggest that his-245 may participate directly in proton conduction. This conclusion is consistent with a hydrogen- bonding circuit model, indeed it adds to the circuit by placing another ionizable group in a vacant area of the circuit (Fig.

B NH~+- Pro'w

Val Asn

(uncB 2019)

W (uncB 2017) trp231-end

,is 27 I

Ala Gly Leu Tr#3 Se?ln

Leu Pro Trp

c 2c6 t

231 235 t t

245

10050 ATPase a Subunit Mutants

6). The effect of the ser-2064eu (uncB2019) mutation can also be explained by a hydrogen-bonding circuit model. While ser-206 may not be directly in the proton circuit, its position places it in close proximity to arg-210 which is conserved in all species (Fig. 6). Loss of the hydroxyl group due to the substitution of leucine for serine could alter the free energy required to protonate the arginine residue, thus impairing the efficiency of the proton channel function.

Further evidence of the importance of the carboxyl-termi- nal region of the a subunit is manifested in a separate series of experiments. The b subunit g l y - h p (uncF) mutation perturbs the subunit interactions in Fo (18, 20). Membranes derived from cells with this mutation do not conduct protons and have reduced F1 binding capacity (20). Partial FIFO- ATPase function is restored by an additional mutation af- fecting pro-240 in the a subunit? Apparently, reduced rigidity in the positioning ofpro-240 mutant helices allows the mutant a subunit to associate with the defective b subunit(s) and assume an a subunit conformation suitable to proton conduc- tion.’

Recently, von Meyenburg et al. (46) suggested that the a subunit alone has protonophoric function based upon in vivo experiments in which the a subunit was synthesized at very high levels. While the physiological complications arising from extensive overproduction of an integral membrane pro- tein such as the a subunit and the indirect nature of their measurements suggest alternative interpretations, the impli- cation of the a subunit in proton translocation suggested in that report remains intriguing.

While much of the discussion above is somewhat specula- tive, the data presented here establish the a subunit as a likely participant in proton translocation and not merely a “struc- tural” subunit as has been suggested (22). A more rigorous approach to demonstrating specific intersubunit interactions in Fo would greatly enhance our arguments. Work is progress- ing here and in other laboratories on biochemical methods for studying the structure of Fo. The hypothesis that the two helices of the a subunit participate directly in proton conduc- tion allows specific predictions regarding the phenotypes re- sulting from altering various amino acids in these helices.

Acknowledgments-We thank Dr. Andrew Porter for his advice and encouragement in initiating this study, and Dr. Carol Kumamoto for the gifts of bacterial strains and for critically reading this manu- script. Drs. John Aris, Daniel Klionsky, and Steven Vik contributed many useful suggestions. We also wish to acknowledge Graeme Cox and Frank Gibson for stimulating discussions and for providing information and speculations in advance of publication.

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