factor b and the mitochondrial atp synthase complexa,b grigory i. belogrudov and

FACTOR B AND THE MITOCHONDRIAL ATP SYNTHASE COMPLEXa,b

Grigory I. Belogrudov and Youssef Hatefic

Division of BiochemistryDepartment of Molecular and Experimental Medicine

The Scripps Research InstituteLa Jolla, California 92037

Running Title: Factor B

aSupported by the United State Public Health Service Grant DK08126

bThis is publication number 14385-MEM from The Scripps Research Institute

cTo whom correspondence should be addressed.Tel: 858-784-8092; Fax: 858-784-2054; E-mail: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 14, 2001 as Manuscript M111256200 by guest on A

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ABSTRACT

Factor B is a subunit of the mammalian ATP synthase complex, whose existence has been

controversial. This paper describes the molecular and functional properties of a recombinant human

factor B, which when added to bovine submitochondrial particles depleted of their factor B restores

the energy coupling activity of the ATP synthase complexes. The mature human factor B has 175

amino acids and a molecular mass of 20,341 Da. The preparation is water-soluble, monomeric, and

is inactivated by monothiol and especially dithiol modifying reagents, probably reacting at its

cysteine residues C-92 and C-94. A likely factor B gene composed of 5 exons has been identified on

chromosome 14q21.3, and the functional role of factor B in the mammalian ATP synthase complex

has been discussed.

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It is generally considered that the mammalian mitochondrial ATP synthase complex is

composed of 16 unlike subunits (1-3). These subunits are α, β, γ, δ and ε in the catalytic F1 domain;

OSCP, a, b, c, d, e, f, g, F6 and A6L in FO and stator; and the ATPase inhibitor protein, IF1, which

binds reversibly to F1 to inhibit ATP hydrolysis. In 1967, Sanadi and coworkers (4) showed that

submitochondrial particles prepared by sonication from bovine mitochondria suspended in 0.25 M

sucrose and 0.6 mM EDTA1 and adjusted to pH 9.0 with ammonium hydroxide lost considerable

activity for respiration-driven ATP synthesis and ATP hydrolysis-driven electron transfer from

succinate to NAD. Addition to the ammonia-EDTA treated particles (AE-SMP) of a partially

purified soluble protein extracted from mitochondrial acetone powders partially restored these

activities (4,5). Sucrose density-gradient centrifugation suggested a molecular mass of 32 kDa for

the active peak of the soluble preparation, which was designated factor B (4,5). Using bovine

mitochondrial acetone powder extracts, we isolated a pure and monodisperse protein, which restored

ATP synthase-coupled activities to AE-SMP (6). Its molecular mass as estimated from

sedimentation equilibrium and gel filtration experiments was 11-12 kDa, and it was

immunoprecipitated by Sanadi’s anti-factor B antiserum in an Ouchterlony double diffusion

experiment (7). Sanadi and coworkers revised the molecular mass of their preparation to 29.2 kDa

and suggested that it is a dimer of monomer molecular mass 14.6 kDa (5,8). They also obtained

preparations of relative molecular mass 13 – 15 and 47 kDa, which exhibited factor B-like activity


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(5,9,10), and in 1990 published the sequence of the 55 amino terminal amino acids of a factor B

preparation with a relative molecular mass of 22 kDa (11).

The existence of factor B as a component of the ATP synthase complex remained

controversial, however. Whereas Sanadi claimed that the ATP synthase complex prepared in his

laboratory contained factor B (5,12), a thorough analysis of the polypeptide composition of an

ATPase complex prepared in Walker’s laboratory demonstrated the existence of the 16 unlike

polypeptides mentioned above, but no factor B (1,2). The multiplicity of Sanadi’s factor B-like

preparations with relative molecular masses ranging from 13 – 15 to 47 kDa, the small yield of our

preparation, which precluded antibody production and chemical analyses, and the compelling

extensive data of Walker’s laboratory discouraged further interest in the pursuit of factor B.

More recently, two nucleotide sequences corresponding in part to Sanadi’s 55 amino acid

sequence of factor B amino terminus were detected by our colleagues in the human genome. The

shorter frame corresponding to 96 amino acid residues was expressed with a histidine tag. The

expressed protein was found in inclusion bodies, and when extracted with 8 M urea and dialyzed

exhibited no factor B-like activity. The longer frame corresponding to 175 amino acid residues was

expressed once with a histidine tag and a second time fused to thioredoxin. Both were recognized

after purification by polyclonal antibodies raised to the shorter polypeptide. The histidine-tagged

preparation was also inactive, but the other, after removal of thioredoxin, exhibited all the functional


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features previously described by Sanadi and coworkers (4,5) and by ourselves (6,7) for factor B.

This paper describes the molecular and functional properties of this human factor B preparation.


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MATERIALS AND METHODS

Materials – NAD, NADH, ATP, oligomycin, and 2,4-dithiothreitol were obtained from

Calbiochem; DCCD, venturicidin, FCCP, tributyltin chloride, NEM, pCMB, sodium succinate, and 2-

mercaptoethanol were from Sigma; DEAE-Sepharose was from Amersham Pharmacia Biotech;

polyacrylamide was from Bio-Rad; Tris was from ICN; and oxonol VI was from Molecular Probes. All

other chemicals were reagent grade. AE-SMP were prepared from heavy beef heart mitochondria (13)

according to (14). After a final wash with 0.25 M sucrose containing 10 mM Tris-HCl, pH 7.8, the

particles were suspended in the same medium at 17 mg of protein/ml and stored at –80°C in small

aliquots. F1-ATPase was prepared according to Senior and Brooks (15) and stored in 50% saturated

ammonium sulfate at 4°C. The catalytic activity of the enzyme was in the range of 90-100 µmol ATP

hydrolyzed (min•mg)-1. OSCP was prepared by Dr. A. Matsuno-Yagi as before (16).

Assays – ATP-driven reverse electron transfer from succinate to NAD was assayed at 38° C

essentially according to Joshi and Sanadi (14), except that after 1-2 min of incubation of AE-SMP with

factor B, succinate, ATP, MgCl2, KCN, and dithiothreitol, the reaction was started by the addition of

NAD. The progress of the reaction was monitored at 340 nm in a Milton Roy 1201 spectrophotometer,

and the rate of NAD reduction was calculated using an extinction coefficient of 6.22 mM-1. Inactivation

of factor B with thiol modifying reagents was performed by incubating factor B for 5 min on ice in 50 mM

Tris-sulfate, pH 7.8, with increasing concentration of CdSO4 dissolved in water, or of phenylarsine oxide,


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pCMB or NEM dissolved in dimethylformamide. The incubation mixtures containing CdSO4 and

phenylarsine oxide also contained 0.25 mM 2-mercaptoethanol. The inhibitor treated samples of factor B

were assayed for reconstitution of the ATP-driven reverse electron transfer activity of AE-SMP in the

absence of dithiothreitol. The ATPase activities of F1 and AE-SMP were measured

spectrophotometrically by the coupled pyruvate kinase/lactate dehydrogenase method as before (17).

SDS-polyacrylamide gel electrophoresis and immunoblotting were performed as described

elsewhere (18). The immune complexes were visualized using the SuperSignal West Pico

chemiluminescence detection kit from Pierce. Protein concentration was determined, using the BCA kit

from Pierce. Standard molecular biology techniques, including cloning, restriction enzyme analysis,

plasmid preparation, and agarose gel electrophoresis were as described in (19). Amino terminal analysis

of purified recombinant human factor B was performed at the Protein and Nucleic Acid Core Facility of

this institution. Tryptic peptide mass fingerprinting and matrix-assisted laser desorption/ionization time-

of-flight (MALDI-TOF) mass spectrometry were performed at the Scripps Center for Mass Spectrometry.

Preparation of recombinant human factor B – The I.M.A.G.E. clone 2663774 containing EST

AW173068 was purchased from Research Genetics, AL. The sequence of the insert was confirmed by

DNA sequence analysis. The forward primer P1: 5’-TTCTGGGGCTGGTTGAATGCA-3’ and reverse

primers P2: 5’-TTACTTCAATTGTAATTTTAGTTC-3’ and P3: 5’-

CTTCAATTGTAATTTTAGTTCCAG-3’, with and without termination codon, respectively, were


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designed to amplify the full length of mature factor B by PCR using Taq DNA polymerase (Qiagen). The

amplicons were subcloned into pBAD/Thio-TOPO plasmid (Invitrogen) according to manufacturer’s

recommendations, and clones containing plasmids, designated pBAD-FB1 and pBAD-FB2, with the

correct sequence of the inserts were selected after DNA sequencing. In both pBAD-FB plasmids, the

factor B nucleotide sequence was located downstream of the nucleotide sequence coding for His-Patch

thioredoxin under regulation of araBAD promoter.

In the present study, a single colony of TOP10 cells transformed with pBAD-FB1 plasmid from a

fresh LB plate containing 100 µg of ampicillin/ml was used to inoculate 5 ml of 2 x YT liquid medium in

the presence of 100 µg of ampicillin/ml and grown overnight in a shaker at 37°C. Then, 0.5 liter of the 2

x YT liquid medium plus 100 µg of ampicillin/ml were placed in a 2 liter flask and inoculated with 3 ml

of the overnight culture and grown in a shaker (250 rpm) at 37°C until OD600 reached 0.5-0.6. The

temperature in the shaker was shifted to 30°C, and the protein expression was induced by adding 20%

arabinose to 0.02% final concentration. The incubation was continued for 4 h, and the culture was

harvested by centrifugation and stored at –80°C until further processing.

The bacterial cells were disrupted by 4 cycles of freeze-thawing, followed by sonication on ice

slurry, using a Branson sonifier. The soluble fraction was recovered by centrifugation for 1 hr at 15,000

rpm in a Beckman SS-34 rotor, and affinity-purified on His-Bind resin (Novagen) preloaded with NiCl2.

After wash with a pH 8.0 buffer containing 50 mM sodium phosphate, 0.3 M NaCl, and 20 mM imidazole,


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the fusion protein was eluted with 50 mM imidazole in the above buffer. The fractions containing the

fusion protein were concentrated using the Centriprep YM-10 centrifugal filter devise (Millipore) and

subjected to size-seiving chromatography on Ultragel AcA 44 (column size 1.5 cm x 90 cm) equilibrated

in a pH 8.0 buffer containing 50 mM Tris-HCl, 0.1 M NaCl, and 5 mM 2-mercaptoethanol.

To remove the thioredoxin moiety, the recombinant protein was cleaved overnight at room

temperature with recombinant enterokinase (Novagen) and purified by chromatography on DEAE-

Sepharose Fast Flow, using a pH 8.0 buffer containing 50 mM Tris-HCl and 5 mM 2-mercaptoethanol.

The fractions were assayed for restoration of ATP-driven reverse electron transfer from succinate to NAD

as catalyzed by AE-SMP and combined based on activity and purity after analysis by SDS-PAGE. The

protein was concentrated on YM-10 and stored in small aliquots at –80°C.

Preparation of cDNA for the ORF of human factor B – To obtain a cDNA encoding the full-

length ORF of human factor B, the oligonucleotide primers P1 and P2 (see above) as well as the

forward primer P4: 5’-CCTGCTCTGTACTCGACCCG-3’ and the reverse primer P5: 5’-

AAGAGTCGGTGGCGTCGAT-3’ were used to amplify by PCR the nucleotide sequences encoded

by inserts in I.M.A.G.E. clones 2663774 (GenBank accession number AW173068) and 24431

(GenBank accession number AF052186) obtained from Research Genetics, AL. The PCR

amplifications were performed in 50 µl volumes, each containing 5 µl of High Fidelity PCR buffer,

2 mM MgSO4, 0.2 mM dNTPs, 0.2 µM of each primer, ~10 ng of template, and 1 unit of


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PLATINUM Taq High Fidelity DNA polymerase (Life Technologies). The PCR conditions used

were: initial denaturation at 94°C for 2 min followed by 30 cycles each of 94°C for 30 s, 55°C for 1

min, 72°C for 1 min, and a final extension at 72°C for 7 min. In a second round, 5 µl from each of

the PCR reaction mixtures were combined and subjected to amplification with primers P2 and P4 as

above. The final PCR product was purified on agarose gel and subcloned into the pDrive vector

(Qiagen). The nucleotide sequence of the insert was verified in both directions by ABI 327

sequencer using BigDye terminator sequencing.


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RESULTS

Molecular properties of factor B – The predicted amino acid sequence of human factor B is

shown in Fig. 1. In the amino terminal 55 residues, there are three differences between the mature

human factor B shown in Fig. 1 and the sequence published by Sanadi and coworkers for the bovine

protein. The human amino acid residues Y-14, C-33 and E-43 are, respectively, H, G and Q in the

Sanadi partial sequence (11)2. Figure 2 shows on SDS polyacrylamide gel the polypeptide pattern of

Escherichia coli total cell lysate before (A) and after (B) induction of fused thioredoxin-factor B

synthesis. Lane C is the pattern of the soluble proteins of the induced cells, and lane D is the

purified thioredoxin-factor B fused preparation. Lane E shows two protein bands due to factor B

(upper) and thioredoxin (lower) after treatment of the fused protein with enterokinase, and lane F

shows a single protein band due to purified factor B. As determined by amino terminal sequence

analysis, this preparation of factor B contained at its amino terminus an extra tripeptide, L-A-L,

which had been retained after cleavage of thioredoxin. Otherwise, mass spectrometric analysis of

tryptic peptide masses of our recombinant factor B matched the calculated masses of the

corresponding peptides expected from its tryptic degradation (data not shown). Thus, the mature

factor B shown in Fig. 1 is composed of 175 amino acids with a calculated molecular mass of 20,

341 Da. The molecular mass derived from MALDI-TOF mass spectrometry of the factor B

preparation containing the amino terminal L-A-L tripeptide was 20,607 Da (calculated value: 20,638


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Da), and gel exclusion suggested a mass of ~ 22 kDa, which indicated that the recombinant human

factor B is monomeric (data not shown). Hydropathy analysis of the human factor B sequence

revealed no hydrophobic clusters of amino acids for possible membrane anchorage.

Functional properties of factor B – The term factor B has been applied to several protein

preparations derived from bovine heart mitochondria with molecular masses of 11 – 12 to 47 kDa,

which partially or largely restored to AE-SMP preparations ATP synthesis and energy-linked

reactions driven by ATP hydrolysis. It was, therefore, assumed that extraction of mitochondria with

ammonia-EDTA partially removed one or more factor B-like molecules from the mitochondrial ATP

synthase complexes. However, the effectiveness and the specificity of this extraction procedure for

factor B removal were unclear. Figure 3 depicts immunoblots of mitochondria (column A), SMP

(column B) and AE-SMP (column C) showing their relative contents of factor B (row 1), F1α

subunit (row 2), OSCP (row 3) and IF1 (row 4). The results clearly show that ammonia-EDTA

extraction is highly effective for the efficient removal of factor B3. The levels of F1 as represented

by its α subunit, and OSCP, which is not anchored to the membrane, were essentially unchanged by

the extraction. The level of IF1 was decreased in AE-SMP (Fig. 3, bottom row) which was

understandable, because of the alkaline pH of the extraction medium (24). Consistent with these

results, the ATPase activity of AE-SMP was shown to be about 40% higher than that of SMP.


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Figure 4 shows on the ordinate the activity of AE-SMP for ATP hydrolysis-driven electron

transfer from succinate to NAD as affected by addition of increasing amounts of recombinant human

factor B. The activity of AE-SMP in the absence of factor B was 4 nmol NADH formed (min • mg

protein)-1. Addition of factor B increased this activity to 225, which was 87% of the activity of the

SMP before ammonia-EDTA extraction. The abscissa intercept of the double reciprocal plot of

these data in the inset indicated half-maximal activation of AE-SMP at 0.33 nmol of factor B/mg of

AE-SMP. Assuming that AE-SMP have roughly the same amount of ATP synthase as SMP, i.e., 0.4

– 0.45 nmol/mg of protein (25), the factor B half-saturation amount of 0.33 nmol/mg of AE-SMP

indicated that this water-soluble recombinant human protein bound specifically and with high

affinity to the site from which its bovine counterpart had been removed. Addition to AE-SMP of F1

plus OSCP, instead of factor B, resulted in a marginal increase of reverse electron transfer activity

(10-12% as compared to 87% when factor B was added), indicating in agreement with the data of

Fig. 3 that the poor coupling activity of AE-SMP was not due to the loss of F1 and/or OSCP.

It has long been known that addition of low concentrations of oligomycin to SMP treated

with EDTA or ammonia-EDTA partially restores their energy-coupled activities. Apparently, when

added at low concentrations oligomycin preferentially reacts with and seals the proton leakiness of

the fraction of the ATP synthase complexes that have been rendered defective, thereby allowing the

remaining intact ATP synthases to function normally (26). Further addition of oligomycin would


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then begin to inhibit the intact ATP synthases, resulting in a decrease of the ATP synthase-coupled

activity tested. Similar results are shown in Fig. 5. It is seen that addition to AE-SMP of oligomycin

up to about 150 ng (0.3 nmol) /mg protein increased the ATP-driven reverse electron transfer

activity of the particles from about 5 to about 54 nmol NADH formed (min • mg protein)-1, and that

further increase in oligomycin concentration resulted in inhibition. As was shown previously (6),

addition of low concentrations of oligomycin (<120 ng/mg protein) to factor B-replenished AE-SMP

caused no activation, but only partial inhibition of ATP-driven reverse electron transfer activity. We

have examined the effect of other FO inhibitors with regard to activation of AE-SMP for ATP driven

electron transfer from succinate to NAD. At concentrations that they caused inhibition of ATPase

activity of AE-SMP from 10 to 70%, venturicidin and tributyltin chloride had no stimulating effect

on its reverse electron transfer activity. Incubation of AE-SMP at 0°C with 6 µM DCCD caused a

time-dependent inhibition of its ATPase activity , as expected. At 10 and 20 min of incubation,

when ATPase activity was inhibited by 30% and 43% respectively, ATP-driven reverse electron

transfer activity of the particles was increased from 4 nmol NADH formed (min • mg)-1 to 18 at 10

min and to 27 at 20 min of incubation. Further incubation of AE-SMP with 6 µM DCCD lowered

this stimulated activity to 22 at 30 min and to 7 at 50 min.

Previous preparations of bovine factor B were shown to be inhibited by thiol and dithiol

modifiers (5-7). The effects of these modifiers on recombinant human factor B are shown in Fig. 6.


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It is seen that factor B was inhibited when treated with the compounds shown, with the dithiol

modifiers phenylarsine oxide and Cd2+ causing 50% inhibition at < 10 µM concentration. As seen in

Fig. 1, human factor B contains 6 cysteine residues, two of which are located at positions 92 and 94.

The strong inhibitory effects of phenylarsine oxide and Cd2+ suggest that these dithiol modifiers bind

to cysteine residues 92 and 94 of factor B.

Effect of factor B on membrane potential – Figure 7 shows the formation of a membrane

potential in SMP (traces A and B), AE-SMP (traces C and D) and AE-SMP plus factor B (traces E

and F) as monitored by the absorbance change of oxonol VI at 630 minus 603 nm. Traces A and B

are controls with SMP, showing membrane energization upon addition of ATP or NADH, and

deenergization by inhibition of ATP hydrolysis by oligomycin, inhibition of NADH oxidation by

rotenone, or by uncoupling with FCCP. Traces C and D show that AE-SMP could not develop a

high membrane potential upon addition of either ATP or NADH, but that addition of oligomycin

repaired this defect and allowed high membrane potential formation upon subsequent addition of

NADH. Similar results were obtained when oligomycin was replaced with venturicidin or tributyltin

chloride, or when the AE-SMP were treated for 60 min at 0°C with 10 µM DCCD. Like oligomycin,

these reagents inhibited ATP hydrolysis, but allowed membrane potential formation, as in Fig. 7C,

upon addition of NADH (data not shown). These results suggested, therefore, that removal of factor

B created a proton leak in the FO of AE-SMP, which could be blocked by the specific FO inhibitors


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mentioned. Traces E and F show that the addition of factor B to AE-SMP repaired the defect shown

in traces C and D, and made it possible for the particles to develop a high membrane potential upon

addition of either ATP or NADH. As seen in traces E and F, the development of the membrane

potential to a static head level in factor B supplemented AE-SMP was a slow process as compared to

the results shown in traces A and B for SMP. It is possible that factor B binding to AE-SMP

requires an energized conformation of FO-F1, apparently best achieved with the addition of ATP.

This possibility would be somewhat analogous to the binding of IF1 to F1, which occurs during ATP

hydrolysis by the enzyme (24).


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DISCUSSION

Molecular properties of factor B – According to the data reported here, mature human factor

B is composed of 175 amino acid residues with a molecular mass of 20,341 Da and the amino acid

sequence shown in Fig. 1. The active, recombinant human factor B purified and used in the studies

reported here contained an extra L-A-L tripeptide at its amino terminus. It was water-soluble,

monomeric, and stable when stored at –80°C in a pH 8.0 buffer containing 5 mM 2-

mercaptoethanol. In its amino terminal 55 residues and molecular mass, this recombinant human

factor B is similar to the latest of a number of bovine mitochondrial factor B preparations reported

previously by Sanadi and coworkers (11). Because rigorous data on the purity and the amino

terminal sequences of the other factor B-like preparations of Sanadi and coworkers are not available,

it is not possible to discuss them further. Nor can we compare our pure protein of molecular mass

11-12 kDa, which was isolated from bovine heart mitochondria and exhibited a factor B-like activity

(6,7), with the recombinant factor B described here. Its low yield precluded in 1976 any attempts at

amino terminal sequencing, but its sensitivity to thiol and dithiol modifiers suggests that it might

have been an active proteolytic fragment of the bovine factor B. This possibility is supported by the

fact that immunoblots of bovine heart mitochondria with our polyclonal antibody used in the present

work recognized only a single protein band corresponding in mobility on SDS-gels to that of our

recombinant human factor B.


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Factor B gene – Figure 8 shows a segment of the human genome on chromosome 14q21.3.

This segment spans ~13 kb and contains 5 exons, of which the nucleotide sequences of the areas

depicted in Fig. 8 by the filled boxes from ATG in exon 1 to TAA in exon 5 correspond to the

sequence of the human factor B cDNA shown in Fig. 1, except that in the draft sequence of the

Human Genome Project (27) T168 is given as C, which would result in the substitution of Pro for Leu

in the corresponding position of the presequence of factor B. As seen in Fig. 8, there are 2 stop

codons and 2 polyadenylation signals in this DNA segment. An mRNA transcript arising from the

processing of pre-mRNA at poly (A) site ATTAAA would encode a hypothetical protein HSU79253

(LocusID 27109). Reverse transcription PCR, using multiple tissue cDNA panels I and II

(Clontech), showed that mRNAs corresponding to both the short (exons 1-3) and the long (exons 1-

5) sequences were present in 16 different human tissues that we examined. As mentioned above, the

short polypeptide containing a His tag was expressed in our laboratory as inclusion bodies and used

to raise polyclonal antibodies. This antibody preparation recognized in bovine heart mitochondria a

single protein with Mr of 22 kDa, which could be extracted from SMP by the procedures used to

remove factor B (Fig. 3). The short polypeptide, expected to have an Mr ~11 kDa , was not detected

in bovine heart mitochondria. This consideration suggests that this short “isoform” of factor B, if

translated, either does not enter mitochondria or is degraded after entering this organelle.


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A second human homologue, the hypothetical protein FLJ10241 (LocusID 55101) encoded

by a gene located on chromosome 19q13.2, with sequence similarity of 49% to human factor B was

identified by BLASTP (28) search of the GenBank database. This search also revealed the existence

of two mouse orthologues (GenBank Accession Nos. NP_080812 and BAB26107) with sequence

similarities of 83 and 47%, respectively, as well as two gene products in Drosophila melanogaster

(GenBank Accession Nos. AAF58055 and AAF51634) and Caenorhabditis elegans (GenBank

Accession Nos. AAK95868 and AAF59541) exhibiting 44-52 % sequence similarity to human factor

B. Neither a prokaryotic homologue nor a counterpart in Saccharomyces cerevisiae has been

identified.

Role of factor B – The results presented here clearly show that factor B is necessary for the

energy transduction activity of the ATP synthase complex. Treatment of well-coupled SMP with

ammonia/EDTA at pH 8.8 resulted in the specific removal of a protein of Mr 22 kDa, and in the

inability of the AE-SMP to develop and maintain a membrane potential as a result of ATP hydrolysis

or NADH oxidation (Fig. 7). This defect could be repaired by addition to the AE-SMP of a

recombinant human protein of Mr 22 kDa at a molar concentration nearly stoichiometric to the ATP

synthases of the particles. The recombinant human protein was homologous in molecular mass and

amino terminal sequence to the purified bovine factor B of Sanadi and coworkers (11), and like all

factor B preparations was sensitive to treatment with monothiol and especially dithiol modifiers.


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The bovine ATP synthase complex contains at least 7 subunits of totally unknown function,

namely subunits ε, F6, A6L, d, e, f, and g. This complexity makes it difficult to employ the E. coli

ATP synthase with only 8 unlike subunits (29) as a model into which to build a role for factor B.

However, the basic operational design of the E. coli ATP synthase, which is composed of a catalytic,

a rotor, and a stator domain (30,31) applies to the more complicated ATP synthases (32,33), and we

will discuss the role of factor B in the light of this basic design. The fact that factor B is not a

component of the catalytic domain is clear and needs no further consideration. It cannot be a

necessary component of the stator either, because unlike the systems lacking OSCP the ATPase

activity of AE-SMP (5 µmol/min/mg) is completely inhibited by FO inhibitors, including DCCD

whose mechanism of inhibition is well known. The sensitivity of the ATPase activity of AE-SMP to

FO inhibitors also indicates that in these particles the rotating part of F1, i.e., γ and δ, is not

disengaged from FO. These considerations allow the conclusion, therefore, that factor B is a

component of FO.

As seen in Fig. 7, AE-SMP are uncoupled and incapable of forming a membrane potential as

a result of respiration or ATP hydrolysis. This defect can be repaired by addition of factor B or by

addition of FO inhibitors at concentrations that these reagents inhibit ATP hydrolysis. Repair by

factor B allows membrane potential formation as a result of ATP hydrolysis or respiration. Repair

by FO inhibitors allows only respiration-dependent membrane potential formation, which indicates


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that the proton leak of AE-SMP involves, at least in part, the normal proton channel of FO. This

leads to the following important question. Since the proton leak of AE-SMP can be blocked by FO

inhibitors, especially DCCD, does the proton leak from the cytosolic (positive, P) side to the matrix

(negative, N) side of the membranes in respiring AE-SMP require the rotation of the c ring? Based

on the generally accepted basic design and operation of the ATP synthase complex, the answer to

this question is no. This is because in respiring AE-SMP the proton leak, which can be blocked by

DCCD, occurs in the absence of F1 substrates. These considerations allow the conclusion that the FO

of mammalian ATP synthase is capable of uncoupled transmembrane proton translocation via a

second path that involves, at least in part, its normal proton channel, possibly including Glu-58 of

subunit c. Proton translocation via this second path does not require the operation of the rotor of the

ATP synthase, and can be blocked by the water-soluble factor B which appears to bind to FO on the

matrix side.

Whether the FO subunits F6, A6L, d, e, f and g, which like factor B do not have prokaryotic

counterparts, are involved in this second proton path remains to be seen. Another mammalian FO-F1

subunit that does not have a prokaryotic counterpart is the ATPase inhibitor protein, IF1, which binds

to F1 β subunits and prevents futile ATP hydrolysis when the protonmotive force is low (34-36).

The fact that factor B can also be easily and reversibly removed from FO-F1, and the fact that its

displacement results in dissipation of membrane potential may be indicative of a regulatory function


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for factor B as well. It is generally assumed that the state 4 rate of respiration is due to a slow proton

leak through the mitochondrial inner membrane at high protonmotive force. However, it is well

known that proteoliposomes of sonicated phospholipids plus a proton pump such as cytochrome

oxidase or nicotinamide nucleotide transhydrogenase are not as proton leaky at high protonmotive

force as SMP. Therefore, considering that over-reduction of the respiratory chain results in an

increased rate of superoxide anion production, which leads to the formation of toxic H2O2 and

hydroxyl radicals (37,38), it is possible that factor B acts as a pressure valve for maintaining the

protonmotive force (hence the reduced level of the respiratory chain) below a damaging threshold

(see also the concept of “mild uncoupling” in 38).

Finally, another point that deserves consideration is the designation “factor B” for the protein

discussed here. Since more than two decades ago, the word “factor” has correctly been replaced by

“subunit,” and already there are among the ATP synthase subunits a β subunit and a b subunit.

Continuing the alphabetical sequence beyond e, f, and g would result in confusion with the yeast

ATP synthase subunit designations. Therefore, it may be appropriate to designate the protein under

consideration here as subunit s, after its original discoverer.


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Acknowledgments

We thank Drs. Akemi Matsuno-Yagi and Mutsuo Yamaguchi for their interest and help in

this work, and C. Munoz for the preparation of bovine heart mitochondria. Synthesis of

oligonucleotides was supported in part by the Sam and Rose Stein Endowment Fund.


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Footnotes

1The abbreviations used are: SMP, bovine heart submitochondrial particles; EDTA,

ethylenediaminetetraacetic acid; AE-SMP, SMP depleted of factor B; OSCP, oligomycin-sensitivity-

conferring protein; DCCD, N,N’-dicyclohexylcarbodiimide; FCCP, carbonyl cyanide p-

trifluoromethoxyphenylhydrazone; NEM, N-ethylmaleimide; pCMB, p-chloromercuribenzoate;

ORF, open reading frame; PCR, polymerase chain reaction; and SDS, sodium dodecyl sulfate.

2The residues G-33 and Q-43 in the partial sequence of the bovine factor B published by Sanadi and

coworkers (11) may in fact be C-33 and E-43 as in the human protein (see GenBank accession

number AV611504, which might be a partial nucleotide sequence of the bovine factor B cDNA).

3The ease with which factor B can be dissociated from the membrane may be related in part to the

fact that most mammalian FO-F1 preparation reported in the literature exhibited low or no ATP-32Pi

exchange activity (see, for example 20-23).


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LEGENDS TO FIGURES

Fig. 1. The nucleotide and the deduced amino acid sequences of human factor B. Arrow

indicates the start of the sequence of the mature human factor B shown in bold letters and numbered

on the right. Star indicates the termination codon. For details see Materials and Methods. This

nucleotide sequence has been submitted to the GenBankTM/EB1 Data Bank with accession number

AY052377.

Fig. 2. Expression and purification of mature human factor B as depicted on SDS-

polyacrylamide gel. Lane A, uninduced total E. coli cell lysate; lane B, total cell lysate after

induction; lane C, soluble E. coli proteins after induction; lane D, purified thioredoxin-factor B fused

preparation; lane E, fused protein of lane D after treatment with enterokinase; lane F, purified human

factor B containing an extra amino terminal L-A-L tripeptide. Lanes A, B and C received 20 µg of

protein each, and lanes D, E and F 5 µg of protein each. The gel (SDS-15% polyacrylamide) was

stained with Coomassie Brilliant Blue. Numbers on the left show an Mr scale.

Fig. 3. Immunoblots of intact bovine heart mitochondria (column A), SMP (column B), and

AE-SMP (column C) blotted with subunit-specific polyclonal antibodies to recombinant human

factor B (row 1), α subunit of bovine F1 (row 2), bovine OSCP (row 3), and bovine ATPase inhibitor

protein IF1 (row 4). For other details, see Materials and Methods.


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Fig. 4. Effect of increasing concentrations of recombinant human factor B on the ATP-

driven reverse electron transfer (succinate to NAD) activity of AE-SMP. Inset shows a double-

reciprocal plot of the data indicating on the abscissa intercept half-maximal activation at 0.33 nmol

of factor B/mg of AE-SMP.

Fig. 5. Effect of increasing concentrations of oligomycin on the ATP-driven reverse electron

transfer activity of AE-SMP. AE-SMP in 0.25 M sucrose, 10 mM Tris-HCl, pH 7.8, were

preincubated for 5 min on ice with oligomycin dissolved in ethanol at the concentrations indicated,

then sampled into the reaction mixture. Ethanol concentration in the reaction mixture was < 1%.

Fig. 6. Inhibition of factor B with NEM, pCMP, phenylarsine oxide (PAO), and CdSO4.

Factor B was preincubated with the indicated reagents as described in Materials and Methods, then

added to the reaction mixture for reconstitution of the ATP-driven reverse electron transfer activity

of AE-SMP. The factor B concentration in the reaction mixture was 17.6 µg of factor B/mg of AE-

SMP. 100% activity was 160 nmol NADH formed (min • mg AE-SMP)-1.

Fig. 7. Effects of factor B and oligomycin on membrane potential formation by AE-SMP.

The reaction mixture at 30°C contained 0.25 M sucrose, 50 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 2

µM oxonol VI, and 0.1 mg of SMP (traces A and B) or AE-SMP (traces C-F) per ml. Where

indicated 4.5 mM ATP, 0.25 mM NADH, 5 µg of oligomycin/ml, 5 µM rotenone, and 5 µM FCCP

were added. In experiments E and F, the reaction mixtures contained 6 µg of factor B. In Trace D,


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oligomycin was added 7.5 min after the addition of NADH (see the 4 min gap shown) to allow

NADH to become completely oxidized. Membrane potential formation was monitored by the

absorbance change of oxonol VI at 630 minus 603 nm in an SLM DW-2000 dual wavelength

spectrophotometer.

Fig. 8. Segment of the human genome on chromosome 14q21.3 containing in exons 1-5

(filled boxes) the nucleotide sequence shown in Fig. 1 for the human factor B cDNA, except for

substitution of T168 for C in the draft sequence of the Human Genome Project (27). The start codon

(ATG), the two stop codons (TGA and TAA), and the two polyadenylation signals are marked. This

figure includes in part (exons 1-3) the DNA sequence encoding the hypothetical protein HSU79253

(see text).


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CCTGCTCTGTACTCGACCCGGCACTGGGTACCGGAAGAACCAGACAGCTCGGTTTTTGCC

ACCATTTATAAGTCTGTGTCCTTTTCTTGAGTACTGAACCGAGATACAGTTTTAAATGTG M C

CTGTGCGGTCTCTGAGCAGCGACTCACCTGTGCAGATCAAATGATGCTGTTTGGAAAAAT C A V S E Q R L T C A D Q M M L F G K I

TTCCCAGCAGTTGTGTGGCGTAAAGAAACTCCCATGGTCATGTGACTCCAGATACTTCTG S Q Q L C G V K K L P W S C D S R Y ↓↓F W 2

GGGCTGGTTGAATGCAGTGTTTAATAAGGTGGATTATGATCGCATCAGGGATGTTGGCCC G W L N A V F N K V D Y D R I R D V G P 22

TGACAGGGCGGCATCCGAGTGGTTGCTGCGCTGTGGGGCCATGGTGCGCTACCATGGCCA D R A A S E W L L R C G A M V R Y H G Q 42

GGAGAGGTGGCAGAAGGACTACAACCACCTTCCAACAGGCCCTCTGGACAAATACAAGAT E R W Q K D Y N H L P T G P L D K Y K I 62

TCAGGCGATCGACGCCACCGACTCTTGTATCATGAGCATTGGATTTGATCACATGGAGGG Q A I D A T D S C I M S I G F D H M E G 82

CCTAGAGCATGTTGAAAAAATAAGGCTGTGCAAGTGTCATTATATCGAGGATGACTGTTT L E H V E K I R L C K C H Y I E D D C L 102

GCTGAGACTTAGTCAACTTGAAAATTTACAAAAAACCATATTGGAAATGGAAATAATATC L R L S Q L E N L Q K T I L E M E I I S 122

CTGTGGGAATATCACAGACAAAGGCATCATTGCTTTGCGTCATTTAAGAAACCTCAAATA C G N I T D K G I I A L R H L R N L K Y 142

TTTGTTGTTAAGTGATCTTCCTGGAGTAAGAGAAAAAGAAAATCTTGTCCAAGCCTTTAA L L L S D L P G V R E K E N L V Q A F K 162

GACAGCACTGCCTTCTCTGGAACTAAAATTACAATTGAAGTAA T A L P S L E L K L Q L K * 175

FIGURE 1


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FIGURE 2


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FIGURE 3


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FIGURE 4


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FIGURE 5


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FIGURE 6


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FIGURE 7


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Grigory I. Belogrudov and Youssef HatefiFactor B and the mitochondrial ATP synthase complex

published online December 14, 2001J. Biol. Chem.

10.1074/jbc.M111256200Access the most updated version of this article at doi:

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factor b and the mitochondrial atp synthase complexa,b grigory i. belogrudov and

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