THE JOURNAL OF B I O ~ I C A L CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269. No. 1, Issue of January I. pp. 421-432, 1994 Printed in U.S.A.

Defective Energy Coupling in &Subunit Mutants of Escherichia coli FIFo-ATP Synthase*

(Received for publication, June 7, 1993, and in revised form, September 1, 1993)

Andrea L. Hazard and Alan E. Senior From the Department of Biochemistiy, University of Rochester Medical Center, Rochester, New York 14642

Membrane vesicles from 13 strains carrying muta- tions in the C-terminal region of the &subunit of Esch- erichia coli FIFo-ATP synthase were characterized in respect to ATPase activity, ATP-driven proton-pumping, dicyclohexylcarbodiimide sensitivity of ATPase, and oxidative phosphorylation. The salient finding was that energy-coupling between F1 and Fo sectors of the en- zyme is impaired by several of the mutations. The SGlSON mutant appeared completely uncoupled in vitro. The data emphasize the role of the C-terminal region of &subunit in integration of the proton conduction ma- chinery in Fo with the three F1 catalytic sites. It is sug- gested that the C-terminal region of &subunit, specula- tively located in the central region of the asps hexagon, acts functionally at the interface between the helical domain of the stalk and the F1 subunits to relay confor- mational signals which alter the affinities of the cata- lytic sites for substrates and products.

The Escherichia coli FIFo-ATP synthase is composed of two sectors, the F1 sector which catalyzes the hydrolysis or synthe- sis of ATP, and the Fo sector which conducts protons across the membrane. ATPase and proton pumping activities are tightly coupled, so that ATP hydrolysis causes the formation of a pro- ton gradient, and in the presence of a proton gradient gener- ated by membrane electron transfer complexes the enzyme catalyzes the synthesis of ATP. F1 is connected to Fo by a nar- row stalk (Gogol et al., 1987; Lucken et d., 1990) which is believed to have an important role in the coupling of proton gradient energy to ATP synthesis and hydrolysis (reviewed in Fillingame (1990), Senior (19901, and Engelbrecht and Junge (1990)).

In E. coli, subunits 6 (an F1 subunit) and b (an Fo subunit) are generally considered to form the stalk connecting F1 and Fo. As discussed in the previous paper (Hazard and Senior, 1994), several lines of evidence support the proposal that the C-ter- minal region of &subunit is important for enzyme function. A recent report (Mendel-Hartvig and Capaldi, 1991a) suggested that this region was involved in energy coupling between F1 and Fo. These workers found that afier digestion of E. coli F1 with trypsin to remove around 20 amino acid residues at the C-terminal end of b-subunit, the F1 retained ATPase activity and could still bind to Fo, but the membrane-bound ATPase was now insensitive to DCCD.l Two other reports (Jounouchi et al., 1992; Joshi et al., 1992) have shown using deletion mutagen- esis that the C-terminal region of &subunit (or the analogous mitochondrial oligomycin sensitivity conferral protein) is im- portant for binding of F1 to Fo.

* This work was supported by National Institutes of Health Grant GM 25349 (to A. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviation used is: DCCD, dicyclohexylcarbodiimide.

The previous paper (Hazard and Senior, 1994) describes the generation of mutations in the C-terminal region of Ssubunit by random, cassette-, and site-directed mutagenesis. Many of the mutations were found to cause impairment of growth on succinate plates or in limiting glucose medium, indicating that oxidative phosphorylation in vivo was defective. The effects on growth ranged from mild to severe, implying possibly different degrees of impairment of enzyme activity or energy coupling between F1 and Fo. In this paper, 13 of these mutants are characterized biochemically. Mutants exemplifying a range of growth characteristics were chosen for analysis, including five mutants which showed zero or very low growth by oxidative phosphorylation (6G150T, sG150P, sG150D, 6G150N, and SL99P/A149D), three mutants with intermediate growth char- acteristics (6L99P, SGlSOA, and SG150CN164L), and five mu- tants with relatively strong growth characteristics (SG150S, 6A149P, SA149S, 6A149T, and 6A149D). Several enzyme activi- ties were assayed to assess the structural integrity, turnover rate, and energy coupling characteristics of FIFO.

MATERIALS AND METHODS Growth of E. coli Cells; Preparation of Membrane Vesicles-Bacterial

strains and growth conditions were as described in the accompanying paper (Hazard and Senior, 1994). Preparation of membrane vesicles was as described by Senior et al. (1979).

Immunoblotting-Immunoblotting of membrane vesicles using rab- bit polyclonal anti-subunit b antibody was done as described by Perlin and Senior (1985). Nonspecific antibodies were removed from the anti-b antiserum by preincubation of 0.5 ml of the antiserum with 7.5 mg of membranes from strain AH3 (which does not express subunits b or 6) and ultracentrifugation as described by Perlin and Senior (1985). The secondary antibody was horseradish peroxidase-coupled donkey anti- rabbit IgG from Amersham, and horseradish peroxidase activity was detected by chemiluminescence using the Amersham ECL kit.

Enzyme Assays-ATPase activity was measured as described by Dun- can and Senior (1985). ATP synthesis by oxidative phosphorylation using NADH as respiratory substrate was measured as described by Wise and Senior (1985). Measurement of proton gradient formation by acridine orange fluorescence quenching was done as in Perlin et al. (1983); stripping of Fl from membranes with 1 M potassium thiocyanate and reconstitution of membranes with wild-type Fl (250 pg of Fl/mg of membranes) was described by the same authors.

Inhibition ofATPase Activity by DCCD-Membranes (0.6 to 1.6 mg of proteidml) were incubated for 30 min at 30 “C in 10 mM HEPES-KOH, pH 7.5,5 m~ MgC12, 100 m~ KC1 (HMK buffer), with varied concentra- tions of DCCD (from a stock solution in ethanol). Control samples con- tained the same amounts of ethanol (51% v/v).

Measurement of Release of Fl from Membranes-Membranes (1 mg/ ml) were incubated in HMK buffer with or without 50 p~ DCCD for 30 min at 30 “C and centrifuged in a Beckman Airfuge (20 min x 120,000 x gmm). ATPase activity and protein concentration in the supernatant fraction were measured. F, release was calculated as total ATPase activity in the supernatant fraction divided by total ATPase activity of a noncentrifuged control sample. The ratio of the specific activity of the ATPase in the supernatant fraction to that in the initial membrane suspension was the same for wild-type and two representative mutants (SG150A and 8G150N; data not shown). Therefore, there was no indi- cation of a differential increase or decrease in the intrinsic turnover rate of F1 after release from the membranes in mutants as compared to wild-type.

427

428 &Subunit Mutants of FIFO Which Cause Uncoupling TABLE I

Membrane ATPase and ATP-driven proton pumping activities of mutants

Genotype ATPase" ATP-driven proton pumping"

Native KSCN-Flb

unc+e uric-' 6G150T 6G150P 6G150D SGl5ON 6A149P 6G150A 6G150S 6G150CN164L 6L99P/A149D 6A149T 6L99P 6A149S 6A149D

% wild-type

100 0 3 3 1

11 10 17 14 14 23 51 49 97 68

% quench of acridine orange fluorescence

88 80 0 0 0 6 0 3 0 3 0 75

17 96 9 80

14 65 18 92 49 92 48 92 71 95 66 87 84 92

a Membrane ATPase of the unc+ (wild-type) was 1.4 unitslmg of mem- brane protein (average of five different preparations). The values for mutants are expressed as percent of wild-type membranes prepared concurrently. All values are means of a t least duplicate assays.

Membranes were extracted with 1 M KSCN, then reconstituted with wild-type F, (see "Materials and Methods").

unc+ (wild-type) was strain pAH31AH3; unc- was strain pUC118I AH3; all mutants were expressed from plasmid pAH3 in strain AH3.

Inhibition of Membrane-bound ATPase by ADP-fluoroaluminate -Membranes (1 mg of proteidml in HMK buffer) were incubated with ADP (25 p ~ ) , AlC13 (0-100 p ~ ) , and NaF (5 mM) a t 30 "C for varying times, then assayed for ATPase activity as described above except that Pi released was estimated by the method of Van Veldhoven and Mann- aerts (1978).

Aurouertin Fluorescence Assays-Membranes were incubated in 20 mM potassium phosphate, pH 7.5,5 m~ MgC12, and 300 m~ KC1 a t 0.5 mg/ml with 05-10 p~ aurovertin. Fluorescence properties were meas- ured in a SPEX Fluorolog 2 spectrofluorimeter, using Aexc = 365 nm and A,, = 495 nm. Succinate (20 mM) was added to generate a proton gra- dient.

Other Techniques-Membrane protein concentration was determined by the method of Lowry et al. (1951), soluble protein was estimated by the method of Bradford (1976).

RESULTS GeneraZ-The mutants to be tested were expressed from

plasmid pAH3 in strain AH3 (Hazard and Senior, 1994). Plas- mid pAH3 expresses the uncE, -F, and -H genes (encoding the c, b, and 6 subunits), and strainAH3 contains a deletion of uncF and -H genes. Haploid levels of FIFo were found in the mem- brane vesicles prepared from strain pAH3/AH3 as indicated by assays of membrane ATPase activity and immunoblotting with anti-subunit b antibody.

Membrane ATPase Activity; NADH- and ATP-driven Proton Pumping-The membrane ATPase activities of wild-type and mutant strains are shown in Table I, column 1. A wide range of activities was seen. Acridine orange fluorescence quenching was assayed as a measure of proton gradient formation. Proton pumping into the (inverted) membrane vesicles can be driven either by electron transport complexes (e.g. upon addition of NADH) or by FIFo (upon addition of ATP). NADH-dependent proton gradient formation was the same for all strains listed in Table I, a t around 90% quenching of acridine orange fluores- cence, and was not increased by preincubation with 50 p~ DCCD, indicating that none of the membrane preparations was unusually permeable to protons (data not shown). ATP-depend- ent proton gradient formation is shown in Table I, column 2, and there was a wide range of activity in the different mutants.

Tests of Correct Assembly of FlF0-ATP Synthase-The very low membrane ATPase and ATP-driven proton-pumping activi-

A

1 1 2 3 4 5 6 7 1

""-

B C

7-

D E

I 1 2 3 4 1 rn "."

FIG. 1. Immunoblota of membrane vesicle preparations wing anti-subunit b antibody. 1 pg of membrane protein was run on an SDS-gel, then immunoblotted with anti-subunit b antibody as described under "Materials and Methods." Mutants are compared to wild-type membranes (pAH3/AH3) which were made concurrently to allow for variation among different preparations. unc- control membranes were from strain pUC118/AH3. A, lanes 1-7 are, respectively, wild-type, 6A149S, 6G150A, %EON, 6G150T, M=150P, and unc-; B, lanes 1 4 are wild-type, 6L99P, 6A149D, 6L99P/A149D; C, lanes 1 and 2 are wild- type, 6G150D; 0, lunes 1-4 are wild-type, 6A149P, &4149T, 6G150C/ V164L; E, lunes 1 and 2, are wild-type, 6G150S.

ties seen in Table I for some of the mutants might have been caused by incorrect assembly of the enzyme subunits into FIFo oligomer. To test this possibility, we carried out two tests. First, we extracted the membranes with 1 M KSCN (which removes F1 subunits), then reconstituted with wild-type F1 and measured ATP-driven proton gradient formation (Table I, column 3). Sec- ondly, we performed immunoblotting tests on the native mem- branes using anti-subunit b antibody (Fig. 1). The mutants 6G150D, 6G150P, and 6G150T each showed very low ATP- driven proton pumping in KSCN-extracted and F1-reconsti- tuted membranes, and low amounts of subunit b in the mem- branes, indicating that incorrect assembly of the FIFO occurred. All of the other 10 mutants had normal or near-normal levels of subunit b and ofATP-driven proton-pumping activity in KSCN- extracted/F1-reconstituted membranes, showing that assembly of the enzyme was not impaired. No further studies were done on the three mutants in which incorrect assembly of FIFo was shown to be the cause of impaired enzyme activity.

DCCD Sensitivity of Membrane ATPase Activity-The inhibi- tor DCCD binds to subunit c and prevents proton movement through Fo. In wild-type enzyme, where ATPase activity and proton movement are tightly coupled, binding of DCCD to Fo inhibits ATPase activity in F1. In order to assess whether ATPase and proton movement were tightly coupled in the mu- tant enzymes, DCCD sensitivity of membrane ATPase activity was measured. The results are shown in Fig. 2,A, B, and C, and the percent inhibition of ATPase activity at 50 p~ DCCD is shown in Table 11, column 1. None of the mutants was inhibited by DCCD to the same extent as wild-type, and in several mu- tants the ATPase activity was significantly insensitive to DCCD.

The ATPase activity of a mutant FIFo might be insensitive to DCCD for one of several reasons. First, DCCD may not react as efficiently with subunit c in the mutant enzyme. Second, F1 may be physically released from Fo more easily in the mutant enzyme, and the activity of F1 released during the assay would

&Subunit Mutants of FIFO Which Cause Uncoupling 429

‘1 64L

0 1 “ c w l _I L99PiA149D

1A\ - Lg9P

A slightly higher (7-13%) but much too low to account for the DCCD insensitivity seen. As noted under “Materials and Meth- ods,” control experiments were done to ensure that there was no differential enhancement ofATPase turnover in mutant ver- sus wild-type on release of F1 from the membranes. To assess whether binding of DCCD to Fo caused release of F1, these experiments were repeated with 50 p~ DCCD present during the preincubation. No increase in release of F1 was observed (data not shown). In summary, these results show that several of the mutants, notably SG150N, SA149P, SGEOA, SG150S, SG150CN164L, and SL99P/A149D, are partly uncoupled, so that ATPase activity of F1 is insensitive, to a greater or lesser degree, to inhibition by DCCD.

Oxidative Phosphorylation in Membrane Vesicles from Mu- B tants-ATP synthesis was measured in the presence of an

NADH-induced proton gradient (Table 11, column 3). The mu- tant 6G150N had zero detectable ATP synthesis activity. The mutants SA149P, 6G150A, SG150S, 6G150CN164L, and SL99P/A149D all had very low ATP synthesis activities (245% of wild-type). The mutants SA149T and 6L99P had intermedi- ate ATP synthesis activities (21 and 28% of wild type, respec- tively), and the mutants 6A149S and SA149D had higher ATP synthesis activities (57 and 66% of wild-type, respectively).

Table 11, column 4, shows the ratio of ATP synthesis to ATPase activity in membranes from wild-type and mutants. This value ranges from 0 (SG150N) up to 0.08 (wild-type). It can be seen that there is a correlation in the various mutants be- tween impairment ofATP-driven proton pumping, sensitivity of ATPase to DCCD, impairment of ATP synthesis, and the ratio ofATP synthesis to ATPase activity (Tables 1 and 11). The latter ratio appears to provide a qualitative measure of the degree of impairment of energy coupling between F1 and Fo.

Overexpression of F,F,Overexpression of FIFo was achieved using plasmid pDP34 harbored in strain AN888 (Mag- gio et al., 1988). This resulted in around a 5-fold increase in membrane ATPase activity (units per mg of membrane protein) in wild-type. The mutations 6G150N and SA149P were trans- ferred into pDP34 using suitable restriction endonuclease frag- ments and expressed in strain AN888. These two mutations were chosen because they appeared to be the most impaired in energy coupling. Membrane ATPase was increased in the two overexpressed mutants, both in absolute terms and as a per- centage of wild-type. Thus, SA149P had the same ATPase as wild-type (compared to 16% in Table I, column l), and SG150N had 41% of Wild-type (compared to 11% in Table I column 1).

FIG. 2. DCCD sensitivity ofATPase activity. The wild-type (wt) is the average of four wild-type membrane preparations.

not be affected by DCCD. Binding of DCCD to Fo may itself cause release of F1 in mutant enzymes. Third, F1 may be firmly attached to Fo, but ATPase activity may be partly or completely uncoupled from proton movement, so that DCCD binding to Fo does not fully inhibit ATPase activity.

The first possible cause of DCCD-insensitive ATPase activity, that DCCD does not bind to Fo, was tested by preincubating mutant membranes with 50 p~ DCCD and measuring ATP- dependent acridine orange fluorescence quenching. ATP-de- pendent proton pumping was completely abolished in all of the mutants (data not shown). The second possible cause of DCCD- insensitive ATPase activity, that F1 is released from the mem- branes, was tested by measuring F1 release directly. Mem- branes were preincubated under the same conditions used for the DCCD sensitivity experiments and then centrifuged in an Airfuge, as described under “Materials and Methods.” ATPase activity in the supernatant was assayed. Under these condi- tions, 6% of F1 was released from wild-type membranes (Table 11, column 2); release of F1 from mutant membranes was

”

The characteristics of ATP-driven proton pumping, however, were essentially unchanged. The values for percent quench of acridine orange fluorescence were: wild-type, 86%; 6G150N, 0%; and 6A149P, 24%; as compared to 88%, 0%, and 17%, re- spectively, in Table I, column 2. These data showed that the observed impairment of energy coupling was independent of the level of expression of the enzyme and was caused by the intrinsic effects of the mutations on the FIFo complex.

Attempts to Develop Assays of Energy Coupling between F, and Fo Using Aurovertin Fluorescence and ADP-puoroalumi- nate Inhibition-Chang and Penefsky (1974) demonstrated that the fluorescence of aurovertin bound to FIFo in submito- chondrial particles from bovine heart was enhanced by succi- nate oxidation, and that the effect was uncoupler-sensitive. Here we found that aurovertin strongly inhibited the ATPase activity in wild-type (overexpressed pDP34/AN888 or haploid- level pAH3/AH3) membranes and gave a small enhancement of fluorescence on binding. However, no enhancement of the fluo- rescence intensity (Aern = 495 nm) or change in emission spec- trum characteristics occurred on addition of succinate. Control experiments showed that there was a strong quench of acridine orange fluorescence under these conditions, i.e. a transmem-

430 &Subunit Mutants of FIFO Which Cause Uncoupling

TABLE I1 Energy coupling in mutant membranes

Genotype Inhibition of ATPase F1 released by 50 p~ DCCD" ATP synthesis by

from membranes" oxidative phosphoq-lation SynthesiefATF'ase Ratio ATP

% % % wild-type unc+ 8G150N fiA149P 8G150A 8Gl50S 8G150CN164L fiL99P/A149D 6A149T 6L99P fiA149S fiA149D

82 22 48 49 69 53 50 72 71 76 78

6 10 13 10 11 11 11 8 9 7 9

100- - 0 2 2 4 6 4

21 28 57 66

0.08 0 0.01 0.01 0.02 0.02 0.01 0.03 0.04 0.07 0.08

made at the same time. The average value for wild-type (four different preparations) was 107 nmol of ATP/min/me of membrane Drotein. This Values shown are means of at least two determinations. For ATP synthesis, mutant membranes are compared with wild-type preparations

activity was reduced to zero by uncoupler carbonyl cyanide m-chlorophenylhydrazone. -

brane proton gradient was generated. Therefore, in these E. coli membrane vesicles, aurovertin fluorescence is not a useful probe of F1-Fo energy coupling. Future experiments will be aimed at use of FIFo reconstituted into proteoliposomes to in- crease the effective concentration of aurovertin-binding sites in the membranes and hence the fluorescence response.

ADP-fluoroaluminate is a strong inhibitor of soluble and membrane-bound mitochondrial Fi-ATPase, and the inhibition of mitochondrial membrane ATPase is reversed substantially (by 60%) by succinate oxidation, in an uncoupler-sensitive fash- ion (Lunardi et al., 1988). These workers also showed that soluble E. coli F1-ATPase activity is also strongly inhibited by ADP-fluoroaluminate. It is very likely that the inhibitor is act- ing as a substrate or transition state analog (Issartel et al., 1991). The reversal of the inhibition of mitochondrial mem- brane ATPase by succinate appears to be caused by proton gradient-induced lowering of catalytic site affinity for the li- gand and could therefore provide a tangible measure of F1-Fo energy coupling.

Here we first demonstrated that ADP-fluoroaluminate is a strong inhibitor of wild-type E. coli membrane-bound F1- ATPase and that the presence of phosphate ions gives substan- tial decrease in inhibition (Table 111). When inhibited mem- branes were passed through a centrifuge column (Penefsky, 1977), then incubated in buffer in the presence or 4 mM EDTA or 0.5 mM deferoxamine to chelate AI3+ ions, no reversal of inhibition was seen even after 4 h. These experiments estab- lished that ADP-fluoroaluminate is a potent, tight-binding, in- hibitor of E. coli FIFO. The membrane ATPase activity of the 6G150A and 6G150N mutants was shown to be inhibited by ADP-fluoroaluminate to about the same final extent as in wild- type (Table 111).

Wild-type membranes that had been inhibited to a varying extent by ADP-fluomaluminate were passed through centrifuge columns then incubated for 15 min with succinate (20 mM) or succinate plus phosphate (0.5 n"), in the presence or absence of deferoxamine, and the ATPase activity was estimated. Only minor (5-15%) and inconsistent reversal of the inhibition of ATPase was seen. It was confirmed, using the acridine orange fluorescence quench test, that a transmembrane proton gradi- ent was sustained for the whole 15 min of incubation by the added succinate. In further experiments, succinate was in- cluded during the preincubation with ADP-fluoroaluminate to ascertain if there was any protection from inhibition, but the time course of inhibition was unchanged. NADH (4 mM) gave similar results to succinate. We also prepared membrane vesicles from E. coli cell suspensions following the procedures of Hertzberg and Hinkle (1974) and Reenstra et a l . (1980). These procedures utilize a relatively low pressure during

TABLE 111 Inhibition of membrane-bound E. coli F,-ATPase by

ADP-fluoroalumimte Membranes (1 mg/ml in HMK buffer) were incubated at 30 "C for 2 h

with ADP (25 p), AlC13 (0-100 pd, and NaF (5 m), then passed through centrifuge columns in HMK buffer and assayed for ATPase activity. Pi release was estimated using the Malachite Green method (Van Veldhoven and Mannaerts, 1978). All results are means of quad- ruplicate assays.

Genotype [AICbI ATPase activity Inhibition of

w 46 unc+n 25 47

50 72 75 79

100 91 100 21b 100 95 100 94

8G150N sG150A

The wild-type strain was pAH3/AH3. The mutants were the corre-

Potassium phosphate (0.5 m) included in incubation. sponding strains.

French pressure cell disruption and provide relatively well- coupled preparations of membrane vesicles. We obtained mem- brane preparations that showed essentially the same results as noted above, i.e. there was strong inhibition of ATPase activity by ADP-fluoroaluminate, lack of consistent or substantial re- versal of inhibition by succinate, and lack of protection against inhibition by succinate.

There could be two possible explanations for the lack of pro- ton gradient-induced reversal of ADP-fluoroaluminate inhibi- tion. First, it may be that in FIFo from E. coli, in contrast to mitochondrial FIFo, the binding of the ADP-fluoroaluminate inhibitor is so tight that the energy-dependent change in cata- lytic site affinity may not be sufficiently large to release it. Second, a significant proportion of the membrane vesicles may be not sufficiently proton-impermeable. Future experiments utilizing FIFO reconstituted in proteoliposomes could help to answer these questions. For the purposes of this study, how- ever, the lack of a sizeable and consistent reversal of inhibition by NADH or succinate in wild-type membranes prevented us from applying this assay to mutants.

DISCUSSION

In the accompanying paper (Hazard and Senior, 19941, we described the isolation of 38 different mutant strains, carrying mutations in the conserved C-terminal region of the &subunit of E. coli FIFo-ATP synthase. The growth characteristics of the strains showed that they were impaired to varying degree in oxidative phosphorylation. Here we characterized a represent- ative group of 13 of those strains biochemically, to ascertain the

&Subunit Mutants of FIFO Which Cause Uncoupling 431 TABLE IV

Categories of mutants based on energy coupling characteristics

Category Mutants in category

I. Mutants which do not assemble FIFO correctly.

11. Mutants which can- not pump protons.

111. Mutants which have low level proton pumping and ATPase activities.

IV. Mutants which have interme-

ATPase activities. diate proton pumping and

V. Mutants similar to wild-type.

6G150T 6G150P 6G150D

SGl5ON

6A149P SG150A SG150S SG150CN164L SL99P/A149D

SL99P SA149T

SA149S SA149D

Characteristics

No ATPase or ATP-driven proton pumping activity; proton pumping cannot be reconstituted with wild-type F1. Very little subunit b present in membranes.

No ATP-driven proton pumping activity, proton-pumping can be fully reconstituted with wild-type F1. Law ATPase activity. Normal amount of subunit b in membranes. ATPase activity is very DCCD-insen- sitive. No ATP synthesis activity in vitro.

Law level ATP-driven proton pumping and ATPase activities. Proton pumping can be fully reconstituted with wild-type F1.

Normal amount of subunit b in membranes. ATPase activity is partially DCCD-insensitive. Very low

ATP synthesis activity.

Intermediate ATP-driven proton pumping and ATPase activity. Proton pumping can be fully reconstituted with wild-type F1. Normal amount of subunit b in membranes. ATPase activity is somewhat DCCD- insensitive.

All characteristics similar to wild-type.

Energy coupling properties

Defective enzyme assembly

Fully uncoupled

Partially uncoupled

Somewhat uncoupled

Almost fully coupled

reasons for impaired oxidative phosphorylation. In order to facilitate description of the 13 strains, we have divided them into five categories, summarized in Table IV.

Mutants in category I caused defective assembly of FIFo-ATP synthase, as shown by immunoblotting experiments and KSCN stripping/F1 reconstitution experiments (Fig. 1, Table I). Thus, it was not unexpected that these mutants (sG150T, SG150D, and 6G150P) showed very low membrane FIFO activities such as ATPase or ATP synthesis (Tables I and 11).

All of the other 10 mutants had apparently normal amounts of FIFo in the membranes (Fig. 1, Table I), and in each case the membrane ATPase activity was lower than in the wild-type strain (see Table I). In addition, it was evident that energy coupling between F1 and Fo was impaired in these mutants. The single mutant in category I1 (sG150N) is extremely inter- esting in that the membrane ATPase activity appeared to be fully uncoupled. There was zero ATP-driven proton pumping (Table I, column 2), zero ATP synthesis (Table 11, column 31, and the ATPase activity was markedly insensitive to inhibition by DCCD (Table 11, Fig. 2).

The category I11 mutants (6A149P, 6G150A, 6G150S, SG150CN164L, and 6L99P/A149D) are partially uncoupled. They showed low levels of ATP-driven proton pumping, par- tially DCCD-insensitive ATPase activity, and low ATP synthe- sis activities. Category IV mutants (6L99P and SA149T) are somewhat uncoupled. They have intermediate ATP-driven pro- ton pumping and ATP synthesis activities and somewhat DCCD-insensitive ATPase activity. Mutants in category V (6A149S and SA149D) are almost fully coupled. They have near-normal DCCD sensitivity of ATPase activity, high ATP- driven proton pumping, and relatively high ATP synthesis ac- tivities. As was pointed out under “Results,” the ratio of ATP synthesis/ATPase in the membranes appears to give a qualita- tive measure of the degree of uncoupling in these mutants.

In general, the biochemical characteristics of the membrane preparations paralleled the in vivo properties of the mutants as reported in the previous paper (Hazard and Senior, 1994), al- though in vitro the energy coupling properties of the mutants appeared consistently to be more impaired than might have been expected from the growth characteristics. For example, the mutant 6G150N was able to grow to some low extent by

oxidative phosphorylation on both solid and liquid succinate media in vivo, but had zero ATP synthesis activity in vitro. It seems likely that the cell disruption and washing procedures necessary for preparation of membrane vesicles could have caused ATP synthesis activities to decrease. The relative level of impairment of energy coupling in vitro did, however, corre- late well with the relative impairment of growth by oxidative phosphorylation in vivo and so the categories of mutants in Table IV are valid groupings.

The data presented here strongly support the idea, discussed in the previous paper, that the C-terminal region of the &sub- unit is important for FIFo function and demonstrate that a major role is to support correct energy coupling between F1 and Fo. This is the first report of mutations in the &subunit being the cause of uncoupling, but previously such an effect has been reported for mutations occurring in the cytoplasmically sided “polar loop” region of subunit c (Mosher et al., 1985; Fraga and Fillingame, 1989; Miller et al., 1989) and in y-subunit (Shin et al., 1992). This indicates that the polar loop of subunit c, the &subunit, and the y-subunit all form part of the conformational signal transmission path between the Fo proton conduction machinery and the F1 catalytic sites. The stalk is expected to feature centrally as “connectodtransmittal device” in this path (Senior, 1990).

The catalytic sites located in the three P subunits of F1 have different affinities for nucleotide; for example in E. coli the first site has a high nucleotide affinity (&(ATp) -0.1 nM), the second site has an intermediate nucleotide affinity (&(ATP) -1 w), and the third site has a lower nucleotide affinity (&(ATP) -60 w) (Senior et aE., 1992; Weber et al., 1993). Reactivity with chemical modification reagents or affinity labels also suggests that the three p subunits exist in different conformations (this extensive body of work is reviewed by Boyer, 1993). There is evidence from labeling studies with lucifer yellow (Nalin et al., 1985) and with N-ethylmaleimide (Lee et al. 1992; Turina et al. 1993) that the three a-subunits also exist in different confor- mations.

The three a- and three P-subunits of F1 alternate to form a hexagon (Boekema et al., 1988; Gogol et al., 1989; Bianchet et al., 1991; Abrahams et al., 1993). The y- and +subunits have been shown to reside in the central region of the hexagon by

432 6-Subunit Mutants of FIFO Which Cause Uncoupling

electron microscopy and appear to be asymmetrically located with respect to the P-subunits (Gogol et al., 1990; Wilkens and Capaldi, 1992; Boekema et al., 1990; Boekema and Bottcher, 1992). The position of the central mass with respect to a specific P-subunit was shown to change under conditions of enzyme turnover (Gogol et al., 1990). Cross-linking studies showed that enzyme turnover or nucleotide binding changes the spatial re- lationship between either y (Aggeler and Capaldi, 1992) or E

(Mendel-Hartvig and Capaldi, 1991b) and the three p subunits; and fluorescence energy transfer measurements demonstrated that substrate or substrate-analog binding induced changes in the pattern of structural asymmetry in catalytic sites (Shapiro and McCarty, 1988, 1990).

The binding change mechanism (Cross, 1981; Boyer, 1993) predicts that, during ATP synthesis, the nucleotide binding affinity of the three catalytic sites changes in a cyclical fashion. The key feature of the mechanism for ATP synthesis is that ATP formed at the high affinity catalytic site is released into the medium by a binding affinity change, as the catalytic sites cycle through three different conformations. We suggest that sub- units b and 6, which are together predicted to form the stalk composed of a six-helix bundle, extend into the central region of the a3p3 hexagon, as discussed in the previous paper. Proton gradient-induced conformational changes in the stalk could cause a shift in the position of the &subunit which would be relayed to the y- and €-subunits, consequently changing the positions of the latter relative to the a& subunits (Cox et al., 1984). This might then alter the affinity of each of the three catalytic sites for nucleotide so that substrate binding and product release steps can occur. The C-terminal region of the &subunit would be intimately involved in this process, as in- dicated by the mutagenic analysis reported in this work, with its most likely role being to act at the interface between the helical part of the stalk and the F1 subunits in the central region of the asp3 hexagon.

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