Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis*

(Received for publication, October 4, 1996, and in revised form, November 26, 1996)

Dongli Hu Dagger , Heike R. Fiedler §, Talila Golan , Marvin Edelman , Heinrich Strotmann §, Noun Shavit Dagger and Stefan Leu Dagger par

From the Dagger  Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel, § Institut für Biochemie der Pflanzen, Heinrich Heine Universität Düsseldorf, Düsseldorf, Federal Republic of Germany, and  Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The participation of the amino acid beta 83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid beta 83 was changed from Glu to Asp (beta E83D) and to Lys (beta E83K), and the highly conserved tetrapeptide beta T82-E83-G84-L85 (Delta TEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15Delta atpB and FUD50 with the mutated atpB genes. The transformants containing the beta E83D and beta E83K mutant genes grew well photoautotrophically. The Delta TEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped Delta pH with thylakoids isolated from cw15 and the two mutants, beta E83D and beta E83K, were similar. However, only the phosphorylation activity of the mutant beta E83D was inhibited by tentoxin with 50% inhibition attained at 4 µM. These results confirm that amino acid beta 83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, beta E83D, and beta E83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the beta E83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the beta E83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the beta E83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF1-ATPase.


INTRODUCTION

The eukaryotic unicellular green algae Chlamydomonas reinhardtii constitutes a powerful experimental model system for the study of the photosynthetic machinery. It is accessible to genetic analysis and grows photoautotrophically on minimal medium or heterotrophically with acetate as the sole carbon source. These properties have been used to isolate numerous photosynthetic mutants which have helped to examine the function of the photosynthetic apparatus (1).

The chloroplast of C. reinhardtii contains approximately 80 copies of its 196-kb1 circular genome (2). Due to recent progress in the molecular genetics of C. reinhardtii, chloroplast proteins can be altered by site-directed mutagenesis of the corresponding genes followed by transformation into the chloroplast (3-7). Chloroplast transformation was first demonstrated in 1988 by Boynton and co-workers (3), by complementation of an atpB deletion mutant with the cloned wild type gene. The transforming DNA integrates into the recipient chloroplast DNA by homologous recombination. Goldschmidt-Clermont (5) has constructed a chimeric selectable marker using the chloroplast atpA promoter and the bacterial gene aminoglycoside adeninetransferase (aadA), which confers resistance to spectinomycin and streptomycin in Escherichia coli.

Tentoxin, a cyclic tetrapeptide (cyclo-L-leucyl-N-methyl-(z)-dehydrophenylalanyl-glycyl-N-methyl-alanyl) produced by the fungus Alternaria tenuis, is a potent inhibitor of the chloroplast ATP synthase of certain sensitive plant species (8, 9), which acts as an uncompetitive inhibitor of the CF1-ATPase with respect to nucleotide substrates. It was proposed that 1 mol of tentoxin/mol of CF1, bound at a site near the interface between the N-terminal domains of subunits alpha  and beta , interferes with the cooperative interaction between nucleotide binding sites inhibiting the enzyme activity (10). Binding of tentoxin to a second, low affinity site stimulates the ATPase activity of CF1 (11-13). Avni et al. (7) have shown that amino acid beta 83 is involved in conferring tentoxin sensitivity. Nicotiana species having a glutamic acid residue at position 83 are resistant, whereas species with an aspartic acid residue in this position are sensitive to tentoxin. The peptide sequence around position 83 in C. reinhardtii beta  subunit was replaced by the corresponding sequence from a tentoxin-sensitive tobacco beta subunit. This exchange of five amino acids including Glu83 to Asp83 converted the normally tentoxin-resistant to a tentoxin-sensitive C. reinhardtii ATP synthase.

We investigated in more detail the role of the acidic amino acid residue at position 83 of the beta  subunit of the ATP synthase by site-directed mutagenesis. The atpB deletion mutant cw15Delta atpB was prepared from the cell wall deficient strain cw15 by replacing the atpB open reading frame by the aadA cassette (14). The use of the cell wall deficient strain cw15 allowed us to isolate well preserved thylakoids and to characterize electron transport and photophosphorylation activities of these membranes (15, 16).


EXPERIMENTAL PROCEDURES

C. reinhardtii cw15 was cultivated in high salt medium (1). The cell wall deficient atpB deletion mutant cw15Delta atpB (14) was cultivated in high salt medium containing 0.4% acetate and 150 µg/ml spectinomycin (high salt medium containing acetate/spectinomycin) at low light intensity (~50 lux). The atpB deletion mutant FUD50 (17) was cultivated in Sager-Granick medium (1) containing 0.2% acetate.

All cloning procedures were done using commercially available restriction endonucleases and DNA-modifying enzymes according to the suppliers instructions. DNA fragments were isolated from agarose gels using the Glass Select DNA Isolation Kit (5 Prime right-arrow 3 Prime, Boulder, CO) or by freezing the sliced agarose fragments in the presence of 80% phenol, followed by chloroform extraction, and ethanol precipitation of the DNA from the supernatant. Transformation into E. coli JM109 or DH5alpha was done according to the instructions supplied in the pAlter site-directed mutagenesis kit (Promega Biotech). Plasmid DNA was isolated by alkaline miniprep as described in the T7 DNA sequencing protocol (Promega Biotech) or by the Qiagen plasmid kit (Qiagen GmbH, Hilden, Germany). DNA sequencing was done using Sequenase version 2' (USB) and denatured plasmid DNA as template. Site-directed mutagenesis was done using the pAlter site-directed mutagenesis kit (Promega Biotech) according to the suppliers instructions, except that T7 DNA polymerase was used for second strand synthesis instead of T4 DNA polymerase. The mutagenesis procedure was performed using single-stranded DNA from plasmid pAlter containing a 2.8-kb HindIII-KpnI fragment with the complete atpB gene (18) and the mutagenic primers shown in Fig. 1. Introduction of the mutation into the atpB gene was confirmed by PCR, restriction analysis, and DNA sequencing using atpB-specific primers. The 0.9-kb EcoRV-PstI fragments containing the mutant sites were cloned back into suitable transformation vectors containing extended 5'- and 3'-flanking regions and antibiotica resistance cassettes as markers for successful transformation.


Fig. 1. Mutagenic primers used for site-directed mutagenesis of the atpB gene. The single-stranded DNA obtained from our pAlter construct was oriented as the atpB mRNA. Therefore the primers were designed in the orientation shown, with the 5' end to the right. The bases changed to give a Asp or Lys codon, respectively, are indicated in bold letters, as well as the single base change introduced to yield a new AseI restriction site. The 12-base pair deletion created in the Delta TEGL primer is indicated by a dashed line.
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Chloroplast transformation was done by vortexing in the presence of glass beads (6) or with the PDS1000-He apparatus of Bio-Rad. Cells were spread in 0.2% soft agar onto high salt medium-agar plates and bombarded with DNA-coated M10 tungsten particles. After incubation overnight at low light, cells were transferred to full light intensity (500-1000 lux) in CO2-enriched atmosphere for selection of transformants. To select for spectinomycin or kanamycin resistance, cells were bombarded on acetate containing medium, incubated overnight at low light intensity, and then spread onto acetate containing plates containing 150 µg/ml spectinomycin or kanamycin. C. reinhardtii total DNA was isolated as described (19) except that cells were scraped from agar plates and directly resuspended in lysis buffer instead of grown in liquid medium. Proteins were separated by SDS-polyacrylamide gel electrophoresis (20) and stained with Coomassie R250 or transferred to nitrocellulose and immunostained using a CF1 specific rabbit antiserum and protein A alkaline phosphatase.

C. reinhardtii CF1 was prepared by chloroform extraction and anion exchange chromatography (21) with some modifications. Cells were harvested, washed, and resuspended in lysis buffer (50 mM Tricine/NaOH, pH 8.0, 10 mM NaCl, 10 mM sodium ascorbate, and 250 mM sucrose) to a concentration of 1 mg/ml chlorophyll. The cells were broken by passage through a Yeda press (2 times at 106 N/m2), and the membranes were precipitated and washed 4 times in 2 mM Tricine/NaOH, pH 8.0, 50 mM NaCl. Membranes were resuspended at room temperature to 3 mg/ml chlorophyll in 20 mM Tricine/NaOH, pH 8.0, 1 mM EDTA, 1 mM ATP, 2 mM dithiothreitol, and 10% glycerol. A 1/2 volume of chloroform was added, and the suspension was vigorously vortexed for 20 s. The suspension was centrifuged for 30 min at 12,000 × g at room temperature, and the upper phase was subjected to a second centrifugation under the same conditions. The chloroform extract was mixed with Fractogel TSK DEAE-650 (M) from Merck preequilibrated with loading buffer (20 mM Tricine/NaOH, pH 8.0, 1 mM NaEDTA, 1 mM ATP, 2 mM dithiothreitol containing 10 mM (NH4)2SO4) at a ratio of 1 ml of gel per 2 mg of chlorophyll extracted and incubated for 30 min at room temperature. This mixture was loaded onto a bed of 5 ml of Fractogel TSK DEAE-650 (M) in a chromatography column, rinsed with loading buffer, and eluted with a 10-300 mM (NH4)2SO4 gradient in loading buffer. The purest fractions, corresponding to a protein peak eluting at about 120 mM (NH4)2SO4, were collected, supplemented with 1 mM ATP, precipitated by addition of (NH4)2SO4 to 50% saturation, and stored at 4 °C. For enzymatic assays, the CF1 pellet was collected by 15 min centrifugation at 12,000 × g, dissolved in 20 mM Tricine/NaOH, pH 8.0, to a concentration of 0.5-1 mg/ml. Salts were removed by column centrifugation through Sephadex G-25 equilibrated with 20 mM Tricine/NaOH, pH 8.0.

Mg-ATPase activity of these preparations was measured by photometric determination of released inorganic phosphate (Pi) as described (22). 5 µg of enzyme were incubated for the indicated times in 20 mM Tricine/NaOH, pH 8.0, with 4 mM MgCl2 and 6 mM ATP present, and the additions indicated in the legends of figures and tables. Reaction mixes lacking either enzyme or ATP were preincubated at the indicated temperature, and the reactions were started by adding enzyme or substrate. The reaction was stopped by addition of 3% trichloroacetic acid, and inorganic phosphate released was determined photometrically.

Active thylakoid membranes were prepared as described (15, 16). Cells were grown at 20 °C in high salt medium containing 0.2% acetate at a light intensity of 900 lux in a 14-h light to 10-h dark cycle, in 400-ml cultures bubbled with sterile air. The cultures were grown to a cell density of 6 × 106 cells/ml and then harvested by centrifugation (10 min, 1000 × g), washed with medium A (0.3 mM sucrose, 10 mM Tricine, pH 8.0, 50 mM NaCl, and 5 mM MgCl2), and resuspended in the same medium at 4 °C. All further steps were carried out at this temperature. The cells were passed twice through a Yeda Press at 5 × 105 N/m2 and 5.5 × 105 N/m2, respectively, and harvested by a 1-min centrifugation step at 3000 × g. The resulting pellet was resuspended and washed twice in 2 mM Tricine, pH 8.0, 50 mM NaCl, 1 mM MgCl2 and resuspended in a small volume of solution A.

ATP formation was measured at clamped proton gradient by employing the Delta pH clamp device (23). The proton gradient was controlled by monitoring the fluorescence quench of 9-aminoacridine and adjusting the light intensity correspondingly. The reaction medium contained in a final volume of 2.5 ml: 25 mM Tricine, pH 8.0, 50 mM KCl, 5 mM MgCl2, 50 µM phenazine methosulfate, 10 mM dithiothreitol, 50 nM valinomycin, 10 mM glucose, 12 units/ml hexokinase, 5 µM 9-aminoacridine, and thylakoid vesicles to give a final concentration of 25 µg Chl/ml. After preillumination at 20 °C for 2 min, the Delta pH was adjusted to 60% fluorescence quench and after 1 min 1 mM ADP and 5 mM [32P]Pi were added. Rates of photophosphorylation were calculated from the organic 32P-labeled phosphate determined in the samples taken after 10, 20, 30, and 40 s according to the method of Sugino and Miyoshi (24).


RESULTS

The oligonucleotide primers presented in Fig. 1 were used to introduce the desired changes (bold letters) into the atpB gene by site-directed mutagenesis as follows: (a) change of codon 83 from glutamic acid to aspartic acid (beta E83D), (b) change of codon 83 from glutamic acid to lysine (beta E83K), and (c) deletion of the tetrapeptide Thr82-Glu83-Gly84-Leu85 (Delta TEGL). In the beta E83D and beta E83K oligonucleotides a second, silent point mutation, creating a new AseI restriction site (underlined), was introduced to facilitate transformant analysis. The mutant atpB genes were cloned into the transformation vectors pT7atpB5'L3'LaadA/Kan (Fig. 2A) or pBSatpB'aadA/Kan (Fig. 2B). The extended 5'- and 3'-untranslated regions of these plasmids significantly increased transformation efficiency. The kanamycin or aadA cassettes were added to select for successful transformation with mutant atpB genes that do not restore photoautotrophic growth. The constructs with the atpB genes containing the beta E83D, beta E83K, and the Delta TEGL mutations shown in Table I were used to transform the cell wall deficient deletion mutant cw15Delta atpB (14) and the atpB deletion mutant FUD50 (17). Selection for successful transformation was done under autotrophic growth conditions on high salt medium or Sager-Granick minimal medium plates or for spectinomycin or kanamycin resistance on acetate-containing media containing 150 µg/ml of the corresponding antibiotica. The beta E83D and beta E83K mutant plasmids yielded photoautotrophic transformants, but the spectinomycin- or kanamycin-resistant transformants obtained by transformation with the Delta TEGL deletion mutant plasmid did not grow photoautotrophically (Table I). The DNA fragments containing the mutant sites were isolated from total DNA by PCR, and in the case of the beta E83D and beta E83K transformants the mutations were confirmed by restriction analysis (Fig. 3). Subsequently, in all three types of transformants, the mutations were verified by sequence analysis of the corresponding PCR products. The presence of the CF0CF1 complex was examined by SDS-polyacrylamide gel electrophoresis of the thylakoid proteins followed by immunostaining of Western blots with a CF1-specific antiserum. The thylakoids from the spectinomycin- and kanamycin-resistant transformants containing the Delta TEGL mutation were completely devoid of the ATP synthase complex (not shown). Thus, this highly conserved peptide segment seems to be important for the assembly or stability of the ATP synthase complex.


Fig. 2. Constructs used for transforming the mutant atpB genes into the atpB deletion mutants. A, the pT7atpB5'L3'LaadA/kan was constructed by supplementing plasmid pT7atpB (18) with a 1.9-kb HindIII fragment to extend the 5'-untranslated region (5'UTR) and with the aadA or kanamycin resistance cassettes (aadA/Kan) joined to the 3.5-kb PstI-BamHI fragment containing the end of the atpB gene and its 3'-untranslated region (36). This construct contains a repeated sequence of 1.3 kb, which reduced the yield of transformants containing the correct inserts. B, pBSatpB'aadA/Kan was produced by a PCR-based cloning strategy. It contains a 1.8-kb 5'-untranslated region and a 2-kb 3'-untranslated region and several unique restriction sites. The aadA- or kanamycin resistance cassettes (aadA/Kan) were inserted into the engineered SmaI restriction site.
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Table I.

Transformants produced with cw15Delta atpB (A) or FUD50 (B) and the indicated constructs

Detailed construction of the transformation vectors and the kanamycin cassette will be described elsewhere. Strains cw-D221 and cw-K221 were used for determination of photophosphorylation activity and for routine preparations of CF1. CF1 preparations from strains D101 and K101 displayed the same catalytic properties as those prepared from strains cw-D221 and cw-K221.
Mutation Transformant strain Construct used for transformation Selection criteria

A, transformants produced in cw15Delta atpB
 beta E83D cw-D221 pBSatpB'-beta E83D Autotrophic growth
 beta E83K cw-K221 pT7atpB(beta E83K)-5'3'L-Kana Autotrophic growth
cw-K222kana (kanr) pT7atpB(beta E83K)-5'3'L-Kana Autotrophic growth + kanamycin resistance
 Delta TEGL cw-Delta 227kana (kanr) pT7atpB(Delta TEGL)-5'L3'L-Kana Kanamycin resistance
B, strains produced in FUD50
 beta E83D D101 pT7atpB(beta E83D)-5'L3'L-aadA Autotrophic growth
D106 (Specr) pT7atpB(beta E83D)-5'L3'L-aadA Spectinomycin resistance
 beta E83K K101 pT7atpB(beta E83K)-5'L3'L-aadA Autotrophic growth
 Delta TEGL  Delta 3 (Specr) pT7atpB(Delta TEGL)-5'L3'L-aadA Spectinomycin resistance


Fig. 3. Identification of the beta E83D and beta E83K transformants by PCR and restriction enzyme digestion. DNA was extracted from two transformants each and from the wild type and subjected to PCR amplification using the primers 5'-TTAGATATCGTATTTGC-3' (834-850 of the atpB sequence (36)) and 5'-TACAAGAGCTCCTTTTG-3' (1465-1448). The resulting 630-base pair fragments were gel-purified and digested with AseI. Uncut and cut PCR products were analyzed by agarose gel electrophoresis. A, lane 1, lambda -DNA digested with HindIII and EcoRI. Lanes 2 and 3, PCR products from two beta E83K transformants; lanes 4 and 5, PCR products from two beta E83D transformants; lane 6, no DNA, control of the PCR reaction; lane 7, PCR product from cw15 DNA. B, lane 1, lambda -DNA digested with HindIII and EcoRI. Lanes 8 and 9, PCR products from two beta E83K transformants, digested with AseI; lanes 10-12, PCR products from three beta E83D transformants, digested with AseI; lane 13, PCR product from cw15 DNA, digested with AseI.
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Thylakoids prepared from cw15 and the transformants cw15D221 and cw15K221 containing the mutations beta E83D and beta E83K, respectively, were assayed for their photophosphorylation capacity. All three thylakoid preparations yielded similar rates at a clamped Delta pH (60% fluorescence quench). Photophosphorylation activity of cw15 and beta E83K was not inhibited by tentoxin, whereas that of beta E83D was sensitive to tentoxin with 50% inhibition attained at 4 µM (Fig. 4A). The degree of inhibition was enhanced by preincubation of thylakoids with tentoxin and maximal inhibition was attained after 1 h with 5 µM tentoxin (Fig. 4C). Fig. 4B shows the response to tentoxin of the ethanol-enhanced Mg-ATPase activity of CF1 isolated from the wild type and the two transformants. The ATPase activity of cw15 and beta E83K CF1 was not inhibited by tentoxin, whereas that of beta E83D was inhibited with 50% inhibition attained at a concentration of 0.5 µM tentoxin. Preincubation with tentoxin was also required for maximal inhibition (Fig. 4 C). Hence, the isolated CF1-ATPase is about 10-fold more sensitive to tentoxin than photophosphorylation.


Fig. 4. A, inhibition of photophosphorylation activity of thylakoids from the different strains by tentoxin. Thylakoids were incubated in a reaction mix without ADP with the indicated concentrations of tentoxin for 45 min at room temperature. Photophosphorylation activity was measured as described under "Experimental Procedures," at clamped Delta pH (60% fluorescence quench). 100% control activities for cw15 (black-diamond ) and beta E83K (black-square) thylakoids were 62 µmol of ATP formed per mg Chl/h and 82 µmol of ATP formed per mg Chl/h for the beta E83D (bullet ) mutant. B, inhibition of the ethanol-stimulated Mg-ATPase activity by tentoxin of cw15 (black-diamond ), beta E83K (black-square), and beta E83D (bullet ) CF1s. 5 µg of enzyme were preincubated in a reaction mix without ATP and ethanol for 45 min at room temperature with the indicated concentrations of tentoxin. ATPase activity was assayed after addition of 20% ethanol and ATP as described under "Experimental Procedures." 100% of control activities of CF1s were as follows: cw15, 32; beta E83K, 36 and beta E83D, 48 µmol of ATP hydrolyzed per mg protein/min. C, preincubation with tentoxin required for inhibition of photophosphorylation with beta E83D thylakoids (black-square) and of the ethanol-enhanced Mg-ATPase activity of isolated beta E83D-CF1 (bullet ). Thylakoids were preincubated in a reaction mix without ADP for the indicated times with 5 µM tentoxin. CF1 was preincubated in a reaction mix, without ATP and ethanol, with 2 µM tentoxin. 100% of control activities were 170 (without preincubation) and 80 (after 60-min preincubation) µmol of ATP formed per mg Chl/h and 51 µmol of ATP hydrolyzed per mg of protein/min.
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We examined whether the mutations in codon 83 had additional effects on the catalytic properties of the mutant ATPases. The general characteristics of all three CF1 preparations were similar to those reported before for C. reinhardtii (21) although the rates with the latent enzyme were lower. As shown in Table II, the low basal rates of the Mg-ATPase activities of all three CF1 preparations were enhanced by elevating the assay temperature, by octylglucoside, by ethanol, and by methanol. In the presence of 20% ethanol the apparent Km MgATP determined for all three enzymes was 1 mM. Full stimulation of the Mg-ATPase activity by octylglucoside was attained at a concentration of about 1% (Fig. 5A). While the basal, heat- and detergent-stimulated ATPase activities were similar for the wild type and mutant enzymes, the ATPase activity of the beta E83K CF1 elicited by alcohols was significantly lower (Fig. 5, B and C). Maximal stimulation of the Mg-ATPase activities of the cw15 and beta E83D enzymes was obtained at an ethanol concentration of 23% (v/v) and a methanol concentration of 30% (v/v), whereas the maximal ATPase activity of the beta E83K enzyme was attained at 20% ethanol and 25% methanol (Fig. 5, B and C). The degree of stimulation of the beta E83K enzyme by alcohols was less than half of that with the cw15 and the beta E83D enzymes (Fig. 5, B and C).

Table II.

Mg-ATPase activity of CF1s from cw15 and beta E83D and beta E83K transformants

Activities were determined after incubation of reaction mixes containing the indicated concentrations of effectors for 2 min at 37 °C as described under "Experimental Procedures." Specific activity is given in µmol of ATP hydrolyzed per mg of protein/min.
CF1 No addition
37 °C
37 °C 65 °C 20% ethanol 25% methanol 1.2% octylglucoside

cw15 1a   21 36 52 46
 beta E83D 1.3a 21 39 63 47
 beta E83K 1.1a 22 32 47 52

a  Assays without effector at 37 °C were run for 10 min.


Fig. 5. Enhancement of the Mg-ATPase activity of cw15 (black-diamond ), beta E83D (bullet ), and beta E83K (black-square) CF1s by octylglucoside (A), ethanol (B), and methanol (C). Activities were determined after incubation of reaction mixes containing the indicated concentrations of effectors for 2 min at 37 °C as described under "Experimental Procedures." ATPase activities are in µmol of ATP hydrolyzed per mg protein/min.
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The lower activity and the shift in the concentration of alcohols needed for maximal stimulation of ATPase with the beta E83K enzyme could be caused by rapid time-dependent inactivation of this enzyme, in particular at higher organic solvent concentrations. We tested this possibility by measuring the time course of the Mg-ATPase activity of all the three preparations at 17 and 25% ethanol. The specific activities of the beta E83K enzyme at both ethanol concentrations were equal and about 30% lower than its maximal activity at 20% ethanol. At 17% ethanol the ATPase activities of all three enzymes were similar (Fig. 6A), but at 25% ethanol the ATPase activities of the cw15 and beta E83D enzymes were increased significantly whereas that of the beta E83K CF1 remained the same (Fig. 6B). At both ethanol concentrations the rates of all three CF1s were linear for several minutes showing that the beta E83K CF1 is in a less active conformation at 25% ethanol and is not subject to rapid denaturation.


Fig. 6. Time course of the Mg-ATPase activity of cw15 (black-diamond ), beta E83D (bullet ), and beta E83K (black-square) CF1s in the presence of (A) 17% and (B) 25% ethanol. Activities were determined after incubation of reaction mixes containing the indicated concentrations of ethanol for the indicated times at 37 °C as described under "Experimental Procedures."
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DISCUSSION

The sensitivity of certain CF0CF1 ATP synthases to tentoxin has been attributed to the presence of aspartic acid in position 83 of the beta  subunit. This was demonstrated by Avni et al. (7) by analysis of resistant and sensitive tobacco strains and subsequent mutagenesis studies with the C. reinhardtii atpB gene. The C. reinhardtii atpB sequence from codons 74-91 (VRAVSMNPTEGLMRGMEV) was replaced by the corresponding tobacco sequences of the resistant (VRAV<UNL>A</UNL>M<UNL>SA</UNL>TEGL<UNL>T</UNL>RGMEV) or sensitive lines (VRAV<UNL>A</UNL>M<UNL>SA</UNL>T<UNL>D</UNL>GL<UNL>T</UNL>RGMEV) by changing four or five amino acids, respectively (underlined). The resulting mutant enzyme containing Glu in position 83 was resistant to tentoxin whereas that of the mutant with Asp in position 83 was inhibited by tentoxin.

In this work we have exchanged a single codon of the C. reinhardtii atpB gene, Glu83 by Asp, to determine whether in fact this change alone determines tentoxin sensitivity. The Mg-ATPase activity of the isolated CF1 containing the Asp residue in position 83 (beta E83D) was indeed inhibited by tentoxin at concentrations similar to those required to inhibit enzymes from sensitive plants and from the sensitive cyanobacterium Anacystis nidulans (Fig. 4B) (7, 8, 25). Effective inhibition by tentoxin required preincubation of the enzyme with tentoxin as reported previously (Fig. 4C) (8, 25). Photophosphorylation of isolated thylakoids of the beta E83D mutant was also inhibited by tentoxin with 50% inhibition obtained at 4 µM (Fig. 4A). The difference of 1 order of magnitude between the sensitivity to tentoxin of photophosphorylation and CF1-ATPase activity (Fig. 4) might be explained by different accessibility of tentoxin to its binding site. It is known that the conformation of isolated CF1 differs from the conformation of the CF1 sector in the membrane-bound CF0CF1 complex (26, 27). On the other hand, photophosphorylation of the mutant enzyme produced by Avni et al. (7) containing a tobacco/C. reinhardtii hybrid beta  subunit was rather sensitive to tentoxin even after a relatively short preincubation time. The apparent higher affinity of this mutant enzyme to tentoxin could be the result of the additional changes made in the sequence of the tobacco/C. reinhardtii hybrid gene (7).

The fact that the enzymes with the bulkier amino acid side chains (Glu and Lys) in position 83, irrespective of their charge, are resistant to tentoxin whereas the enzyme with Asp in position 83 is sensitive indicates that in the resistant species the access of tentoxin to its binding site is sterically hindered. According to the three-dimensional structure of F1 (28), the amino acid corresponding to position 83 of the beta  subunit of C. reinhardtii is located in the lower region of the crown structure at the interface where the alternating alpha  and beta  subunits are in close contact. Tentoxin contains several hydrophobic residues that might be important for the interaction with a hydrophobic pocket partly screened by amino acid beta 83. The spatial structure of tentoxin is wedge shaped and could fit into the alpha beta interface domain. The more extended Glu and Lys residues in position beta 83 may sterically hinder the access of tentoxin to this binding domain inducing resistance to tentoxin. Tentoxin was shown to inhibit the AMP-PNP-induced release of tightly bound ADP from CF1 (10), suggesting that tentoxin may bind at the interface between an alpha  and beta  subunit and thereby preclude conformational changes required for catalytic turnover. Tentoxin acting as a wedge can prevent the transfer of information between different nucleotide binding sites on the ATP synthase and inhibit the enzymatic activity.

The transformants where the highly conserved tetrapeptide beta T82-E83-G84-L85 was deleted could not grow photoautotrophically probably because the CF0CF1 complex was not assembled. As revealed by the crystal structure of mitochondrial F1 (28), the four amino acids deleted in the beta  subunit form a loop connecting two beta  sheets of the beta  subunit in the stabilizing crown structure, and their deletion would prevent proper folding of the mutant beta  subunit. Changing only one residue, a Glu to a Lys, in the beta E83K mutant yielded an active ATP synthase which was not sensitive to tentoxin and exhibited similar rates of photophosphorylation to those of the wild type and the beta E83D mutant.

The Mg-ATPase activities of the cw15, beta E83D, and beta E83K CF1's were quite similar at elevated assay temperature (Table II) or in the presence of octylglucoside (Fig. 5A), but significant differences were found with the alcohol-enhanced Mg-ATPase activity (Fig. 5, B and C). The higher methanol versus ethanol concentrations required to attain maximal rates of ATP hydrolysis indicate that the dielectric properties of the assay medium plays an important role in the activation process by alcohols. Denaturation by alcohols was ruled out (Fig. 6, A and B) as a possible reason for the lower activity of the beta E83K enzyme as compared with the cw15 and beta E83D enzymes. We propose that the presence of a Lys residue in position 83, which according to the three-dimensional structure of F1 is in close vicinity to residue Argbeta 52, alters the active conformation of the CF1-ATPase induced with alcohols. On the other hand, the octylglucoside-induced activation of the CF1-ATPase does not seem to be affected in the same manner by the introduction of Lys in position 83.

It is very interesting that both the sensitivity of CF1 to tentoxin and the ATPase activity enhanced by alcohols are affected by mutations at codon 83. It is likely that the primary effect of organic solvents is to alter the conformation of the enzyme by lowering the dielectric constant of the medium (29, 30). The release of tightly bound, inhibitory ADP, which is a rate-limiting step in catalysis, is stimulated by alcohols (31) and by octylglucoside (32). In the presence of ethanol the Mg-ATPase activity of CF1 is no longer inhibited by ADP (29). Ethanol also removes the epsilon  subunit from the CF1 complex and stimulates the ATPase in this way (33). It will be interesting to determine which of these effects is affected by the mutations in codon 83. Our results demonstrate that the stimulatory effects of octylglucoside and ethanol are based on different mechanisms. The ATPase activity of the beta E83K enzyme is stimulated to the same extent as that of the cw15 and beta E83D enzymes by octylglucoside but affected differently by methanol and ethanol. Tentoxin inhibits multisite catalysis by binding at one of three alpha /beta interfaces of the enzyme (probably near amino acid beta 83), interferes with the interactions between catalytic sites, and inhibits the release of ADP from the catalytic site (10). The results presented here demonstrate that the domain around amino acid beta 83 in the crown domain participates in activation and inhibition of the catalytic activity of CF1, probably via long range conformational effects that affect the affinity of the tight sites for ADP. Other mutants in the crown domain (alpha E38K (34) and beta C63W (35)) were shown to affect the coupling efficiency of ATP synthase, probably by interfering with conformational signaling. These results imply that the crown domain is involved in determining the conformational states of the catalytic sites by transmitting conformational signals essential for catalysis.


FOOTNOTES

*   This work was supported by Grants from the German-Israeli Foundation GIF-No. I 0251-129.03/92 (to N. S. and H. S.), Deutsche Forschungsgemeinschaft SFB 189, and Fonds der Chemischen Industrie (to H. S.), by the Doris and Bertie Black Center for Bioenergetics in Life Sciences (to N. S. and S. L.), and by the Wilstatter and Forchheimer Centers (to M. E.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: The Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel. Tel.: 972 7 6461 325; Fax: 972 7 6472 890.
1    The abbreviations used are: kb, kilobase; PCR, polymerase chain reaction; Chl, chlorophyll; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; AMP-PNP, adenosine 5'-(beta ,gamma -imino)triphosphate.

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