©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of the Chlamydomonas reinhardtii Coupling Factor 1 -Subunit Cysteine Bridge in the Regulation of ATP Synthase(*)

Stuart A. Ross (§) , Michael X. Zhang (¶) , Bruce R. Selman

From the (1) Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The -subunit of coupling factor 1 (CF) contains a cysteine bridge that is thought to be involved in the redox control of enzymatic activity. In order to test the regulatory significance of this disulfide bond, genetic transformation experiments with Chlamydomonas reinhardtii were performed. C. reinhardtii strain atpC1 ( nit1-305, cw 15, mt), which is null for the -subunit, was transformed and complemented with -subunit constructs containing amino acid substitutions localized to the cysteine bridge between Cysand Cys. Successful complementation was confirmed by phenotypic selection, Northern blot analysis, reverse transcription polymerase chain reaction, and cDNA sequencing. CFATPase activities of the soluble enzymes were measured in the presence and absence of dithiothreitol (DTT). Mutant CFenzymes showed no effect of DTT although increased activity was observed for the wild-type enzyme. In vitro, phenazine methosulfate-dependent photophosphorylation assays revealed that wild-type CFexhibits a 2-fold stimulation in the presence of 25 mM DTT, whereas each of the mutant enzymes has activities that are DTT-independent. Growth measurements indicated that despite the absence of a regulatory disulfide/dithiol, the mutant strains grew with the same kinetics as wild type. This study provides evidence to illustrate the involvement of the -subunit in the redox regulation of ATP synthesis in vivo. This work is also the first demonstration in C. reinhardtii of stable nuclear transformation using mutated genes to complement a known defect.


INTRODUCTION

The light reactions of photosynthesis generate reducing equivalents in the form of NADPH and energy in the form of ATP. During photosynthetic electron transfer, protons are unidirectionally deposited into the lumen of the thylakoid membrane. The chloroplast ATP synthase (CFCF)() couples the chemical potential energy of the protonmotive force to the synthesis of ATP (1) . ATP synthase contains two functionally distinct domains, designated CFand CF. CFis an integral thylakoid membrane protein complex that functions as a proton channel. CFis a peripheral membrane complex that is located on the stromal side of the thylakoid; it contains the catalytic sites for ATP synthesis (2, 3) .

ATP synthases from all species are highly regulated (4) . One possible mode of regulation that appears to be unique for plant enzymes is the redox state of the -subunit (5, 6, 7, 8, 9, 10, 11, 12, 13) . The Chlamydomonas reinhardtii CF-subunit, like other plant -subunits, contains an intrapeptide disulfide bond between positions 198 and 204 of the mature polypeptide. This is the only disulfide bond within the entire multisubunit CFcomplex (5) . Protein sequence comparisons between the known plant CF-subunits and their Fmitochondrial and bacterial counterparts reveal that this disulfide bond is a unique feature of the plant enzymes (14) . Several in vitro chemical modification studies have indicated that the disulfide bond may be important for regulatory control of ATP synthesis. In the presence of reducing agents, such as DTT, CFexhibits elevated ATPase activity (5, 6, 7, 8, 9, 10, 11, 12, 13) .

In contrast to other coupling factor complexes, CFhas not been fully reconstituted from its component subunits (4) . Furthermore, in plants, it is difficult to perform genetic manipulations that affect the photosynthetic apparatus without causing lethal effects on the plants. However, Ort and colleagues (15) have reported experiments where they screened a large population of Arabidopsis thaliana to look for mutant phenotypes affiliated with the redox state of coupling factor. A few candidate strains have been obtained, and the characterization of these mutant strains is currently under way. ()In this study we utilized the green algae C. reinhardtii, which can be grown photoautotrophically or heterotrophically on acetate, thereby rendering the photosynthetic apparatus dispensable. Recently, a strain of C. reinhardtii designated atpC1, which is null for CF-subunit, was isolated and characterized (17) . Here we describe nuclear transformation of atpC1 with mutated CF-subunit genes. We believe that this is the first demonstration of stable nuclear transformation and complementation of a defective nuclear gene in C. reinhardtii using mutant genes. With this model system, we were able to test, by site-directed mutagenesis of the -subunit, its putative role in redox control of coupling factor 1.


MATERIALS AND METHODS

Generation of CF -Subunit Mutations

Fig. 1 shows the cloning strategy utilized. Site-directed mutageneses of the cysteine bridge region were achieved by four subcloning steps. The genomic CF-subunit clone, P, was utilized. This clone, a SalI restriction fragment isolated from a C. reinhardtii EMBL-3 phage library, contains the transcription unit plus 762 bp of upstream promoter sequence of the CF-subunit gene cloned into the SalI site of pBluescript SK(Stratagene, La Jolla, CA) (18) . Three mutations were generated within the 168-bp BglII region located between positions 1811 and 1979 of the -subunit transcription unit. Due to the presence of a BglII site within the promoter region at position 122, P was subcloned by digestion with SacI into two halves, designated plasmids P1 and P2, with the latter containing the BglII region of interest. Using PCR mutagenesis, we generated three different mutations within the BglII cassette, all of which are localized to the cysteine bridge between positions 198 and 204 of the -polypeptide. The PCR products were restricted with BglII and inserted into P2 to yield plasmid P3. Proper sequence and orientation were confirmed by dideoxy sequencing (19) and restriction digestion analyses. The P3 inserts were restricted with SacI and subcloned into P1 to yield P4 plasmids. In total, three mutations were generated at each or both of the cysteine residues at positions 198 and 204 of the CF-subunit polypeptide. They are designated C198S, C204S, and C198S/C204S to indicate that a serine residue has replaced the cysteine at position 198 or 204, or both 198 and 204. In generating site-directed mutants by PCR, the 168-bp region in P, which is flanked by BglII restriction sites, served as the template. The noncoding strand primers were designed to introduce the mutations into the targeted region. They are listed in .


Figure 1: Cloning strategy used to make CF -subunit mutants for cotransformation into atpC1 cells. P, the plasmid containing the wild-type atpC gene, was subcloned by SacI digestion into two segments designated P1 and P2. PCR primers were used to generate each of the three mutants by amplifying a 168-bp fragment that was restricted with BglII and inserted into P2 to yield P3 constructs. P3 constructs were restricted with SacI and inserted into P1, which yielded P4 constructs.



Introduction of Plasmids C198S, C204S, and C198S/C204S into atpC 1 Cells by Cotransformation

Because we could not a priori predict the phenotypes of atpC1 cells complemented with mutant CF-subunit genes, we employed a cotransformation protocol. This approach takes advantage of the fact that strain atpC1 is also null for nitrate reductase. For the first selection, the atpC gene constructs were cotransformed along with plasmid PMN24 (20) , which encodes C. reinhardtii nitrate reductase, into nit1-305, cw-15, mt, atpC1 cells. Cells were grown in liquid Sager-Granick II-NH (SGII-NH) medium (21, 22) under continuous light to midlogarithmic phase (1-2 10cells/ml) and concentrated to 3 10cells/cotransformation experiment by centrifugation at 4,000 g for 5 min. The cells were washed two times and finally suspended in 0.5 ml of SGII-NO. atpC1 cells were cotransformed according to the glass bead protocol described by Kindle (23) . The cells were uniformly plated onto 150 15-mm Petri plates containing SGII-NO, 1% agar (previously washed six times with double distilled water) and cultured under continuous illumination of approximately 120 µmol/ms from cool white fluorescent bulbs. Nitcolonies appeared in about 2 weeks. They were reselected two times in 96-well microtiter plates in liquid SGII-NO medium. Surviving colonies were replica plated two times into liquid SGII-NHacetate minus medium and selected for photoautotrophic growth, again under low light of 120 µmol/ms. In each case about 15% of the nitcolonies were able to grow photoautotrophically, and they were thus labeled as putative cotransformants. Photoautotrophic colonies were grown in SGII-NH medium prior to isolation of DNA, RNA, or protein.

DNA and RNA Isolation, Electrophoresis, and Blotting

Cells were grown in SGII-NH media to midlogarithmic phase and pelleted by centrifugation at 4,000 g for 5 min. DNA isolation, RNA isolation, gel electrophoresis, and Southern and Northern blotting were all performed as described previously (17, 24) . Blots were probed with CF-cDNA (or -subunit cDNA) excised from host plasmid by EcoRI digestion (25) , gel-purified, and randomly primed with [P]dCTP. All blots were exposed to x-ray film with an intensifying screen.

Reverse Transcription PCR

Five µg of total RNA were reverse transcribed with 50 ng of random hexamers by following the manufacturer's protocol (Life Technologies, Inc.). The cDNA products were phenol/chloroform-extracted, precipitated, and resuspended in 80 µl of Tris/EDTA (pH 8.0). Two µl were used for each PCR reaction. PCR reactions were carried out as described previously (17) . The reaction conditions were 1 cycle at 94 °C for 5 min, 35 cycles at 94 °C for 2 min, 57 °C for 1 min, and 72 °C for 2 min.

Preparation of Thylakoid Particles

1 liter of cells were grown to midlogarithmic phase, collected by centrifugation (5 min at 200 g), washed in 50 mM Tricine-NaOH (pH 8.0) buffer, and resuspended in 50 mM Tricine-NaOH (pH 8.0) to an equivalent concentration of 1 mg of chlorophyll/ml. This suspension was sonicated in 3-ml batches at room temperature in a water bath-type sonicator (Laboratory Supplies Co., Inc., Hicksville, NY; power output, 80 kHz, 80 watts) for 20 s. The resulting suspension was centrifuged at 200 g for 1 min in order to pellet unbroken cells. Thylakoid particles were obtained by centrifugation of the supernatant at 30,000 g for 10 min. When used to prepare ATPase, the particles were resuspended (at 0.5 mg of chlorophyll/ml), washed three times in 10 mM NaPP(pH 7.8), and centrifuged at 30,000 g for 10 min. When used for photophosphorylation experiments, the thylakoid particles were resuspended in a buffer that contained 20 mM Tricine-NaOH (pH 8.0), 0.5 mM MgCl, 0.3 M sucrose, and 10 mM NaCl and maintained at 4 °C until they were used.

Growth Curves

About 1 10cells for each strain were placed into 250 ml of SGII-NH acetate minus medium and were cultured in an 8-h light/16-h dark photoperiod for several days in a growth chamber kept at 18 °C. The light intensity during the 8-h light period was 120 µmol/ms. At the indicated times duplicate 1 ml samples of cells were harvested, immobilized by the addition of 50 µl of a solution containing 1 mM iodine dissolved in 95% ethanol, and counted with a hemacytometer.

ATPase Assays

Cells were grown to midlogarithmic phase. Isolation of enriched fractions of CFwas performed as described previously (18, 26) . The ATPase reaction mixture, in a total volume of 0.1 ml, contained the following: 20 mM Tricine-NaOH (pH 8.0), 1 mM EDTA, 5 mM MgCl, 10 mM [-P]ATP (2 10cpm), 50 mM dithiothreitol, and 0.5 µg of protein. Reaction mixtures were incubated for 6 min at 37 °C. The reactions were terminated as described previously (26) , and 1 ml of released [P]phosphate was counted in a Packard 460C liquid scintillation counter.

Phenazine Methosulfate (PMS)-dependent Photophosphorylation

Reaction mixtures for PMS-dependent photophosphorylation experiments contained, in a total volume of 0.1 ml, 50 mM Tricine-NaOH (pH 7.8), 10 mM NaCl, 0.5 mM MgCl, 3 mM ADP, 2 mM [P]phosphate (2 10cpm), and 0.08 mM PMS. Thylakoid particles (3-10 mg of chlorophyll/ml) were added to the reaction mixture, and the reactions were initiated by the addition of DTT to a final concentration of 25 mM or ddHO and illuminated at 20 °C under white light isolated from a 150-watt halogen projection light pipe (Ealing Optics, South Natick, MA; 2.4 10µmol/ms) for 0, 15, 30, 45, 60, and 90 s. Esterified phosphate was extracted as described previously (27) and counted in a Packard model 460 C liquid scintillation counter.


RESULTS

Introduction of Nitrate Reductase and Mutant CF -Subunit Genes into atpC1 Cells and Identification of Cotransformed Strains

Introduction of mutant CF-subunit genes into atpC1, which is null for both -subunit and nitrate reductase, was accomplished by cotransformation using the glass bead protocol (23) . After about 10 days small, green nitcolonies appeared on SGII-NO plates. The nittransformation efficiency was about 25 cells/2 µg of circularized DNA for each experiment. The nitcolonies were reselected two times by transferring them into a 96-well microtiter plate, which contained liquid SGII-NO medium. Surviving colonies were then replica-plated two times and selected for photoautotrophic growth in SGII-NH minus acetate medium. In each case about three to five colonies were positive for photoautotrophic growth. Therefore, about 15-20% of the nittransformants were cotransformants.

DNA was isolated from parental strain nit1-305, atpC1, and each of the photoautotrophic colonies and subjected to Southern blot analysis with PstI digestion. Photoautotrophic colonies were labeled as putative cotransformants if they contained DNA fragments that hybridized to P-labeled CF-cDNA that were in addition to the endogenous atpC PstI gene fragments. All putative cotransformants displayed additional bands, and in no case was there any indication of a reversion event (data not shown).

Northern Blot Analysis of atpC mRNA from Cotransformed Cells

To determine whether the transformed atpC mutant genes were being expressed, total, steady-state RNA was isolated from parental nit1-305, atpC1, and each of the cotransformed strains and subjected to Northern blot analysis. All of the photoautotrophic colonies appear to have accumulated atpC mRNA to a level similar to that of the parental nit1-305 strain (Fig. 2). As previously demonstrated (17) , strain atpC1 does not accumulate a -subunit message. In contrast, all the cell strains accumulated mRNA for the CF-subunit to levels similar to nit1-305 (wild type). Reverse Transcription PCR, Cloning, and Sequencing of nit1-305, atpC1, and Cotransformant cDNAs-In order to determine whether the expressed mRNAs in the putative cotransformants contained the sequences we engineered and introduced into atpC1, we utilized a reverse transcription PCR technique. Samples containing 5 µg of RNA from nit1-305, atpC1, and each of the cotransformants were reverse transcribed in order to generate cDNA. The cDNA from each reaction was then subjected to PCR amplification. In order to ensure that the PCR products arose from cDNA and not from the inherent, albeit nonfunctional, -subunit gene within strain atpC1, we chose to perform PCR using two primers that flank intron 4 of the genomic -subunit gene. The presence of contaminating DNA could then be detected by the presence of a 1015-bp band that would co-migrate with a control PCR product amplified from P, the CF-subunit genomic clone. As a size control we amplified the -subunit cDNA using the same two primers. Fig. 3B shows that the parental nit1-305, C198S, C204S, and C198S/C204S strains each amplify a 308-bp fragment that co-migrates with the positive control PCR product from cloned -cDNA. Since this 308-bp band contains the 168-bp BglII region of interest, each band was gel-purified, digested with BglII, inserted into the BamHI site of pBluescript SK, and screened by blue/white detection. Plasmid DNA from white colonies was isolated and sequenced for each cell line. The RT-PCR reactions yielded the expected 0.3-kilobase product from nit1-305 and each of the cotransformant lines but not from strain atpC1. The lack of an RT-PCR product from atpC1 is consistent with the Northern blot results for this cell strain. The dideoxy cytosine track of each construct is shown in Fig. 3C, and the arrows indicate where each of the base pair changes has been engineered. Thus, the data from RT-PCR clearly demonstrate that each of the engineered CF-subunit constructs is expressed in its respective cell line.


Figure 2: Northern blot analysis of total RNA from nit1-305, atpC1, and putative cotransformants. Samples of (5 µg) RNA were separated in a 1% denaturing agarose gel, blotted onto a nylon membrane, and probed with randomly primed P-labeled CF-subunit cDNA ( lower panel) and -subunit cDNA ( upper panel). Lane 1, nit1-305; lane 2, atpC1; lane 3, C198S; lane 4, C204S; lane 5, C198S/C204S.




Figure 3: Reverse transcription PCR and cDNA sequencing of nit1-305, atpC1, and atpC1 cotransformants. Samples (5 µg) of each RNA were reverse transcribed by following the Life Technologies, Inc. protocol and suspended in 80 µl of Tris/EDTA, pH 8.0, and 2 µl were used for each PCR reaction. A, map depicting the area of the CF-subunit cDNA subjected to PCR. Primers A and B were designed to amplify through intron 4 of the genomic -subunit gene. The black box represents the 168-bp BglII region, which contains the cysteine bridge. B, ethidium bromide-stained 1% agarose gel, which shows RT-PCR results. For nit1-305 and each of the putative cotransformants there is a 308-bp product that comigrates with the cDNA control. atpC1 does not yield a cDNA product. The map shows both of the PCR primers relative to intron 4 of the -subunit genomic clone. kb, kilobase. Lane 1, nit1-305; lane 2, atpC1; lane 3, C198S; lane 4, C204S; lane 5, C198S/C204S; lane 6, -cDNA; lane 7, -genomic clone. C, partial cDNA sequence that shows only the dideoxycytidine track. The arrows indicate the engineered sequences that were introduced into atpC1. Lane 1, nit1-305; lane 2, C198S; lane 3, C204S; lane 4, C198S/C204S.



Photoautotrophic Growth in an 8-h Light/16-h Dark Photoperiod

In order to examine the effects, if any, that the mutations within the CF-subunit disulfide bond might have on photoautotrophic growth, strains nit1-305, atpC1, and each of the cotransformants were subjected to an alternating light/dark growth regime under conditions that demanded photoautotrophic growth (that is without acetate in the growth medium). At specific time points 1 ml of each cell type was counted in duplicate using a hemacytometer. Fig. 4demonstrates that each of the cotransformant strains grows with kinetics similar to the wild-type nit1-305 cells and that each reaches the same cell density. In contrast, strain atpC1 dies within 12 h after plating into medium without acetate, presumably because it fails to accumulate the CF-subunit polypeptide, which apparently prevents the assembly of CF(18) .


Figure 4: Growth curves of nit1-305, atpC1, C198S, C204S, and C198S/C204S grown in an 8-h light/16-h dark period in the absence of acetate. At the indicated times, 1 ml of cells were harvested and counted using a hemacytometer. The data points represent the average of the duplicates where the standard deviation was less than 10% of the average.



Soluble ATPase Assays

Thylakoid-associated proteins were obtained from chloroform-extracted membranes, and this ``enriched'' fraction was utilized for ATPase studies. The catalytic activities of parental nit1-305, atpC1, and mutant enzymes were measured as Mg-dependent ATPases (26) . The enzyme from the parental strain, nit1-305, has manifest activity that is stimulated about 5-fold by the addition of 20% ethanol to the reaction mixture () (18, 26) . demonstrates that each of the mutant CFenzymes behaves in a similar fashion. It should be noted, however, that the ethanol-induced nit1-305 ATPase activity is further increased by 50 mM DTT. In comparison, the ATPase activities of the mutant enzymes are DTT-independent, and furthermore, the rates are similar to the rate of ATP hydrolysis of the wild-type enzyme in the presence of both ethanol and DTT. The presence of a D. salina anti-CFantibody (18, 28) completely inhibits ATPase activity with all of the coupling factors.

PMS-dependent Photophosphorylation

Fig. 5 shows the results of photophosphorylation measurements for parental nit1-305 and each of the three mutant CFenzymes. Panel A demonstrates that the rate of photophosphorylation with nit1-305 is stimulated approximately 2-fold by 25 mM DTT. In contrast, DTT has no effect on the rate of PMS-dependent photophosphorylation with thylakoids isolated from any of the three mutants ( Panels B-D). Moreover, the rate of phosphorylation activity with all of the mutant thylakoids is the same as the rate of phosphorylation with wild-type thylakoids assayed in the presence of DTT. Thus, it appears that the site-directed mutants of CF-subunit have been rendered DTT-independent.


Figure 5: PMS-dependent photophosphorylation of nit1-305 and atpC1 cotransformants. Dark-relaxed thylakoids (3-10 µg of chlorophyll ( chl.)) were assayed in the presence or absence of 25 mM DTT for ATP synthesis from ADP and P-labeled orthophosphoric acid for 0, 15, 30, 45, 60, and 90 s. The data points are the means of the duplicate experiments, each with a standard deviation not greater than 10%.




DISCUSSION

The -subunit of CFis believed to play an important role in the regulation of ATP synthase (4, 29) . Using thiol reducing agents such as DTT, the redox state of the -subunit disulfide bond has been correlated with the activation of soluble ATPase activity (5, 6, 7, 8, 9, 10, 11, 12, 13) . Sequence comparisons between F-subunits from various sources illustrate that the cysteine bridge domain is unique to the enzyme in Chlamydomonas and other plants (12) . Recent developments in C. reinhardtii genetics have made it possible to address the functional significance of the CF-subunit disulfide bond in vivo in this model plant system.

Previous studies have shown that a wild-type CF-subunit gene can complement the defect in C. reinhardtii mutant strain atpC1 (18) . However, in order for strain atpC1 to be useful for these studies, we had to demonstrate that we could successfully perform transformations with mutant CF-subunit genes, which would be inserted into the nuclear genome and be expressed in a stable fashion. In this study we have succeeded in doing this by using a cotransformation protocol that takes advantage of the fact that the atpC1 cell line is also null for nitrate reductase (17, 30) . This procedure was advantageous, because if the mutant -subunit genes failed to complement the defect in atpC1 cells, potential cotransformants could still have been screened by Southern blot analysis. In the three cases studied, we were able to demonstrate that each of the mutant CF-subunit genes was stably expressed and assembled into apparently functional CFcomplexes. Using RT-PCR, we were also able to demonstrate that the expressed -subunit mRNAs each contained mutant sequences that we engineered and cotransformed into atpC1. Furthermore, Southern blot analysis indicated that complementation and not a reversion event restored photoautotrophic growth to atpC1. Indeed, to date we have never observed a reversion event in atpC1 in transformed or untransformed cells.() The results from the reverse transcription PCR studies coupled with the photoautotrophic phenotypes strongly suggest that each strain analyzed expressed mutant CF-subunits.

There are already several examples of stable nuclear transformation in C. reinhardtii (30, 31, 32, 33, 34, 35) . However, we believe that this is the first example of stable nuclear transformation in which gentically engineered genes have been used to complement a known defect. That we were successful demonstrates the utility of atpC1 as a model system to study the CF-subunit. This genetic system allows us to alter almost any amino acid within the -subunit and study its possible effect(s) on CFassembly, function, and regulation. In addition, this work with an enzyme involved in photosynthesis provides a strategy for site-directed mutagenesis studies of other nuclear encoded genes involved in other cellular processes.

A growth curve for wild type, atpC1, and each of the three mutants was performed in order to determine the effects, if any, of each of the -subunit mutations on photoautotrophic growth. McCarty and colleagues (6) have shown that reduced CFcan catalyze ATP synthesis in the light and ATP hydrolysis in the dark. We therefore reasoned that because the mutant enzymes may be locked in a ``reduced'' form that they too might catalyze ATP hydrolysis in the dark. If this were the case, then one might expect that the cells would become sick or even die since ATP synthesis activity would be balanced by ATP hydrolysis activity in a futile cycle. The data from the photoautotrophic light-dark growth curves argue against ATP hydrolysis in the dark because the mutant cells grew with the same kinetics and reached the same cell density as wild type. Therefore, deactivation of CF in vivo probably involves an alternative form of regulation such as the binding of ADP to the -subunit, which has previously been shown to deactivate CFenzymatic activity in the dark (16) .

Examination of enzymatic activities of each of the mutants using a partially purified, enriched cell lysate (18) revealed that in each case the mutant enzymes behaved in similar fashion to parental nit1-305 with respect to manifest activity and stimulation by 20% (v/v) ethanol. However, nit1-305 showed an increase in ATPase activity when 20% ethanol and 50 mM DTT were included in the reaction mixture when compared with the same experiment minus DTT. The soluble CFenzyme from C. reinhardtii does not exhibit the marked DTT-stimulatory effects associated with the spinach enzyme (5, 6, 7, 8, 9, 10, 11, 12, 13, 26) . Nevertheless, we believe the increase in nit1-305 ATPase activity in the presence of DTT to be noteworthy because this effect is not seen in any of the mutants. In comparison, there is no detectable ATPase activity in partially purified atpC1 cell extracts, which is in agreement with previously published results (18) .

We believe that the data from the PMS-dependent photophosphorylation experiments support the model of Nalin and McCarty (5) , which implicates the disulfide bond within the CF-subunit in the redox control of ATP synthesis. In the nit1-305 (wild type) assay the amount of Pesterified is increased about 2-fold by 25 mM DTT. The DTT effect was abolished in each of the three CFmutants assayed. We believe that the effect of DTT is on the CFcomplex because Mills and Mitchell (7) have previously shown that DTT has no effect on photosynthetic electron transfer. The three mutant enzymes in this study involve changes in the cysteine bridge by replacement of each or both of the cysteine residues with serines. In these mutants the CFenzymes have no redox state by way of a disulfide bond, and therefore, if the model is correct, they would not be affected by DTT reduction. Indeed this is the case, since C. reinhardtii strains C198S, C204S, and C198S/C204S all exhibited high rates of PMS-dependent photophosphorylation independent of DTT. Furthermore, the rates of ATP synthesis for the mutant enzymes, regardless of the presence of DTT, were identical to that of wild type in the presence of DTT.

The data from the growth curves may also suggest that the in vivo form of coupling factor in the light is the reduced form of the enzyme. The PMS-dependent photophosphorylation experiments indicate that the mutant enzymes have activities that are virtually identical to that of the reduced form of the wild-type enzyme. In wild type, the reduced form of CFcatalyzes rates of ATP synthesis that are about two times as fast as the oxidized form of the enzyme. Because the wild-type nit1-305 cells grow with the same kinetics as the cells harboring mutations in the -subunit, which effectively ``reduce'' the -subunit disulfide bond, this implies that the in vivo form of the wild-type enzyme is in fact the reduced conformation.

In the work presented in this report it was important for us to first demonstrate that we could obtain viable transformants and functional CFenzymatic activity from our mutant enzymes before proceeding to further biochemical characterization. The molecular data strongly indicate that the restoration of photoautotrophic growth to cotransformed atpC1 cells was accomplished by complementation with mutant -subunit genes. The data from the soluble ATPase assays and PMS-dependent photophosphorylation experiments suggest that the mutant C. reinhardtii strains harbor CFenzymes, which behave differently from wild type in that they are not affected by DTT reduction and have activities identical to wild type in the presence of DTT. Furthermore, growth curves may imply that the in vivo form of the wild-type enzyme is the reduced form but that reoxidation of the CF-subunit disulfide bond may not be required to shut down the enzyme in vivo. This work represents the first complementation of a known nuclear defect with mutated genes. It also provides data that support the redox model for control of ATP synthesis (5) as mediated by the CF-subunit.

  
Table: Noncoding strand PCR primers

The coding strand primer for all reactions was 5`-GGAGGAAGGGGGTGTTGCGGGGAGGTCCTG-3`.


  
Table: C. reinhardtii CF ATPase assay with an enriched protein fraction isolated from parental nit1-305, atpC1, and atpC1 cotransformants

Listed reactions contained 20% (v/v) ethanol, 50 mM DTT, and 330 µg of chicken egg yolk antibodies prepared either prior to (preimmune) or after (immune) immunization of a chicken with purified CFfrom Dunaliella salina. This antibody has been shown to cross-react with C. reinhardtii CF.



FOOTNOTES

*
The work was supported by Grant 144CW52 from the United States Department of Agriculture (to B. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844. Tel.: 603-650-1615; Fax: 603-650-1128.

Recipient of the Sam C. Smith Fellowship.

The abbreviations used are: CF, coupling factor o; CF, coupling factor 1; DTT, dithiothreitol; Tricine, N-tris(hydroxymethyl)methylglycine; PMS, phenazine methosulfate; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; bp, base pair(s); SGII, Sager-Granick II.

D. R. Ort, personal communication.

S. A. Ross, M. Z. Zhang, and B. R. Selman, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Pete Lefebvre for plasmid PMN24, helpful hints about Chlamydomonas transformation, and continued interest and support. We are also grateful to Susanne Selman-Reimer for assistance with the ATPase assays, to Tracy Baas for synthesizing oligonucleotides, to Adam Steinberg and Laura Vanderploeg for artwork, and to Drs. Milo Aukerman and Michael Sussman for helpful suggestions about the manuscript.


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