©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chloroplasts Can Accommodate Inclusion Bodies
EVIDENCE FROM A MUTANT OF CHLAMYDOMONAS REINHARDTII DEFECTIVE IN THE ASSEMBLY OF THE CHLOROPLAST ATP SYNTHASE (*)

Susan L. Ketchner (1)(§), Dominique Drapier (1), Jacqueline Olive (2), Sophie Gaudriault (1), Jacqueline Girard-Bascou (1), Francis-André Wollman (1)(¶)

From the (1)Service de Photosynthèse, URA/CNRS 1187, Institut de Biologie Physico-Chimique, Paris, France and the (2)Laboratoire de Microscopie Electronique, Institut CNRS Jacques Monod, Université Paris VII, Paris France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We identified two neighboring missense mutations in the chloroplast atpA gene which are responsible for the defect of ATP synthase assembly in the FUD16 mutant from Chlamydomonas reinhardtii. The two corresponding amino acid substitutions, Ile Asn and Asn Tyr, occurred at strictly conserved sites among the and subunits of (C)F complexes from bacteria, mitochondria, and chloroplasts. The altered region in the polypeptide chain is located 7 amino acids downstream of the P-loop, which forms most of the conserved nucleotide binding site. Although the resulting chloroplast mutant fails to accumulate most of the ATP synthase subunits, it displays an increased intracellular content in both the and subunits. We demonstrate that the two subunits do not bind to the thylakoid membranes but associate and overaccumulate in the chloroplast stroma as inclusion bodies. Increased rates of synthesis of the two subunits in the mutant point to an early interaction between the two subunits during their biogenesis.


INTRODUCTION

The H-ATP synthase, located on bacterial, mitochondrial, and chloroplast inner membranes, is highly conserved from procaryotes to eucaryotes (Nelson, 1992). It consists of two functionally independent components, the extrinsic soluble portion, (C)F, which is the site of nucleotide binding and ATP catalysis, and the intrinsic membrane sector, (C)F, which is responsible for the proton flux across the membrane. The (C)F sector consists of three copies each of two large subunits, and and single copies of three smaller subunits, , , and . The crystal structure of the F-ATPase from bovine heart mitochondria has been recently solved at atomic resolution (Abrahams et al., 1994). Three heterodimers are arranged around a central -helix formed by the C-terminal domain of the subunit. The (C)F sector forms a pore with 1-2 copies of subunit I, 1 copy of subunits II and IV, and 10-12 copies of subunit III. In the chloroplast, both CF and CF sectors are comprised of nuclear- and chloroplast-encoded subunits. In Chlamydomonas reinhardtii, as in higher plants, the CF subunits , , , and the CF subunits I, III, and IV are chloroplast-encoded while CF subunits and , and CF subunit II are nuclear-encoded (Lemaire and Wollman, 1989a; Herrmann et al., 1993). Therefore the two compartments of the cell must cooperate to regulate the proper assembly of the CF and CF complexes.

Our primary interest has been to elucidate the assembly process of the chloroplast ATP synthase in C. reinhardtii by utilizing the numerous mutants available that are defective in correct ATP synthase assembly. Assembly mutants are useful for the dissection of the steps involved in the proper interaction of the nuclear and cytoplasmic derived components of the complex. The extent to which the assembly process can proceed when one of the ATP synthase subunits is not synthesized or is synthesized in an altered form can provide valuable information concerning the assembly process as well as the cellular functions which are necessary to cope with defective complex formation. In most cases of ATP synthase assembly mutants, improper assembly of the ATP synthase results in an instability of the unassembled or partially assembled complexes and a loss of subunits normally found in ATP synthase (Lemaire and Wollman, 1989b). An exception to this case is the mutant FUD16, defective in ATP synthase, which was previously characterized by Woessner et al.(1984) as bearing a chloroplast mutation, which belongs to complementation group II when compared with a series of ATP synthase mutants of chloroplast origin. In a subsequent biochemical analysis, Lemaire and Wollman (1989b) showed that the FUD16 mutant displayed improper assembly of CF1 on the thylakoid membranes together with increased intracellular levels of the and subunits. The complementation group II was then tentatively ascribed to the atpA locus (Lemaire and Wollman, 1989b).

The FUD16 mutant has a 2-fold interest. It provides a means to further understand a process of protein assembly but it should also help to determine the outcome of overexpressed proteins in organellar compartments. Quite often the overexpression of proteins in procaryotes and eucaryotes results in the formation of substructures, termed inclusion bodies (Mitraki and King, 1989). These structures have been found in the cytosol and are composed of high molecular weight aggregates of the overexpressed protein. Whether this process occurs in organellar compartments has not been observed until now. The expression of organelle-encoded proteins is blocked or down-regulated in a number of mutants from yeast or Chlamydomonas (see reviews of Tzagoloff and Dieckmann(1990) and Rochaix(1992)). In contrast, isolation of mutants overexpressing an organelle-encoded protein is difficult to achieve. Several nuclear-encoded proteins, which are targetted to the mitochondria, have been overexpressed in yeast and in some cases displayed an increased steady-state concentration in the mitochondria but their state of assembly or self-aggregation was not characterized (van Loon et al., 1983a, 1983b).

In this study we have identified the mutation in FUD16 as a 2-base pair substitution in the atpA gene coding for the subunit, which is located immediately downstream from the conserved region involved in nucleotide-binding (Walker et al., 1982). We show that the overaccumulation of the mutated subunit is accompanied by the formation of inclusion bodies in the chloroplast. This process requires the interaction of the mutated subunit with the subunit which also accumulates in the inclusion bodies. We discuss the molecular basis for the altered interactions between the two subunits.


MATERIALS AND METHODS

Growth Conditions, Strains, and Genetic Characterization

Wild-type (WT)()and mutant strains were grown at 300 lux (5.86 µE/m s PAR) in Tris acetate-phosphate (TAP) medium. The WT strain is derived from strain 137c. The nuclear mutants F54, thm24, and ncc1 and the chloroplast mutant FUD16, have been previously described (Lemaire and Wollman, 1989b; Drapier et al., 1992). The chloroplast mutant 10-6C has been shown to bear a mutation in the rbcL gene by Dron et al.(1983).

Induction of gametes, crosses, maturation of zygotes, and dissection of tetrads were carried out according to Levine and Ebersold(1960). Chloroplast recombination tests were achieved according to Girard-Bascou(1987).

The thm24.FUD16 double mutant was selected from tetrads obtained after FUD16.mt+ thm24.mt- crosses. Two clones, out of four per tetrad, were selected as double mutants based on the absence of atpB transcripts as determined by mRNA hybridization analysis. FUD16.mt+ ncc1.mt- crosses yielded two double mutant clones per tetrad. These were selected as affected in their atpA transcript accumulation upon mRNA analysis.

Protein Analysis

Thylakoid membranes were isolated as described by Chua and Bennoun(1975). Protein content was analyzed after urea/SDS-polyacrylamide gel electrophoresis according to Piccioni et al.(1981). Gels were either silver-stained as in Rabilloud et al.(1988) or used for immunoblotting experiments as in de Vitry et al.(1989). Antibody labeling was detected with I-protein A (Amersham, France) or the enhanced chemiluminescence method (ECL, Amersham, France).

Anti-, anti-, and anti- were kindly provided by C. Lemaire (Centre de Genetique Moleculaire, Gif sur Yvette, France) and anti-CF subunit III by O. Vallon (Institut Jacques Monod, Paris, France) and B. Lagoutte (CEN, Saclay, France). Anti-P10 was kindly provided by R. Bassi (Universita di Verona, Verona, Italy) and anti-LHC (light-harvesting complex) II was prepared in the laboratory as described in de Vitry et al.(1989).

Pulse labeling experiments were carried out according to Delepelaire (1983). Cells were labeled for 5 min with 5 µCi/ml [C]acetate (56 mCi/mM, Amersham, France) in the presence of 6.6 µg/ml cycloheximide which inhibits translation in the cytoplasm. Quantification of the C labeling or I labeling was performed using a PhosphorImager (Molecular Dynamics).

Separation of Protein Complexes on Sucrose Gradient

16 10 cells, resuspended in 4 ml of buffer (25 mM HEPES, pH 7.5, 10 mM EDTA, 0.3 M sucrose) were broken in a French press, directly loaded on a continuous 20-80% (w/v) sucrose gradient, containing 10 mM HEPES, pH 7.5, and centrifuged at 38,000 rpm for 60 or 1 h in a Beckman SW-41 Ti rotor. Fractions of 0.5 ml were collected and analyzed by immunoblotting as described above.

Assays of Dissociation of the Inclusion Bodies in FUD16 Strain

Fractions from the sucrose gradient containing the inclusion bodies were diluted four times with 10 mM HEPES, pH 7.5, and centrifuged at 50,000 rpm for 15 min in a Beckman TLA-100.3 rotor. The pellet was resuspended in 25 mM HEPES, pH 7.5, 10 mM EDTA, 0.3 M sucrose, then incubated, at 4 °C for 30 min, with either 2% Triton X-100 or 8 M urea with gentle stirring. Samples were loaded on a continuous 20-80% (w/v) sucrose gradient centrifuged at 54,000 rpm for 40 min in a Beckman TLS-55 rotor. Fractions of 300 µl were collected and analyzed on urea/SDS-acrylamide gels which were silver-stained.

CF1 Analysis

Chloroform-release extracts of purified thylakoid membranes were prepared and ATPase activity tested as in Piccioni et al.(1981). Sedimentation of WT CF, prepared by the chloroform release procedure was carried out on a continuous sucrose gradient as described above.

DNA Analysis

Chloroplast DNA was isolated according to Rochaix et al.(1988). For isolation of the R7 and R15 EcoRI fragments (nomenclature according to Rochaix, 1978), chloroplast DNA was digested with the restriction enzyme EcoRI and subcloned in the pUC21 vector by standard protocols (Sambrook et al., 1989). After transformation into Escherichia coli, tetracycline-resistant colonies were hybridized against WT C. reinhardtii atpA R7 and R15 gene fragments. Plasmid DNA from the selected colonies was isolated and analyzed by DNA filter hybridization with WT fragments. Fragments were subcloned in pBSKS- (Stratagene) for DNA sequence analysis. Double-stranded plasmids were sequenced using Sequenase dideoxy nucleotide sequencing (U. S. Biochemical Corp.) according to the manufacturer's instructions. Sequence from both strands was obtained and compared with the atpA sequence obtained from Leu et al.(1991) and Dron et al.(1982).

Transformation Protocols and Plasmids

Chloroplast transformation was carried out according to Kuras and Wollman(1994). Cells were grown up to 2.5 10 cells/ml in liquid TAP medium after treatment for about 6 generations (48 h) with 3-fluorodeoxyuridine. Cells were then plated on TAP medium containing 500 units/ml penicillin at a density of 10 cells/plate for transformation. Cells were bombarded with 1.2-µm tungsten particles containing appropriate DNA. Cells were then plated on TAP medium for 5 days under dim light (300 lux), then transferred to high light (4000 lux) on minimal medium for recovery of phototrophic transformants.

Electron Microscopy

Thin sections of intact cells were performed on samples fixed in 4% paraformaldehyde and 2% glutaraldehyde, postfixed in 1% osmium tetroxyde and embedded in Epon-Araldite resin.

For the immunocytochemistry analysis, cells were fixed in 2% paraformaldehyde and 1% glutaraldehyde in 4 mM potassium phosphate buffer plus 10 mM MgCl, rinsed in the same buffer plus 0.1 M glycine and embedded in Lowicryl K4M resin. Lowicryl thin sections were labeled with antisera (1/200 dilution) as described in Vallon et al.(1985), using colloidal gold-labeled protein A to reveal binding of primary antibodies.


RESULTS

FUD16 Mutant Strain Bears Two Base Substitutions in the atpA Gene

The atpA gene is located between the atpH and rbcL genes on the C. reinhardtii chloroplast genome, with the rbcL gene being transcribed in the direction opposite to atpA. The genetic lesion in FUD16 mapped next to the rbcL gene, yielding 1-2% recombinants upon crosses with the rbcL mutant 10-6C (experiments not shown). This result strongly suggested that the mutation was in atpA.

A restriction map of the Chlamydomonas chloroplast DNA region containing the atpA gene is presented in Fig. 1A. Transformation rescue experiments were performed on the FUD16 mutant, with various restriction fragments from WT atpA gene region. After particle bombardment, transformants with a WT phenotype were selected by their ability to grow in phototrophic conditions. A restriction fragment containing the atpB gene was used in control transformations. As shown on Fig. 1B, all fragments which restored phototrophic growth mapped in the coding region of the atpA gene. The smallest fragment of this series was a 940-base pair EcoRI-PstI fragment which mapped within the first half of the atpA coding region.


Figure 1: Determination of the 2-bp substitutions conferring the FUD16 phenotype. A, schematic restriction map of the atpA gene region in the chloroplast DNA. The nomenclature of the chloroplast fragments (R15,R7) is from Rochaix (1978); arrows point to directions of transcription for rbcL and atpA. The adenine additions between the two start sites of the transcripts are indicated by , whereas the two substitutions responsible for FUD16 phenotype are indicated by *. H3, HindIII; P, PstI; RI, EcoRI; X, XbaI. B, results from the transformation of the FUD16 strain with wild-type chloroplast DNA fragments. The fragments listed, with the exception of Bam5 which contains the atpB gene, are shown diagramatically in A. Selection was based on recovery of phototrophic transformants as discussed in the text. C, location of the two base substitutions in the atpA gene with the two corresponding amino acid changes in the polypeptide sequence. Nucleotide numbers start by +1 at the first A in the AUG start codon of atpA.



We then cloned the choroplast DNA fragments comprising the atpA gene from the FUD16 strain (see ``Materials and Methods''). A comparison of the atpA gene sequences in WT (as determined by Leu et al.(1991) and Dron et al.(1982)) and FUD16 strains, revealed two base changes at positions 551 and 556 (with nucleotide number 1 being the A of the AUG start codon for atpA) in the coding region of the atpA gene (Fig. 1C). The two base changes are located 1930 and 1935 base pairs upstream from the rbcL mutation 10-6C, at the distance we expected for the 1-2% recombination frequency (Girard-Bascou et al., 1987). The observed T551A and A556T substitutions resulted in amino acid changes Ile Asn and Asn Tyr. We also found a few adenine additions at positions -453, -524, and -553 in the extragenic region between the atpA and rbcL genes. As mentioned above, FUD16 transformation with the EcoRI-PstI fragment, which restored only the two substituted bases from the coding region of atpA, proved sufficient to restore a WT phenotype. We thus conclude that the chloroplast mutation responsible for the FUD16 phenotype corresponds to two amino acid substitutions located within a tripeptide segment of the polypeptide chain.

Rates of Synthesis of the and Subunits Are Increased in FUD16

Lemaire and Wollman (1989b) previously reported that the FUD16 mutant displayed an increased synthesis of the subunit (hereafter referred to as ). This conclusion was drawn from a comparison of pulse-labeled FUD16 cells with a control strain which was subsequently demonstrated to bear a nuclear mutation (ncc1) altering the rates of synthesis of the and subunits (Drapier et al., 1992). We therefore re-examined, by [C]acetate pulse labeling experiments, the rates of synthesis of these two subunits in exponentially growing cells of the FUD16 and WT strains. Labeled proteins (equivalent loads) from cell extracts were separated on urea/SDS-PAGE and dried gels were exposed to autoradiography for 1-4 weeks. As shown in Fig. 2, the resulting labeling of both the and subunits was higher in the FUD16 strain as compared to that in the WT strain. Quantification of the respective labeling in the two strains by phosphoimaging showed that FUD16 displayed a 4- and 3-fold increase in the rates of synthesis for the and subunits, respectively (mean value of three distinct experiments). We detected no parallel changes in the amount of atpA and atpB transcripts in FUD16 cells compared to WT cells (results not shown).


Figure 2: Chloroplast translates in the 50-60 kDa region from WT and FUD16 cells, pulse-labeled with [C]acetate for 5 min. Cells were labeled in the presence of cycloheximide to inhibit synthesis of proteins in the cytoplasm. Polypeptides were separated by urea/SDS-polyacrylamide gel electrophoresis and viewed by autoradiography. LS, the large subunit of ribulose-P carboxylase, migrates in the same region of the gel as the and subunits.



and Subunits Overaccumulate in FUD16

The relative amounts of and subunits, present in exponentially growing cells or purified thylakoid membranes from FUD16, were estimated by immunoblotting using specific antibodies conjugated with iodinated protein A (Fig. 3A). We used combined antisera for both the and subunits, which show a higher titer for antibodies against the subunit (Lemaire and Wollman, 1989b). Therefore the : stoichiometric ratio of 1:1 in WT samples corresponds to a stronger labeling of the subunit.


Figure 3: Accumulation of and subunits in whole cells or thylakoid membranes from WT strain and mutant FUD16. Polypeptides were separated by urea/SDS-PAGE, transferred to nitrocellulose, and detected with specific antibodies. A, immunoblots of whole cells (grown at 2 10 cells/ml) and thylakoid membranes from WT strain and mutant FUD16 treated with antibodies against , , or subunits of chloroplast ATP synthase and antibody against P10, an antenna polypeptide (loading control). Binding of the antibodies was detected using radioiodinated protein A. B, immunoblot reacted with antibody against subunit III (suIII) of CF; a diluted sample of WT membranes was used to estimate the sensitivity of the method. Binding of the antibody was detected by ECL, using a goat anti-mouse horseradish peroxidase-conjugated antibody.



Most strikingly, the FUD16 mutant showed an increased intracellular accumulation in both the and subunits whereas purified thylakoid membranes displayed only trace amounts of these subunits: FUD16 membranes displayed one-tenth of the subunits and 1/35 of the subunits present in WT membranes (). In contrast, FUD16 intracellular contents in and subunits were, respectively, three and two times higher than that of and subunits in the WT. We note that these values depended on the state of the cell culture for FUD16: the content in subunits increased in the late phase of growth, whereas that in subunits decreased ().

We also examined the steady-state concentrations of the subunit of CF (Fig. 3A) and subunit III of CF (Fig. 3B) with specific antibodies. These two subunits remained below detection in the FUD16 samples. Thus the mutation in FUD16 strain prevents the intracellular accumulation of both CF and CF.

FUD16 Thylakoid Membranes Still Accommodate Minor Amounts of CF-like Complexes

Chloroform extraction is a selective tool to extract CF bound to biological membranes (Beechey et al., 1975). Although the rationale for this purification procedure is poorly understood, its use with the WT of C. reinhardtii proved to be selective for both chloroplast CF (Piccioni et al., 1981) and mitochondrial F (Atteia, 1994). Although the FUD16 mutant failed to accumulate significant amounts of CF, we applied the chloroform treatment to purified FUD16 thylakoid membranes in an attempt to increase the sensitivity of our immunological detection. We used a large amount of highly concentrated membranes purified from cultures at 2 10 cells/ml. The chloroform-release extract contained a low level of and subunits (Fig. 4A). Surprisingly, the two subunits now displayed the same stochiometric ratio as in the WT extract, although the starting membranes did not (). Moreover, the chloroform-release extract from FUD16 contained detectable amounts of subunit (Fig. 4B). These two criteria suggested that chloroform extracted CF-like complexes from FUD16 thylakoid membranes. Based on the respective amounts of WT and FUD16 membranes extracted with chloroform, we could estimate that assembled CF-like complexes in FUD16 represent approximately 3% of the genuine CF bound to WT thylakoids.


Figure 4: Analysis of CF from WT and CF-like complexes from FUD16 by immunoblotting. Polypeptides recovered in the aqueous phase after chloroform treatment of thylakoid membranes were separated by urea/SDS-PAGE, transferred to nitrocellulose, and probed with antibodies. A, immunoblot of CF from the two strains. Anti- and anti- binding was detected with radioiodinated protein A. Samples corresponding to 50 µg of chlorophyll for WT and 500 µg of chlorophyll for FUD16 were loaded. B, subunit was detected using a specific antibody on the same immunoblot as in A revealed by the ECL method.



CF released by chloroform extraction of WT thylakoid membranes still showed some Ca-ATPase activity as measured by CaCl precipitation on native gels (Piccioni et al., 1981). Such an ATPase activity was also observed with the FUD16 sample at the same migration position as that of the WT sample (results not shown). No CaCl precipitate was observed in the control FUD50 strain, totally devoid of CF complexes (Lemaire and Wollman, 1989b), which supports the assignment of this ATPase activity to assembled CF-like protein complexes in mutant FUD16.

Overaccumulated and Subunits Form Aggregates of High Molecular Weight in FUD16 Cells

Because of the discrepancy between the amount of assembled CF-like complexes bound to FUD16 thylakoid membranes, and the amount of and subunits present in total cell extracts, we expected to find either soluble - complexes with abnormal stochiometry, or at least individual and subunits in the chloroplast stroma of FUD16. Therefore we separated cellular extracts of the WT and FUD16 strains by differential centrifugation on 20-80% (w/v) sucrose gradients. We first chose a centrifugation time of 60 h which was long enough for both membrane vesicles and soluble oligomeric proteins to reach their equilibrium density. The fractions were separated by urea/SDS-PAGE and analyzed by Western blotting. Combined antisera against and subunits were used to determine their location in the gradient and antibodies against LHCII proteins were used to monitor the location of thylakoid membranes. The data presented in Fig. 5result from phosphoimaging analysis of the antibody labeling patterns. Antibodies against subunit were also used to determine the possible state of assembly of the CF (data not shown).


Figure 5: Distribution profiles of and subunits, released from WT and FUD16 cells, upon sucrose gradient centrifugation. Cells were broken with a French press, then directly loaded on a 20-80% (w/v) sucrose gradient and centrifuged at 240,000 g for 60 h. Fractions were collected from bottom (fraction 1) to top (fraction 25) of the gradient. Graphs were constructed using the and immunolabeling quantification of the fractions by phosphoimaging. Also shown is the labeling of LHCII as a marker of thylakoid membrane distribution in each gradient. All profiles were normalized to the fraction showing maximal LHCII content (fractions 9 or 10). A correction factor was applied to the subunit labeling in order to provide a 1:1 ratio between the and subunits in CF1 from WT thylakoid membranes fractions. +, subunit; , subunit; &cjs0800;, LHCII.



In the WT profile, the and subunits co-localized with the subunit, in the same fractions as the LHCII (fractions 8-11). This reflects the association of CF with the thylakoid membranes. CF subunits were also detected in a minor peak (fraction 13), slightly above the bulk of the thylakoid membranes, with an / subunit ratio similar to that in fractions 8-11. Fraction 13 most likely corresponds to a subset of thylakoid membranes rather than to free CF since (i) it displayed the same amount of LHCII relative to that of cytochrome f as in fractions 8-12 (data not shown) and (ii) chloroform-extracted CF from WT thylakoid membranes was not recovered at the level of fraction 13 but in fraction 10, i.e. at 40% sucrose density (results not shown).

In the FUD16 profile, most of the and subunits migrated to a higher density (fractions 3-5) than that of the LHC-containing thylakoid membranes (fractions 8-11 as in WT). The : ratio in these higher density fractions was 2.3, as calculated by phosphoimaging. These fractions contained neither the subunit nor any other protein components (see the silver-stained gel on Fig. 6A). Some and subunits still sedimented to fractions 13-15 (Fig. 5), which corresponds to the minor and peak in the WT gradient. It is of note that, in spite of the higher accumulation of than subunits in FUD16, all the fractions displaying a significant content in the subunit also showed the presence of subunit. Extracts from older FUD16 cells displayed the same profile as those from younger cells, although in an : ratio of 5, instead of 2.3, in fractions 3-5 (results not shown), which was consistent with the changes in their relative accumulation described in .


Figure 6: Identification of the inclusion bodies in FUD16. A, fractions 3 and 4 from the sucrose gradient (Fig. 5) were pooled, diluted in buffer, and centrifuged at 100,000 g. The pellet was resuspended and analyzed by silver staining after urea/SDS-gel electrophoresis; B, after treatment with 8 M urea, the and subunits from A are recovered at the top of a 20-80% (w/v) sucrose gradient. Note that the faint high molecular weight band (*), detected in fractions 3-4 in A, remains close to the bottom of the gradient in B.



Thus, the experiment of Fig. 5clearly points to the presence, in the FUD16 mutant, of a membrane-free protein complex made of and subunits. This - complex equilibrated to a 55% sucrose concentration which corresponds to a density of 1.26. Surprisingly, in a similar experiment in which the time of centrifugation was shortened to 1 h, the same distribution of the major - peak was observed, peaking at 55% sucrose concentration (result not shown). This latter observation further suggested that the - oligomer was of larger molecular mass than expected from a regular oligomeric protein complex, well above 1000 kDa. Indeed, when we diluted the --containing fractions to a sucrose concentration lower than 0.3 M, we recovered the - oligomers in the pellet, after low speed centrifugation at 5000 g for 15 min.

We then attempted to disrupt the - association by various treatments. Whereas 2% Triton X-100 had no effect, incubation of - oligomers from fraction 3 with 8 M urea (see ``Materials and Methods'') totally destroyed their association: when loaded on a second sucrose gradient, the subunits were recovered in the two fractions close to the top of the gradient (Fig. 6B). These observations strongly suggested that - oligomers behaved as an inclusion body, a subcellular structure observed, for example, when foreign proteins are overexpressed in E. coli (Williams et al., 1982).

Direct Observation of an - Inclusion Body in FUD16 Chloroplasts

We looked at the ultrastructure of FUD16 cells by electron microscopy. Fig. 7shows a thin section across a cell region where the cytosol can be distinguished from the stroma of the chloroplast by its granular and darker aspect. A striking ultrastructural characteristic of the chloroplast in FUD16 cells was the presence of a round-shaped amorphous and dark mass which is typical of an inclusion body (labeled ib on Fig. 7). It was unambiguously localized in the stroma next to the thylakoid membranes (arrows on Fig. 7) but not enclosed within a membrane vesicle. Its average diameter was 1 µm, as measured on a preparation of inclusion bodies purified from FUD16 broken cells by sucrose gradient centrifugation (experiment not shown). It was similar to that of the majority of the inclusion bodies recovered after purification (Taylor et al., 1986).


Figure 7: Thin section of a FUD16 cell. The cell contains a round-shaped and amorphous electron dense mass corresponding to an inclusion body (ib). Note the presence of thylakoid membranes (arrows) in the vicinity of the inclusion body pointing to its chloroplast localization.



An immunocytochemical characterization of the composition of these inclusion bodies was undertaken, in situ, using various antibodies conjugated with gold-labeled protein A (Fig. 8). The inclusion bodies (white arrows), but not the thylakoid membranes (black arrows), were heavily labeled with antibodies against the or subunits (Fig. 8, a and b). We observed no labeling of the inclusion bodies with antibodies against the subunit of CF (Fig. 8c) or against LHCII subunits (Fig. 8d). The LHCII antibody, but not the antibodies against the various ATP synthase subunits, heavily labeled the thylakoid membranes in FUD16 (black arrows). This labeling pattern of the membranes is consistent with their unaltered content in peripheral antenna proteins but with extensive deficiency in ATP synthase.


Figure 8: Immunocytochemical characterization of the inclusion bodies in the FUD16 mutant. Inclusion bodies were densely labeled with antibodies directed against (a) and (b) subunits (white arrows) but not with antibodies directed against the subunit (c). Thylakoid membranes (black arrows) were labeled only with anti-LHCII (d).



Nuclear Mutations, Which Block Synthesis of the Subunit or Alter the Rates of Synthesis of the Subunit, Prevent Inclusion Body Formation

That the overexpressed subunit in FUD16 was found in association with the subunit in inclusion bodies led us to suspect that an early recognition of the mutated subunit by a neighboring subunit may be required for its stable accumulation. In addition, this early recognition may critically depend on the respective rates of synthesis of the two subunits. Therefore we looked at the synthesis and accumulation of and subunits in nuclear contexts which markedly altered their synthesis. Two nuclear mutants were used in this study: the thm24 mutant which is unable to synthesize the subunit, because of a destabilization of the atpB transcript, and the ncc1 mutant which shows a decrease in the rate of synthesis of the subunit because of a decreased stability of the atpA transcript (Drapier et al., 1992). We placed the FUD16 chloroplast mutation in each of these nuclear backgrounds by selecting double mutants from FUD16.mt+ thm24.mt- or FUD16.mt+ ncc1.mt- crosses (see ``Materials and Methods'').

We characterized these double mutants for their rates of synthesis of the and subunits by pulse labeling (Fig. 9A) and for the extent of intracellular accumulation of the two subunits by Western blotting (Fig. 9B). The double mutant FUD16.thm24 had characteristics similar to that of the thm24 single mutant. In vivo pulse labeling of chloroplast translates showed the absence of synthesis and no overexpression of the subunit (Fig. 9A). Pulse labeling of chloroplast translates in the double mutant FUD16.ncc1 showed no differences with that in the single mutant ncc1. In each case, the rate of synthesis of the (respectively ) subunit was severely decreased as indicated by its much lower labeling than that of the subunit (Fig. 9A) at variance with the situation observed in the WT or FUD16 cells. The immunoblots on Fig. 9B show that neither the FUD16.thm24 nor the FUD16.ncc1 accumulated the subunit. The FUD16.ncc1 strain thus behaves very differently from the single mutant ncc1 which accumulates and subunits in CF complexes. The trace amount of subunit visible in whole cells of FUD16.ncc1 is similar to the phenotype of the F54 strain which totally lacks synthesis of the subunit but accumulates small amounts of the subunit (Drapier et al., 1992). Thus, the absence of inclusion bodies in the two double mutants suggests that their formation critically depends on the rates of association between - oligomers.


Figure 9: Characterization of the double mutants FUD16.thm24 and FUD16.ncc1. A, 50-60-kDa region of a urea/SDS-acrylamide gel loaded with mutant cells pulse-labeled for 5 min. Same experimental conditions as described in the legend to Fig. 2. The WT and single nuclear mutants thm24 and ncc1 are shown for comparison with the double mutants. B, immunoblots of whole cells from the same mutant strains as in A. and subunits were detected with specific antibodies and radioiodinated protein A, as in Fig. 3. The ncc1 accumulates the two subunits in the same ratio as the wild type (Drapier et al., 1992).




DISCUSSION

The Substitutions in FUD16 Occur at Strictly Conserved Sites in Subunits from (C)F

In the present study we have identified the mutational event which prevents ATP synthase assembly in the chloroplast mutant FUD16 of C. reinhardtii. Two neighboring point mutations in the atpA gene result in the expression of an subunit with two amino acid substitutions: the Ile Leu Asn tripeptide segment from the WT subunit is converted into Asn Leu Tyr in the FUD16 . The IXN motif found in the WT subunit is strictly conserved among the 40 sequences of subunits from bacterial, mitochondrial, and chloroplast (C)F available in the NBRF data bank. This motif is also present in subunits from bacterial, beef heart, and yeast mitochondrial F and higher plants and C. reinhardtii CF (Walker et al., 1985). These two amino acid changes in are located 7 and 9 amino acids downstream the conserved nucleotide-binding fold GXXXXGKT (Walker et al., 1982), a protein motif also present in all and subunits of (C)F-ATP synthase. According to the three-dimensional structure of the F-ATPase from bovine heart mitochondria, which is now resolved at 2.8-Å resolution (Abrahams et al., 1994), the nucleotide-binding motif forms the ``P-loop'' between strand 3 and helix B in the nucleotide-binding domain of either the or subunit. The two amino acid substitutions in are thus located next to the end of helix B, toward the periphery of the complex, and opposite the region facing the internal cavity where the helical domains of the subunit are located.

Misfolded - Complexes Accumulate in the Chloroplast as Inclusion Bodies

Most strikingly, the conversion of the Ile Leu Asn tripeptide segment from the WT subunit into Asn Leu Tyr in FUD16 results in the overproduction of the subunit. The intracellular concentration of the subunit is also increased in the mutant but to a more limited extent than that of the subunit. In contrast, the other subunits of the chloroplast ATP synthase, such as the subunit of CF or subunit III of CF, were hardly detectable in this mutant strain. The bulk of the and subunits in FUD16 copurified in membrane-free, high molecular weight aggregates which sedimented upon low speed centrifugation. These urea-sensitive aggregates had morphological features and a density of 1.26 on sucrose gradients, which are typical of previously characterized inclusion bodies from E. coli (Taylor et al., 1986). Although chloroplast chaperonins can associate with subunits in some instances (Lubben et al., 1989; Chen and Jagendorf, 1994), we found no evidence for their presence in the inclusion bodies: silver-stained gels showed that they contained no other proteins than the and subunits.

The FUD16 phenotype is thus remarkable since it demonstrates that the chloroplast compartment can accommodate inclusion bodies which, up to now, were observed only in the cytosol upon expression of abnormal proteins or overexpression of endogeneous or foreign proteins, in bacteria (Nicolas et al., 1993; Marston, 1986) or in Saccharomyces cerevisiae (Binder et al., 1991).

Inclusion Bodies Are Made of Partially Folded Intermediates

It is of note that the mutation in FUD16 preserved - interactions but almost totally prevented further interactions with the subunit. Although the two point mutations in alter amino acids located toward the periphery of CF complexes, as viewed from the three-dimensional structure of F (Abrahams et al., 1994), one could consider that these mutations should interfere with the folding of the whole P-loop region thereby preventing interaction with the subunit. However, the study of Smart and Selman(1993) showed that the mere absence of interactions between the subunit and - oligomers does not lead the latter to aggregate in inclusion bodies. Instead the and subunits were rapidly degraded in a nuclear mutant of C. reinhardtii disrupted in the atpC gene encoding the subunit.

In a thorough survey of the various reports on inclusion body formation, Mitraki and King(1989) concluded that inclusion bodies result from the aggregation of partially folded intermediates. This view is consistent with our observation that some CF, although in marginal amounts, were properly assembled in the FUD16 mutant and displayed some ATPase activity in nondenaturing gels. In addition, as we previously reported (Lemaire and Wollman, 1989b), the FUD16 strain displays some ATP synthase activity in vivo, as deduced from the increase in the decay rates of the flash-induced transmembrane potential upon preillumination of dark-adapted cells. This treatment is known to activate the membrane-bound ATP synthase (Joliot and Delosme, 1974). Thus the two point mutations in the FUD16 strain, instead of destroying a site required for proper folding of the subunit, would rather alter its folding pattern during CF biogenesis. Newly synthesized subunits still interact with subunits and are thus protected from proteolytic degradation as demonstrated by their destruction in the FUD16.thm24 mutant in which the subunit is not synthesized. The subsequent assembly pathway in FUD16 would drive most, but not all, of the - folding intermediates toward inclusion body formation rather than toward CF assembly. This explains why the inclusion bodies in FUD16 comprise two components, the and subunits, instead of one only as usually observed (Mitraki and King, 1989). Our study is consistent with the folding pathway recently proposed by Chen and Jagendorf(1994) based on a chaperonin-assisted in vitro system for reconstitution of , , and subunits from CF: several - dimers would assemble up to an hexamer complex able to interact with the subunit. The FUD16 mutation would prevent formation of most of the hexamers competent for binding. A similar situation most likely prevails after import of the and subunits of F in the mitochondria: Ackerman and Tzagoloff(1990) showed that the proper folding pathway of - oligomers in the organelle requires assistance of at least two nuclear-encoded proteins which are not part of mitochondrial F. Yeast mutants defective in either of these two proteins displayed a phenotype which strikingly resembles that of FUD16. They accumulated - aggregates of high molecular weight in the mitochondria. We believe that further analysis of these mutants would show genuine intramitochondrial inclusion bodies made of - aggregates.

Increased Rates of Synthesis of the and Subunits in FUD16 Point to an Early Interaction during their Biogenesis

Five-min pulse labeling experiments showed that there was a 4-fold increase in the rate of synthesis of in FUD16 as compared to that of in the WT. As we discussed elsewhere (Kuras and Wollman 1994), these rates of synthesis do not reflect the subsequent state of accumulation of a given polypeptide and result from a combination of the rates of translation with the rates of possible co-translational or early post-translational modifications. According to the codon usage in the chloroplast of C. reinhardtii (Rochaix, 1987) we could exclude that either of the 2-base pair substitutions corresponded to a conversion of a rare codon in a more frequently used codon, a situation which may have accounted for an increased rate of translation of . We can also exclude an increased translation due to an increased availability in atpA transcripts since mRNA hybridization experiments showed no changes in their accumulation between the WT and FUD16 strains.

We observed that the rate of synthesis of the subunit was also increased in FUD16. It suggests that the biogenesis of the two subunits are intimately related. Aschenbrenner et al.(1993) working with mitochondrial yeast F had observed an increased synthesis of a mutated subunit with a concomitant increase in synthesis of the subunit. Other evidence for an early interaction in the synthesis of the two subunits came from our previous study of several nuclear mutants of C. reinhardtii (Drapier et al., 1992). These mutants displayed changes in the rates of synthesis associated with changes in the rates of synthesis of the subunit. Here, we observed that FUD16.thm24 and FUD16.ncc1 double mutants no longer displayed an increased rate of synthesis of the subunit. In the two cases, the translated subunits were rapidly degraded. This suggests that the increased synthesis of subunit observed in the single mutant FUD16 is controlled by an early interaction with neighboring subunits.

At the moment we cannot discriminate between co-translational interactions and early post-translational interactions. The nucleotide fold of newly synthesized or subunits could act as a translational regulator on the atpA or atpB transcripts as recently suggested for other (di)nucleotide binding proteins (Hentze, 1994). Changes in ribosome pausing during translation, at positions where proper folding intermediates would otherwise accommodate cofactor-binding or further interactions between nascent subunits and chaperonins (Kim et al., 1991; Stollar et al., 1994), could also account for our observations.

  
Table: Comparative accumulation of the and subunits in cells and membranes preparations from WT and FUD16 strains

Polypeptides were separated by urea/SDS-PAGE, transferred to nitrocellulose, detected with antibodies against and subunits, and radioiodinated protein A; quantification was performed by phosphoimaging. Values in mutant FUD16 are given relative to those in WT.



FOOTNOTES

*
This work was supported in part by the CNRS/URA 1187 and Human Capital and Mobility network European Economic Community contract ERB CHRX CT 920045. S. L. K. and D. D. contributed equally to the work. 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.

§
Recipient of a Direchon de la Recherche et des Ehides Doctorales postdoctoral fellowship. Present address: Dept. of Plant Biology, University of California, Berkeley, CA.

To whom correspondence should be addressed: Service de Photosynthèse, Institut de Biologie Physico-chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France. Tel.: 33-1-43-25-26-09; Fax: 33-1-40-46-83-31; E-mail: Wollman@citi2.fr.

The abbreviations used are: WT, wild-type; LHC, light harvesting complex; TAP, Tris acetate-phosphate; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank R. Kuras for numerous suggestions in the course of this work, D. Picot for comments on the F0 structure, F. Lacqueriere and M. Recouvreur for expert technical assistance, and the members of the Service de Photosynthèse for critical reading of the manuscript.


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