Identification of a Nuclear Gene (FMC1) Required for the Assembly/Stability of Yeast Mitochondrial F1-ATPase in Heat Stress Conditions*

Linnka Lefebvre-LegendreDagger, Jacques Vaillier, Houssain Benabdelhak, Jean Velours, Piotr P. Slonimski§, and Jean-Paul di Rago

From the Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 33077 Bordeaux cedex, France and the § Centre de Génétique Moléculaire du CNRS, Laboratoire propre associé à l'Université P. et M. Curie, 91190 Gif-sur-Yvette, France

Received for publication, October 19, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37 °C) for the formation/stability of the F1 sector of the mitochondrial ATP synthase. Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix. At elevated temperatures in yeast cells lacking Fmc1p, the alpha -F1 and beta -F1 proteins are synthesized, transported, and processed to their mature size. However, instead of being incorporated into a functional F1 oligomer, they form large aggregates in the mitochondrial matrix. Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F1 sector (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986-4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p. However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28-30 °C). We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

F1Fo-ATP synthases play a major role in cellular energy production. They are found in the plasma membranes of bacteria, thylakoid membranes of chloroplasts, and in the inner membrane of mitochondria. They use a proton gradient across their host membrane to produce ATP from ADP and inorganic phosphate (1, 2). This enzyme contains two distinct parts, called Fo and F1. The Fo mediates the transmembrane transport of protons, and the synthesis of ATP takes place on the F1.

The F1 contains five different types of subunits in the stoichiometric ratio alpha 3beta 3gamma delta epsilon (3, 4). The three-dimensional structures of F1 from bovine heart (5), rat liver (6) and yeast (7) show that the alpha - and beta -subunits alternate in a hexagonal array with a central cavity occupied by the amino and carboxyl termini of the gamma -subunit. The interfaces between the alpha - and beta -subunits form three catalytic and three noncatalytic nucleotide binding sites.

In the yeast Saccharomyces cerevisiae, the F1 subunits are encoded in the nucleus (8-12), synthesized in the cytoplasm, imported into mitochondria as unfolded polypeptide chains (13), and then folded in the mitochondrial matrix with the help of Hsp60p and Hsp10p (14). The oligomerization of the F1 monomers is assisted by two proteins called Atp12p and Atp11p. These interact directly with the alpha -F1 and beta -F1 proteins, respectively (15, 16). In yeast strains lacking either Atp11p or Atp12p, the alpha -F1 and beta -F1 proteins aggregate in the mitochondrial matrix (17). Thus it is believed that Atp12p and Atp11p facilitate the formation of alpha beta heterodimers by protecting these two F1 subunits from non-productive interactions (16).

We report in this study the identification of Fmc1p, a novel protein required for the formation or stability of the F1 oligomer. Like Atp11p and Atp12p, its absence also results in the aggregation of the alpha -F1 and beta -F1 proteins. However, this is seen only at elevated temperatures (37 °C), whereas Atp11p and Atp12p are required both in normal (28-30 C°) and heat stress conditions. Interestingly, the formation/stability of the F1 oligomer was restored in cells lacking Fmc1p by increasing the expression of Atp12p. We propose that Fmc1p assists the folding/stability or functioning of Atp12p and that this role becomes essential at elevated temperatures.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Strains and Media

The S. cerevisiae strains used are listed in Table I. Escherichia coli XL1-Blue (Stratagene) was used for the cloning and propagation of plasmids. Yeast strains were grown in either YPGA (1% yeast extract, 1% bactopeptone, 2% glucose, 30 mg/liter adenine), YPGALA (1% yeast extract, 1% bactopeptone, 2% galactose, 30 mg/liter adenine), or synthetic complete media SC (2% glucose, 0.67% yeast nitrogen base, 20 mg/liter adenine containing either 1% casamino acids or appropriate drop-out mix (CSM series from BIO 101) at the concentration recommended by the manufacturer. The SC media with casamino acids were always supplemented with 50 mg/liter tryptophan and 20 mg/liter methionine. The growth of yeast strains on nonfermentable carbon sources was tested on N1 (1% yeast extract, 1% bactopeptone, 2% ethanol, 0.05 M sodium-potassium phosphate, pH 6.2) and N3 (1% yeast extract, 1% bactopeptone, 2% glycerol, 0.05 M sodium-potassium phosphate, pH 6.2). All media were solidified by adding 2% Bacto-Agar Difco.

Construction of Yeast Strains Carrying a Null Mutation in FMC1

FMC1 was deleted according to the procedure described by Wach (18). The 297- and 470-base pair DNA sequences located, respectively, upstream of the ATG initiator codon and downstream of the stop codon of FMC1 were PCR1-amplified from yeast genomic DNA (W303-1B) with the primers P1 (5'-GTATGCTTGATACGTTTGGACAG), P2 (5'-CAATCTATACGTGTCATTCTGAACGATTCCCTGGCACTCTCTCTTTCTCTCTC), P3 (5'-GAGTGTACTAGAGGAGGCCAAGAGCCGACATAGTATCTAATCAATTTATAATATC), and P4 (5'-CATTTGGGAAATGACGAAGGCTATTTG). DNA fragments carrying HIS3 were generated by PCR from plasmid pUC18-HIS3 (19) with the primers Pa (5'-TCGTTCAGAATGACACGTATAGAATG) and Pb (5'-CTCTTGGCCTCCTCTAGTACACTC). The three PCR products (P1-P2, P3-P4, and Pa-Pb) were mixed, and PCR with the primers P1 and P4 gave a DNA fragment in which HIS3 was flanked by sequences adjacent to the FMC1-coding region. The strain W303-1B was transformed with the deletion cassette, and resulting His+ clones were analyzed by Southern blot. Out of 12 His+ transformants analyzed, 11 gave the expected hybridization signals (not shown).

Plasmid Constructions

Cloning of FMC1-- A DNA fragment carrying FMC1 was amplified by PCR from the genomic DNA of W303-1B with the primers 5'-ATTCCTCGTCAGATAATCACC and 5'-GGGCAACGTAAAAACCTCGATAG. The PCR product was cut by RsaI to produce a blunt end fragment containing the FMC1 coding sequence with 579 base pairs upstream of the initiator ATG and 498 base pairs downstream from the Stop codon. This fragment was cloned at the HincII site of pUC19 to give pJR22. From this plasmid, the HindIII-BamHI fragment containing FMC1 was cloned into the centromeric plasmid pRS316 (20) cut with the same pair of enzymes to give pLL1.

Cloning of ATP12 in a Low Copy Number Vector-- The ATP12-containing HindIII-HindIII fragment of pLE10 was inserted at the HindIII site of pRS316 to give pLL5.

Isolation of ATP12 as a Multicopy Suppressor of the Null Allele Delta fmc1

The Delta fmc1 strain MC6 was transformed with a partial HindIII digest of yeast chromosomal DNA cloned into the URA3-containing multicopy vector pEMBLYe23 (a gift of Dominique Thomas, Gif-sur-Yvette). About 100,000 Ura+ transformants were selected on SC medium lacking uracil and were then replica-plated onto glycerol medium (N3) prewarmed for at least 2 h at 37 °C. After an incubation of 5 days at 37 °C, clones that showed good growth were retested for their growth on glycerol at 37 °C after curing the plasmids they contained on SC medium supplemented with 0.1% of 5-fluoroorotic acid. When growth on glycerol at 37 °C was plasmid-dependent, the suppressor plasmids were recovered in E. coli and tested again by retransformation of Delta fmc1 cells. Several rescuing plasmids contained the same insert, a HindIII-HindIII 1599-base pair fragment corresponding to a segment of chromosome X between coordinates 87249 and 88848 (this plasmid has been called pLE10). Since ATP12 was the only gene present on this fragment, it can be concluded that it was responsible for the suppressor activity of pLE10.

Genetic and Molecular Biology Methods

Genetic experiments were carried out as described by Rose et al. (21). Standard recombinant DNA techniques were used as described by Sambrook et al. (22). Yeast transformations were performed using described procedures (23). Yeast genomic DNA was isolated as described previously (24). Yeast mitochondrial RNAs were isolated as described by di Rago et al. (25). The Northern hybridizations were performed with pSCM511 (21 S rRNA, Ref. 26), p14 (cytochrome b, Ref. 27), pYGT21 (CoxI, Ref. 28) as DNA probes labeled with [alpha -32P]dCTP using the nick translation kit from Roche Molecular Biochemicals.

Biochemical Techniques

Mitochondria were prepared by the enzymatic method described by Guérin et al. (29). Alkaline extraction of mitochondrial proteins with sodium carbonate at pH 11.5 was performed as described by Rouillard et al. (30). Protein concentrations were determined by the procedure of Lowry et al. (31) in the presence of 5% SDS using bovine serum albumin as standard. The specific ATPase activity was measured at pH 8.4 as described by Somlo (32). Oxygen consumption rates were measured in the respiratory medium (0.65 M mannitol, 0.3 mM EGTA, 3 mM Tris phosphate, 10 mM Tris maleate, pH 6.75) as described by Rigoulet and Guérin (33). Variations in transmembrane potential (Delta Psi ) were evaluated in the same medium by measurement of the fluorescence quenching of rhodamine 123 with an SFM Kontron fluorescence spectrophotometer (34). Immunoprecipitation experiments were made from 2 mg of mitochondrial proteins as described by Todd et al. (35). Polyclonal antibodies raised against the alpha -F1 subunit were added to the 100,000 × g supernatant of the 0.375% Triton X-100 mitochondrial extract. The immunoprecipitated proteins were washed with a buffer containing 0.1% Triton X-100 (w/v), 150 mM NaCl, 10 mM sodium phosphate, pH 7.0. The final pellet was dried under vacuum and dissolved in 20 µl of dissociation buffer devoid of reducing agent. SDS-PAGE was performed according to Laemmli (36). The slab gel was silver-stained as described by Ansorge (37). Western blot analyses were performed as described previously (38). Sedimentation analysis in sucrose gradients was performed as described by Ackerman and Tzagoloff (17), except that the gradient was centrifuged at 48,000 rpm for 3 h in a Beckman SW55 Ti rotor. Polyclonal antibodies raised against yeast ATP synthase subunits, Aac2p, Atp12p, and Fmc1p were used after dilution 1:10,000; 1:10,000, 1:2,500, and 1:5,000, respectively. ProBlott membranes were incubated with peroxidase-labeled antibodies at a dilution of 1:10,000 and revealed with the ECL+ reagent of Amersham Pharmacia Biotech. The chloroform/methanol extraction of subunits 6, 8, and 9 of ATP synthase was as described by Michon et al. (39). Mitochondrial proteins were labeled in cycloheximide-blocked cells according to Claisse et al. (40). Mitoplasts were prepared according to Daum et al. (41).

Production of anti-Fmc1p Antibodies

Anti-Fmc1p antibodies were prepared by Eurogentec (Seraing, Belgium) with the synthetic peptide NH2-YNPGNKLTQDEK as an immunogen.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During a systematic program of functional analysis of newly discovered yeast genes, we found that a null allele of the ORF YIL098c (which we have called FMC1 for formation of mitochondrial complexes 1) impairs the ability of yeast to grow at 37 °C on media containing a nonfermentable carbon source such glycerol or ethanol (in W303-1B and with HIS3 as the inactivation marker) (Fig. 1). Three lines of evidence show that this phenotype is due to the inactivation of FMC1 as follows. (i) The correct integration of the deletion cassette at the FMC1 locus was confirmed by Southern blot analysis (not shown); (ii) the mutant phenotype and histidine prototrophy cosegregated 2:2 in tetrads originating from heterozygous Delta fmc1/+ cells (not shown); and (iii) after transformation with a plasmid containing FMC1 (pLL1), Delta fmc1 cells recovered a normal growth on ethanol or glycerol at 37 °C. As oxidative phosphorylation is essential for growing yeast on glycerol or ethanol, these data indicated that FMC1 may be needed for the formation or functioning of the yeast energy transducing system at elevated temperatures.



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Fig. 1.   Yeast cells carrying a null mutation in the FMC1 gene fail to grow at elevated temperatures on media containing a non-fermentable carbon source. Wild type FMC1+ and mutant Delta fmc1, containing either an intronless mtDNA or a mtDNA with 13 introns, were grown overnight at 28 °C in glucose (YPGA). The cultures were diluted and 5 µl of each dilution spotted on glucose (YPGA) and ethanol (N1) media. The plates were photographed after 3 days of incubation at 37 °C.

We first aimed to determine whether FMC1 is needed for pre-mRNA splicing or the propagation of mtDNA at elevated temperatures. To this end, we constructed a Delta fmc1 strain containing an intronless mtDNA (strain MC6, see Table I). This strain was still unable to grow on glycerol or ethanol at 37 °C (Fig. 1), showing that the absence of respiratory growth is not due to a defect in mitochondrial pre-mRNA splicing.


                              
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Table I
Yeast strains used in this study

The propagation of the mtDNA by the mutant was analyzed by determining the production of cytoplasmic petites2 (rho-/rho° cells) during fermentation at elevated temperatures. Fresh cells were grown at 28 °C in glycerol (selecting for a rho+ mitochondrial genome), then grown for 5-6 generations in glucose at 28 or 37 °C. The cultures were then plated at 28 °C on a glycerol medium containing 0.1% glucose (on this medium cells that fail to produce ATP by oxidative phosphorylation give very small colonies). This analysis was performed for wild type and Delta fmc1 strains, with either a wild type (intron-containing) or intronless mitochondrial genome. The results presented in Table II show that deletion of FMC1 gene leads to a 5-7-fold increase in petite production in the presence of both an intron-containing and intronless mitochondrial genome at 37 °C. However, it is clear that the level of petite production seen at 37 °C, especially in the presence of an intronless mitochondrial genome, is not sufficient to explain the respiratory-deficient phenotype. We conclude that the increased production of petites is probably a secondary effect of the Delta fmc1 mutation.


                              
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Table II
Production of petite (rho-/rhoo) cells by the Delta fmc1 mutant
The strains were cultivated for five to six generations in glucose (YPGA) at the indicated temperature (28 or 37 °C). The cultures were then diluted and spread for single colonies on a glycerol medium containing 0.1% glucose. The petite (rhoo/rho-) and grande (rho+) colonies were scored after 4 days of incubation at 28 °C.

When the Delta fmc1 strain was grown for a longer time on glucose at 37 °C (up to 15 generations), higher levels of petites were not observed. This suggests that the Delta fmc1 petites grow poorly at 37 °C, preventing them from completely taking over the cultures. To test this, Delta fmc1 cells were made rho° by growing them in the presence of ethidium bromide at 28 °C and then transformed with a plasmid bearing FMC1 or with the corresponding empty vector. The resulting transformants were then tested for their growth on glucose at 37 °C. At 37 °C growth of the Delta fmc1 rho°, cells transformed with the empty vector was severely affected compared with that of the Delta fmc1 rho° cells transformed with the cloned FMC1 gene (Fig. 2). Consistent with this, we found that rho+ Delta fmc1 cells grew poorly at 37 °C in the presence of ethidium bromide (not shown). Thus when S. cerevisiae lacks FMC1, it needs a wild type mtDNA to grow normally by fermentation at elevated temperatures.



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Fig. 2.   In the absence of mtDNA, the Delta fmc1 mutant grows poorly by fermentation at elevated temperatures. The Delta fmc1 mutant was made rho° by growing at 28 °C in the presence of ethidium bromide and was then transformed with a plasmid containing the FMC1 gene (pLL1) or the corresponding empty vector (pRS316). The transformants were grown for 2 days at 28 °C in a glucose medium lacking uracil (SC-ura). The cultures were diluted, and 5 µl of each dilution were spotted on SC-ura. The plates were incubated for 3 days at 37 °C and then photographed.

Next we analyzed the expression of the mtDNA in the Delta fmc1 mutant with an intronless mtDNA (to reduce problems associated with petite production). In a Northern blot analysis, probes specific for the genes CoxI, CoxII, ATP9, ATP6, and 21 S RNA produced similar radioactive signals in the mutant and wild type, indicating that FMC1 is not involved in transcription of the mitochondrial genes at elevated temperatures (Fig. 3A). We then determined the ability of Delta fmc1 cells to synthesize the mitochondrially encoded proteins at 37 °C. This was done with whole cells grown by fermentation for five to six generations at 37 °C. These were incubated at 37 °C for varying times (5, 15, or 30 min) with radiolabeled Met + Cys in the presence of cycloheximide (an inhibitor of cytoplasmic protein synthesis that has no effect on the mitochondrial ribosomes). Mitochondrial membranes were then prepared and analyzed by SDS-PAGE and autoradiography. This revealed that the mutant grown at 37 °C was able to normally synthesize six of the eight proteins encoded by the mtDNA (cytochrome b, Cox1, Cox2, Cox3, Var1, and ATP6), whereas the radioactive signals corresponding to ATP9 and ATP8 were significantly weaker in comparison to the wild type (even when the time of incorporation of the radiolabeled amino acids was reduced to 5 min) (Fig. 3B). In the experimental conditions used, it is difficult to determine whether the rate of synthesis of ATP9 and ATP8 was reduced in the mutant and/or whether they were less efficiently incorporated into mitochondrial membranes or more prone to proteolytic degradation (see below).



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Fig. 3.   Analysis of the expression of mitochondrially encoded genes in the Delta fmc1 mutant at elevated temperatures. Panel A, Northern blot analysis of mitochondrial transcripts. Total mitochondrial RNAs were isolated from wild type FMC1+ (strain MC1) and mutant Delta fmc1 (strain MC6) grown for 6-8 generations in galactose (YPGALA) at 37 °C. The RNAs were separated in a formaldehyde-containing agarose gel and then transferred to a nitrocellulose membrane. The same blot was hybridized successively with 32P-radiolabeled DNA probes specific for cytochrome b (Cyt b), CoxI, CoxIII, ATP6, and 21 S rRNA genes. Panel B, mitochondrial protein synthesis. Wild type FMC1+ (strain MC1) and mutant Delta fmc1 (strain MC6) cells were grown in galactose (YPGALA) for 6-8 generations at 37 °C and then incubated as indicated for 5' or 15' at 37 °C with [35S]methionine and [35S]cysteine in the presence of cycloheximide. Mitochondrial membranes were then isolated and analyzed by SDS-PAGE and an autoradiograph of the gel (500,000 cpm were loaded on each lane). The photograph on the right is from a long run gel to increase the resolution of Cox3 and ATP6.

Respiratory, ATPase, and Proton-Pumping Activities of Delta fmc1 Mitochondria-- To further understand the role of FMC1 in oxidative phosphorylation, we analyzed the energy conversion capacity of mitochondria isolated from cells lacking this gene. For this, we used a Delta fmc1 strain containing an intronless mtDNA. Mitochondria isolated from the mutant and wild type strains grown at 28 °C exhibited the same oxygen consumption rates (see Table III). By contrast, when the mutant was grown at 37 °C, its respiratory activity was substantially reduced (by 70%) in comparison to the wild type strain cultivated at the same temperature.


                              
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Table III
Respiratory and ATPase activities
Mitochondria were isolated from wild type rho+ (strain MC1), mutant Delta fmc1 rho+ (strain MC6), and wild type rhoo [derived from strain W303-1B) grown for six to eight generations in galactose (YPGALA) at the indicated temperature (28 or 37 °C). Respiration rates are the average of two different experiments. The ATPase activities are the average of three different experiments presented with the S.D. For the cells grown at 28 °C, the zymolyasae treatment for the preparation of mitochondria was made at 28 °C. For those grown at 37 °C, the zymolyasae treatment was performed at 37 °C. All the ATP hydrolytic assays were performed at 37 °C. CCCP, carbonyl cyanide m-chlorophenylhydrazone; TMPD, N,N,N,N-tetramethyl-p-phenylenediamine.

Mitochondria from the mutant and wild type strains cultivated at 28 °C had approximately the same DCCD-sensitive ATP hydrolytic activity (Table III). By contrast, Delta fmc1 cells grown at 37 °C had a very low mitochondrial ATPase activity (less than 10% of the control). This residual activity was essentially insensitive to DCCD and also about two times lower than that of mitochondria isolated from wild type rho° cells cultivated at 37 °C. Thus, the F1 sector of the ATP synthase is severely affected in yeast cells lacking FMC1 grown at 37 °C, whereas it is fully active when these cells are grown at 28 °C.

It should be noted that the ATPase activity measured for the wild type grown at 37 °C was much less sensitive to DCCD than that of wild type cells grown at 28 °C (see Table III). This suggests that a substantial part of the F1 sector is not physically or functionally coupled with the Fo sector when wild type yeast is cultivated at elevated temperatures. This observation has been reported previously (42).

The proton-pumping activities of Delta fmc1 mitochondria were probed by fluorescence quenching of rhodamine 123. The Fig. 4 shows the results obtained with mitochondria isolated from mutant and wild type cells grown at 37 °C. For the wild type, the addition of ethanol produced a fluorescence quenching of the dye, which was transiently decreased by adding 50 µM ADP, thus reflecting an electrogenic exchange of internal ATP against external ADP and a proton influx through the Fo during phosphorylation of the added ADP. By contrast, with mutant mitochondria, although ethanol was still able to energize the membrane, a subsequent addition of ADP could not substantially decrease the membrane potential (Delta Psi ). Changes in Delta Psi mediated by the ATPase proton-pumping activity were analyzed after energizing mitochondria by ethanol, an activation step necessary to remove the natural inhibitory peptide (IF1) of the mitochondrial ATPase, which would otherwise inhibit ATPase activity (43). As expected, with wild type mitochondria, inhibition by KCN of the proton pumping by the respiratory chain resulted in a collapse of Delta Psi , and subsequent addition of ATP promoted a fluorescence quenching of the dye that was DCCD-sensitive. With mutant mitochondria, an addition of ATP after collapsing Delta Psi by KCN promoted a lower increase in Delta Psi that was almost insensitive to DCCD.



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Fig. 4.   Proton-pumping activities of mitochondria. Proton-pumping activities were monitored by fluorescence-quenching of rhodamine 123 with intact mitochondria isolated from wild type FMC1+ (strain MC1) and mutant Delta fmc1 (strain MC6) grown in galactose (YPGALA) at 37 °C for 6-8 generations. The additions were 0.3 mg of mitochondrial proteins (mito), 10 µl of ethanol (EtOH), 50 µM ADP, 3 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 6 µg of DCCD, and 200 µM KCN; 1 mM ATP was added 1 min after KCN.

Taken together, the results of these different analyses show that oxidative phosphorylation is severely affected at elevated temperatures when the FMC1 gene is absent, with the most dramatic consequences seen at the level of the ATP synthase.

FMC1 Is Required for the Assembly or Stability of the ATP Synthase at Elevated Temperatures-- As described above, the respiratory growth defect of the Delta fmc1 strain is probably due to a failure in mitochondrial ATP synthesis. To determine whether this was caused by a block in the assembly of the ATP synthase, we examined the steady state levels of several subunits of this enzyme in the mutant grown at elevated temperatures. This was done either by Western blot analysis of whole mitochondria or by silver staining of mitochondrial proteins extracted with a mixture of chloroform and methanol (Fig. 5, A and B). The levels of the F1-alpha and F1-beta proteins were apparently not affected by the absence of Fmc1p. By contrast, the amounts of all the other subunits tested (gamma , delta , su.4, su.d, oligomycin sensitivity-conferring protein (OSCP), ATP6, ATP8, ATP9, su.i, su.g, and su.f) were strongly reduced in the mutant. Since synthesis of the mitochondrially encoded ATP6 is normal in the mutant (see above), the near complete absence of this protein at the steady state is probably due to proteolytic degradation. A higher susceptibility to proteolysis is probably also responsible for the virtual absence of the two other subunits of mitochondrial origin (ATP9 and ATP8), although these two proteins may be synthesized at a slower rate (see above). We do not know whether the missing subunits of nuclear origin are synthesized in the Delta fmc1 mutant, but it is reasonable to assume that their absence also results from proteolytic degradation after to a block in the assembly of the enzyme.



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Fig. 5.   Delta fmc1 cells grown by fermentation at 37 °C exhibit severe structural defects at the level of the ATP synthase. Mitochondria were isolated from wild type FMC1+ (strain MC1) and mutant Delta fmc1 (strain MC6) grown in galactose (YPGALA) for 6-8 generations at 37 °C. Panel A, SDS-PAGE and Western blot of mitochondria with specific antibodies against the indicated subunits of the ATP synthase. Panel B, the mitochondria were treated with a 1:1 mixture of chloroform:methanol according to the procedure of Michon et al. (39). The proteins of the organic phase were separated on a 15% SDS-PAGE and silver-stained. Panel C, the mitochondria were sonicated and then centrifuged through a discontinuous sucrose gradient as described by Ackerman and Tzagoloff (17). 15 µl of each fraction were analyzed by SDS-PAGE and Western blot with antibodies against the alpha -F1, beta -F1, and Aac2p proteins. OSCP, oligomycin sensitivity-conferring protein.

It has been shown previously that in the absence of either Atp12p or Atp11p, two chaperones specifically involved in the assembly of the F1 sector of the ATP synthase, the F1-alpha and F1-beta proteins accumulate normally, but instead of being incorporated into a functional F1 oligomer, they form large aggregates in the mitochondrial matrix (17). Interestingly, a significant portion of the F1-alpha and F1-beta proteins also behaved as large protein aggregates in the Delta fmc1 mutant, as shown by sucrose gradient analysis (Fig. 5C).

Immunoprecipitation with antibodies against the F1-alpha protein were made to see whether this protein was associated with the remaining ATP synthase subunits in the Delta fmc1 mutant. The immunoprecipitates obtained from mitochondria isolated from the mutant grown at 37 °C contained only the F1-alpha and F1-beta subunits (not shown). This indicates that these two proteins are part of an entity that is not, or is poorly, associated with the remaining subunits of the enzyme.

Fmc1p Is a Soluble Mitochondrial Protein-- An analysis of the 155-amino acid (18,352 Da) sequence deduced from FMC1 using the P-sort program of Nakai and Kanehisa (44) indicates that Fmc1p is a mitochondrial protein. This prediction was confirmed with the use of antibodies raised against a 14-amino acid peptide corresponding to the nucleotide sequence of FMC1 between positions 390 and 432. A Western blot analysis of mitochondrial proteins isolated from wild type cells using these antibodies produced a specific 14-kDa signal that could not be detected in the Delta fmc1 mutant (Fig. 6A). The apparent size of the protein indicates that Fmc1p is synthesized as a precursor containing an amino-terminal presequence of ~4 kDa. After an osmotic disruption of both the outer and inner mitochondrial membranes in the presence of carbonate, the protein was recovered in a water-soluble form (Fig. 6B). After disruption of just the outer membrane by a mild osmotic treatment of mitochondria, a portion of the immunological signal was recovered in the mitoplast fraction and preserved after treatment of the mitoplasts with proteinase K (the recovery of a part of the signal in the supernatant fraction is probably due to partial damage of the mitoplasts) (Fig. 6C). These data indicate that Fmc1p is a protein of the mitochondrial matrix, either free or loosely bound to the inner face of the inner mitochondrial membrane.



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Fig. 6.   Localization of Fmc1p. Panel A, SDS-PAGE and Western blot analysis of total mitochondrial proteins from wild type FMC1+ and mutant Delta fmc1 with antibodies against Fmc1p. Panel B, intact wild type mitochondria (300 µg) were disrupted osmotically in the presence of carbonate as described by Rouillard et al. (30). One-tenth of the pellet (lane 2) and supernatant (lane 3) and 20 µg of proteins from intact mitochondria (lane 1) were analyzed by SDS-PAGE and Western blot with antibodies against Fmc1p, Aac2p, and cytochrome b2 (Cyt b2). Panel C, mitoplasts were prepared from fresh wild type mitochondria (4 mg) as described by Daum et al. (41). The pellet and supernatant fractions were each divided in two parts. One part of each fraction was treated with 20 µg/ml proteinase K at 4 °C for 15 min. Equivalent amounts of proteins from the four samples were analyzed by SDS-PAGE and Western blot with the indicated antibodies. Lane 1, mitoplasts; lane 2, supernatant; lane 3, mitoplasts plus proteinase K; lane 4, supernatant plus proteinase K.

The Delta fmc1 Mutant Can Be Rescued by Increasing the Copy Number of ATP12-- The results described above show that Fmc1p is a mitochondrial protein needed at elevated temperatures for the assembly or stability of the F1 sector of the ATP synthase. To gain more insight into its function, we decided to determine whether the loss of Fmc1p can be compensated for by overexpressing another yeast protein(s), a current approach to identifying proteins with related cellular functions. To this end, the Delta fmc1 mutant was transformed with a yeast wild type genomic library in a high copy number vector, and the resulting transformants were tested for their growth on glycerol at 37 °C. In that way, we found that overexpression of Atp12p efficiently rescued the Delta fmc1 phenotype (Fig. 7).



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Fig. 7.   The Delta fmc1 mutant is rescued by overexpressing Atp12p. The mutant Delta fmc1 (strain MC6) was transformed with pEMBLYe23 (2-µm empty plasmid), pLL1 (FMC1 in CEN plasmid pRS316), pLL5 (ATP12 in pRS316), and pLE10 (ATP12 in pEMBLYe23). Pregrown transformed cells were diluted, and 5 µl of each dilution were spotted on glucose (YPGA) and ethanol (N1) media. The plates were photographed after 3 days of incubation at 37 °C.

Somewhat surprisingly, Delta fmc1 cells transformed with ATP12 on a low copy number vector also show a nearly wild type growth on glycerol at 37 °C. Thus, a small increase in the expression of Atp12p may be apparently sufficient to overcome the absence of Fmc1p at elevated temperatures. However, in vitro, the F1-ATPase activity was found to be restored only partially (50% in comparison to the wild type control; data not shown). By contrast, this activity was fully restored with ATP12 on a high copy number vector. It is well known that a substantial decrease of the ATP synthase activity (up to 80%) has only marginal effects on the growth of yeast on non-fermentable carbon sources at temperatures above 20 °C (45). This could explain that despite their reduced F1-ATPase activity, Delta fmc1 cells transformed with ATP12 on a low copy number vector have a wild type growth on glycerol at 37 °C.

These results are particularly interesting. First, given the very specific action of Atp12p in the assembly of the F1 oligomer (16, 17), they strongly suggest that Fmc1p has a function also confined to this process. Second, because the Delta fmc1 mutant exhibits at elevated temperatures all the characteristics of the Delta atp12 mutant and because the absence of Fmc1p can be overcome by increasing the production of Atp12p, a logical view is that the function of Atp12p is impaired at elevated temperatures when Fmc1p is missing. Consistent with this, we found that the abundance of Atp12p was significantly reduced in mitochondria isolated from Delta fmc1 cells cultivated at 37 °C (Fig. 8). We have also shown that the multi-copy suppressor relationship between FMC1 and ATP12 is not reciprocal, as the Delta atp12 mutant remained unable to grow on glycerol after transformation with FMC1 cloned in a high copy number vector. Altogether these data suggest that Fmc1p may be required for the formation/stability or functioning of Atp12p at elevated temperatures.



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Fig. 8.   The level of Atp12p is decreased in the mutant Delta fmc1. Mitochondria of wild type FMC1+, and mutant Delta fmc1 were isolated from cells grown at 37 °C in galactose (YPGALA) for 6-8 generations. 70 µg of proteins from these mitochondria were analyzed by SDS-PAGE and Western blot with the indicated antibodies.

Since Atp11p cooperates with Atp12p in the assembly of the F1 oligomer (15-17), we decided to determine directly whether Atp11p could, like ATP12, rescue the Delta fmc1 mutant. ATP11 may have been poorly represented in the library we used and, hence, not isolated in the search of multicopy suppressors. We therefore constructed a high copy number vector containing ATP11. After transformation with this plasmid, the Delta fmc1 mutant remained unable to grow on glycerol at 37 °C (not shown). Also, we found that the Delta atp11 mutant was not rescued by overexpression of Fmc1p. Thus, contrary to Atp12p, it seems that the proper folding/stability or functioning of Atp11p at elevated temperatures does not require the presence of Fmc1p.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have identified a novel nuclear-encoded yeast mitochondrial protein and showed that its presence is required for the assembly/stability of the F1 sector of the ATP synthase in heat stress conditions. In its absence and at elevated temperatures, the alpha -F1 and beta -F1 proteins are synthesized, transported, and processed to their mature size, but instead of being incorporated into a functional F1 oligomer, they form large aggregates in the mitochondrial matrix. Identical defects in the assembly of the F1 oligomer were observed previously for yeast cells lacking either Atp12p or Atp11p (17). However, unlike Fmc1p, these two latter proteins are required for the formation of the F1 oligomer not only at elevated temperatures but also in normal growth conditions.

The oxygen consumption rate of mitochondria isolated from the Delta fmc1 mutant grown at elevated temperatures was strongly reduced (by 70%) in comparison to the wild type. This is probably a secondary consequence of the defect in the assembly of the F1 oligomer. Indeed a decreased respiratory activity was also seen for strains carrying null mutations in the genes of alpha -F1, beta -F1 (8, 9, 46), Atp11p, and Atp12p (17).

Interestingly, the Delta fmc1 mutant recovered the ability to assemble the F1 oligomer at elevated temperatures by increasing the expression of Atp12p, suggesting that the function of Atp12p may be compromised at elevated temperatures when Fmc1p is lacking. Consistent with this, the steady state level of Atp12p was found to be substantially reduced in mitochondria isolated from Delta fmc1 cells grown at elevated temperatures. Based on these observations, a reasonable hypothesis is that Fmc1p helps the folding/stability or functioning of Atp12p at elevated temperatures.

The molecular mass of native Atp12p, estimated from its sedimentation properties in sucrose gradients, is at least twice as great as that of the monomer (47). Chemical modifications and two-hybrid genetic studies argue against the formation of oligomers of Atp12p (48). Furthermore, in a strain unable to express Atp11p, the native size of Atp12p was not found to be modified, indicating that Atp11p and Atp12p are not part of the same complex (49). Based on the results reported here, it will be particularly interesting to see whether Fmc1p belongs to or is required for the formation of the Atp12 oligomer. Experiments to resolve this question are in progress.

Atp12p and Atp11p interact directly with the F1-alpha and F1-beta proteins, respectively, and this is presumed to facilitate the formation of alpha beta heterodimers by protecting these two subunits of the ATP synthase from non-productive interactions (15, 16). If Fmc1p actually mediates the alpha beta dimerization by assisting Atp12p at elevated temperatures, how can we explain that antibodies against the F1-alpha protein coimmunoprecipitated the F1-beta protein in mitochondrial extracts of the Delta fmc1 mutant. As will be presented elsewhere, the F1-alpha and F1-beta proteins were found by immunocytochemistry to be part of the same inclusion bodies in mitochondria of the Delta fmc1 mutant.3 Thus, even when they fail to interact properly, the F1-alpha and F1-beta proteins still remained associated physically, which could explain their coimmunoprecipitation from the Delta fmc1 mutant.

Severe perturbations at the level of the Fo sector of the ATP synthase were seen in the Delta fmc1 mutant. It is not known whether or not the Fo sector is assembled in the Delta atp11 and Delta atp12 mutants. We believe that Fmc1p is not directly required for the assembly of this part of the enzyme. Rather the perturbations at the level of the Fo sector in the Delta fmc1 mutant were probably a secondary consequence of the defect in the assembly of the F1 sector. Indeed as in the Delta fmc1 mutant, the three mitochondrially encoded subunits of the ATP synthase, which are all essential components of the Fo sector, failed to accumulate in a mutant carrying a null mutation in the F1-beta protein gene (42). Thus, whereas the F1 sector can assemble independently of the Fo sector, for example in yeast cells lacking mtDNA, it appears that the formation or stability of the Fo sector may depend on the presence of an assembled F1 sector. A sequential assembly of the F1 and Fo sectors could be of critical importance in preventing the depolarization of the inner mitochondrial membrane during the assembly of the ATP synthase complex.

At 37 °C, the growth of the Delta fmc1 mutant on glucose was severely affected when it lacked wild type mitochondrial DNA. This phenotype is most likely related to the defect in the assembly of the F1 oligomer. Indeed, null mutations in the genes of alpha -F1, beta -F1, Atp12p, and Atp11p also impair the growth of yeast lacking mtDNA (for review, see Ref. 50). The basis of the so-called "petite negativity" conferred by mutations affecting the F1 sector is not totally understood, but evidence suggest that in the absence of a functional respiratory chain, the ATP hydrolytic activity of the F1 sector would be needed for a correct polarization of the inner mitochondrial membrane and, hence, for the biogenesis of mitochondria (50, 51).

Taken together, the data reported in this study suggest that Fmc1p functions only at elevated temperatures. If this is true, one might expect that the protein would only be expressed in such conditions. However, this was not found to be case (accumulation of the mRNA and the protein are about the same at 28-30 °C and 37 °C, data not shown). There are cases known where a protein whose accumulation is not influenced by temperature plays a critical role in heat stress conditions. For example, subunit 6 of yeast complex III is dispensable at 28 °C, whereas at 37 °C, its absence has dramatic consequences on the assembly/stability of this complex (52). Thus it is possible that Fmc1p is involved in the assembly/stability of the F1-ATPase at all temperatures but that this role becomes critical only in heat stress conditions.


    ACKNOWLEDGEMENTS

We are very grateful to A. Tzagoloff for the gift of Delta atp12 and Delta atp11 strains and antibodies against Atp12p and to G. Lauquin for antibodies against Aac2p. We thank A. Tzagoloff and our colleagues B. Guérin, M. Rigoulet, G. Dujardin, C. Herbert, O. Groudinski, M.-F. Giraud, and D. Brèthes for their interest and helpful discussions. We are very grateful to C. Herbert for a critical reading of the manuscript and English revisions. We are grateful to X. Grandier-Vazeille for help in preparing anti-Fmc1p antibodies.


    FOOTNOTES

* This work was supported by the CNRS, the Conseil Régional de la Région Aquitaine, the Fondation pour la Recherche Médicale, and Groupement de Recherches et d'Etudes sur les Génomes Grant 8/95 (to J.-P. dR.).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.

Dagger Recipient of a fellowship from the French Ministère de la Recherche et de l'Enseignement.

To whom correspondence should be addressed: Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 1 Rue Camille Saint-Saëns, 33077 Bordeaux cedex, France. Tel.: 33 5 56 99 90 43; Fax: 33 5 56 90 99 51; E-mail: jp.dirago@ibgc.u-bordeaux2.fr.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009557200

2 A petite designates a mutant yeast cell that fails to grow on a non-fermentable carbon source (glycerol or ethanol). On a 2% glycerolmedium containing low amounts of glucose (0.1%), such cells can makeonly small size colonies and are therefore called petites.

3 B. Coulary, L. Lefebvre-Legendre, D. Savouré, J. Schaëffer, and J.-P. di Rago, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; Fo and F1, integral membrane and peripheral portions of ATP synthase; Delta Psi , transmembrane electrical potential; DCCD, dicyclohexylcarbodiimide; PAGE, polyacrylamide gel electrophoresis; mtDNA, mitochondrial DNA.


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