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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 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 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 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 The 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 [ 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 ( Production of anti-Fmc1p Antibodies
Anti-Fmc1p antibodies were prepared by Eurogentec (Seraing,
Belgium) with the synthetic peptide NH2-YNPGNKLTQDEK as an immunogen.
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 -F1 and
-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3
3
(3, 4).
The three-dimensional structures of F1 from bovine heart
(5), rat liver (6) and yeast (7) show that the
- and
-subunits
alternate in a hexagonal array with a central cavity occupied by the
amino and carboxyl termini of the
-subunit. The interfaces between
the
- and
-subunits form three catalytic and three noncatalytic
nucleotide binding sites.
-F1 and
-F1
proteins, respectively (15, 16). In yeast strains lacking either Atp11p
or Atp12p, the
-F1 and
-F1 proteins
aggregate in the mitochondrial matrix (17). Thus it is believed that
Atp12p and Atp11p facilitate the formation of
heterodimers by
protecting these two F1 subunits from non-productive interactions (16).
-F1 and
-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
fmc1
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
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.
-32P]dCTP using the nick
translation kit from Roche Molecular Biochemicals.
) 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
-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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
fmc1/+ cells (not shown); and (iii) after transformation
with a plasmid containing FMC1 (pLL1),
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.
View larger version (59K):
[in a new window]
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 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 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.
|
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
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
fmc1 mutation.
|
When the 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
fmc1 petites grow poorly at 37 °C, preventing them
from completely taking over the cultures. To test this,
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
fmc1
rho°, cells transformed with the empty vector was severely
affected compared with that of the
fmc1 rho° cells
transformed with the cloned FMC1 gene (Fig.
2). Consistent with this, we found that
rho+
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.
|
Next we analyzed the expression of the mtDNA in the 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
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).
|
Respiratory, ATPase, and Proton-Pumping Activities of 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
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.
|
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, 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 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 (
). Changes in
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
, 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
by KCN promoted a lower increase in
that
was almost insensitive to DCCD.
|
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 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-
and F1-
proteins were apparently not affected
by the absence of Fmc1p. By contrast, the amounts of all the other subunits tested (
,
, 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
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.
|
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- and
F1-
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-
and F1-
proteins also behaved as large protein aggregates in the
fmc1 mutant, as shown by sucrose gradient analysis (Fig. 5C).
Immunoprecipitation with antibodies against the F1-
protein were made to see whether this protein was associated with the remaining ATP synthase subunits in the
fmc1 mutant. The
immunoprecipitates obtained from mitochondria isolated from the mutant
grown at 37 °C contained only the F1-
and F1-
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 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.
|
The 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
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
fmc1
phenotype (Fig. 7).
|
Somewhat surprisingly, 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,
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 fmc1 mutant exhibits at elevated temperatures all the characteristics of the
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
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
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.
|
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 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
fmc1 mutant remained unable to grow on glycerol at
37 °C (not shown). Also, we found that the
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -F1 and
-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
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
-F1,
-F1 (8, 9, 46), Atp11p, and Atp12p
(17).
Interestingly, the 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
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- and
F1-
proteins, respectively, and this is presumed to
facilitate the formation of
heterodimers by protecting these two
subunits of the ATP synthase from non-productive interactions (15, 16). If Fmc1p actually mediates the
dimerization by assisting Atp12p at elevated temperatures, how can we explain that antibodies against the F1-
protein coimmunoprecipitated the
F1-
protein in mitochondrial extracts of the
fmc1 mutant. As will be presented elsewhere, the
F1-
and F1-
proteins were found by
immunocytochemistry to be part of the same inclusion bodies in
mitochondria of the
fmc1 mutant.3 Thus, even when
they fail to interact properly, the F1-
and F1-
proteins still remained associated physically, which
could explain their coimmunoprecipitation from the
fmc1 mutant.
Severe perturbations at the level of the Fo sector of the
ATP synthase were seen in the fmc1 mutant. It is not
known whether or not the Fo sector is assembled in the
atp11 and
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
fmc1 mutant were probably a secondary consequence of the defect in the assembly of the F1 sector.
Indeed as in the
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-
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 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
-F1,
-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 atp12 and
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.
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;
, transmembrane
electrical potential;
DCCD, dicyclohexylcarbodiimide;
PAGE, polyacrylamide gel electrophoresis;
mtDNA, mitochondrial DNA.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Pedersen, P. L. (1996) J. Bioenerg. Biomembr. 28, 389-395[Medline] [Order article via Infotrieve] |
2. | Weber, J., and Senior, A. E. (1997) Biochim. Biophys. Acta 1319, 19-58[Medline] [Order article via Infotrieve] |
3. | Penefsky, H. S., and Cross, R. L. (1991) Adv. Enzymol. 64, 173-214[Medline] [Order article via Infotrieve] |
4. | Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215-250[Medline] [Order article via Infotrieve] |
5. | Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628[CrossRef][Medline] [Order article via Infotrieve] |
6. | Bianchet, M., Medjahed, S., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1994) Biochim. Biophys. Acta 1187, 163-164[Medline] [Order article via Infotrieve] |
7. |
Stock, D.,
Leslie, A. G.,
and Walker, J. E.
(1999)
Science
286,
1700-1705 |
8. |
Takeda, M.,
Vassarotti, A.,
and Douglas, M. G.
(1985)
J. Biol. Chem.
260,
15458-15465 |
9. |
Takeda, M.,
Chen, W. J.,
Salzgaber, J.,
and Douglas, M. G.
(1986)
J. Biol. Chem.
261,
15126-15133 |
10. |
Guélin, E.,
Chevallier, J.,
Rigoulet, M.,
Guérin, B.,
and Velours, J.
(1993)
J. Biol. Chem.
268,
161-167 |
11. | Giraud, M.-F., and Velours, J. (1994) Eur. J. Biochem. 222, 851-859[Abstract] |
12. |
Paul, M. F.,
Ackerman, S.,
Yue, J.,
Arselin, G.,
Velours, J.,
and Tzagoloff, A.
(1994)
J. Biol. Chem.
269,
26158-26164 |
13. |
Tokatlidis, K.,
and Schatz, G.
(1999)
J. Biol. Chem.
274,
35285-35288 |
14. | Hendrick, J. P., and Hartl, F.-U. (1993) Annu. Rev. Biochem. 62, 349-384[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Wang, Z.-G.,
and Ackerman, S. H.
(2000)
J. Biol. Chem.
275,
5767-5772 |
16. |
Wang, Z.-G.,
Sheluho, D.,
Gatti, D. L.,
and Ackerman, S. H.
(2000)
EMBO J.
19,
1486-1493 |
17. | Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986-4990[Abstract] |
18. | Wach, A. (1996) Yeast 12, 259-265[CrossRef][Medline] [Order article via Infotrieve] |
19. | Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330[Medline] [Order article via Infotrieve] |
20. |
Sikorski, R. S.,
and Hieter, P.
(1989)
Genetics
122,
19-27 |
21. | Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
22. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
23. | Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425-1426[Medline] [Order article via Infotrieve] |
24. | Hoffman, C. S., and Winston, F. (1987) Gene 57, 267-272[CrossRef][Medline] [Order article via Infotrieve] |
25. | di Rago, J. P., Netter, P., and Slonimski, P. P. (1990) J. Biol. Chem. 265, 3323-3339 |
26. | Jacquier, A., and Dujon, B. (1983) Mol. Gen. Genet. 192, 487-499[Medline] [Order article via Infotrieve] |
27. | Labouesse, M., and Slonimski, P. P. (1983) EMBO J. 2, 269-276[Medline] [Order article via Infotrieve] |
28. | Szczepanek, T., and Lazowska, J. (1996) EMBO J. 15, 3758-3767[Abstract] |
29. | Guérin, B., Labbe, P., and Somlo, M. (1979) Methods Enzymol. 55, 149-159[Medline] [Order article via Infotrieve] |
30. | Rouillard, J. M., Dufour, M. E., Theunissen, B., Mandart, E., Dujardin, G., and Lacroute, F. (1996) Mol. Gen. Genet. 252, 700-708[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 |
32. | Somlo, M. (1968) Eur. J. Biochem. 5, 276-284[Medline] [Order article via Infotrieve] |
33. | Rigoulet, M., and Guérin, B. (1979) FEBS Lett. 102, 18-22[CrossRef][Medline] [Order article via Infotrieve] |
34. | Emaus, R. K., Grunwald, R., and Lemasters, J. J. (1986) Biochim. Biophys. Acta 850, 436-448[Medline] [Order article via Infotrieve] |
35. |
Todd, R. D.,
Griesenbeck, T. A.,
and Douglas, M. G.
(1980)
J. Biol. Chem.
255,
5461-5467 |
36. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
37. | Ansorge, W. (1983) in Electrophoresis; 82 (Stathakos, D., ed) , pp. 235-242, Walter de Gruyter & Co., Berlin |
38. |
Arselin, G.,
Vaillier, J.,
Graves, P. V.,
and Velours, J.
(1996)
J. Biol. Chem.
271,
20284-20290 |
39. | Michon, T., Galante, M., and Velours, J. (1988) Eur. J. Biochem. 172, 621-625[Abstract] |
40. | Claisse, M., Slonimski, P. P., Johnston, J., and Malher, H. R. (1980) Mol. Gen. Genet. 177, 375-387[Medline] [Order article via Infotrieve] |
41. |
Daum, G.,
Böhni, P. C.,
and Schatz, G.
(1982)
J. Biol. Chem.
257,
13028-13033 |
42. | Paul, M. F (1992) Disruption et Mutagénèse du Gène ATP4, Codant pour une Sous-Unité du Secteur fo de l'ATP Synthase Mitochondriale de Saccharomyces cerevisiae. Ph.D. thesis , Université de Bordeaux II |
43. |
Vaillier, J.,
Arselin, G.,
Graves, P. V.,
Camougrand, N.,
and Velours, J.
(1999)
J. Biol. Chem.
274,
543-548 |
44. | Nakai, K., and Kanehisa, M. (1992) Genomics 14, 897-911[Medline] [Order article via Infotrieve] |
45. | Mukhopadhyay, A., Uh, M., and Mueller, D. M. (1994) FEBS Lett. 343, 160-164[CrossRef][Medline] [Order article via Infotrieve] |
46. | Tzagoloff, A., Akai, A., and Needleman, R. (1975) J. Biol. Chem. 250, 8228-8235[Abstract] |
47. |
Bowman, S.,
Ackerman, S. H.,
Griffiths, D. E.,
and Tzagoloff, A.
(1991)
J. Biol. Chem.
266,
7517-7523 |
48. |
Wang, Z. G.,
and Ackerman, S. H.
(1998)
J. Biol. Chem.
273,
2993-3002 |
49. |
Ackerman, S. H.,
Martin, J.,
and Tzagoloff, A.
(1992)
J. Biol. Chem.
267,
7386-7394 |
50. | Chen, X. J., and Clark-Walker, G. D. (1999) Int. Rev. Cytol. 194, 197-238 |
51. | Giraud, M. F., and Velours, J. (1997) Eur. J. Biochem. 245, 813-818[Abstract] |
52. |
Yang, M.,
and Trumpower, B. L.
(1994)
J. Biol. Chem.
269,
1270-1275 |