From the Institut de Biochimie et Génétique Cellulaires du CNRS, Université Victor Ségalen, Bordeaux 2, 33077 Bordeaux cedex, France
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ABSTRACT |
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Two subunits of the yeast ATP synthase have been
isolated. Subunit e was found loosely associated to the complex. Triton
X-100 at a 1% concentration removed this subunit from the ATP
synthase. The N-terminal sequencing of subunit i has been performed.
The data are in agreement with the sequence of the predicted product of
a DNA fragment of Saccharomyces cerevisiae chromosome XIII. The ATP18 gene encodes subunit i, which is 59 amino acids
long and corresponds to a calculated mass of 6687 Da. Its pI is 9.73. It is an amphiphilic protein having a hydrophobic N-terminal part and a
hydrophilic C-terminal part. It is not apparently related to any
subunit described in other ATP synthases. The null mutant showed low
growth on nonfermentable medium. Mutant mitochondria display a low
ADP/O ratio and a decrease with time in proton pumping after ATP
addition. Subunit i is associated with the complex; it is not a
structural component of the enzyme but rather is involved in the
oxidative phosphorylations. Similar amounts of ATP synthase were
measured for wild-type and null mutant mitochondria. Because 2-fold
less specific ATPase activity was measured for the null mutant than for
the wild-type mitochondria, we make the hypothesis that the observed
decrease in the turnover of the mutant enzyme could be linked to a
proton translocation defect through F0.
The mitochondrial ATP synthase is the major enzyme responsible for
aerobic synthesis of ATP. ATP synthase exhibits a tripartite structure
consisting of a head piece (the catalytic sector), a base piece (the
membrane sector), and a connecting stalk. However, the enzyme can be
resolved into two parts: the first part is the catalytic sector called
F1, which is a water-soluble entity, composed of subunits
The E. coli ATP synthase and the bovine enzyme contain 8 and
16 different types of subunits, respectively (9). In the case of
Saccharomyces cerevisiae, the ATP synthase is composed of at least 13 different kinds of subunits involved in the structure of the
enzyme (10); the disruption of each of their structural genes leads to
a lack of assembly of the complex. Additional subunits are involved in
the regulation of the catalytic sector, such as the natural inhibitory
peptide of the mitochondrial ATPase (11) and stabilizing factors in
yeast (12, 13). Other additional subunits (e-g), which have been
identified in the beef enzyme (9, 14) and found to be associated with
the membranous sector (15), have their homologue in the yeast enzyme.
With the exception of subunit f (16), the two other components, e and
g, appear as being not essential for the structure of the mitochondrial enzyme, because their respective gene disruption does not significantly alter yeast growth on nonfermentable carbon sources (17-19). In this
paper, we describe a new component of the yeast ATP synthase, the gene
disruption of which leads to a decrease in oxidative phosphorylation yield.
Biochemical Procedures--
The S. cerevisiae strain
D273-10B/A/H/U (MAT Purification of the ATP Synthase--
Immunoprecipitated samples
of ATP synthase were prepared from 2 mg of mitochondrial proteins as
described by Todd et al. (28). Polyclonal antibodies raised
against the Purification of Subunits e and i--
The ATP synthase was
concentrated by the centrifugation method described above, and the
pellet was dissolved in 6 M guanidinium chloride, 0.1 M NaCl, 0.05 M Tris-HCl, pH 8.0. After
centrifugation at 100,000 × g for 20 min, the
supernatant was submitted to gel permeation chromatography on a
Superdex 75 (HR 10/30) column (Amersham Pharmacia Biotech) eluted with
the same buffer at a flow rate of 0.2 ml/min. Protein-containing
fractions of relative molecular masses of 10 and 5 kDa were immediately
submitted to reverse phase HPLC on a Vydac C18 column (15 × 0.46 cm). Elution was done by a linear acetonitrile gradient (24-71%)
containing 0.1% trifluoroacetic acid that was developed for 40 min.
Protein-containing fractions were dried under vacuum and stored at
Other Biochemical Procedures--
SDS-PAGE was performed
according to the method of Schägger and Von Jagow (30). The slab
gel was then silver-stained according to the method of Ansorge (31).
Western blot analyses were performed as described previously (29). The
polyclonal antibodies anti-TIM11 raised against subunit e (18) were
kindly provided by Dr. Neupert (Institut für Physiologishe Chemie
der Universität Munchen, Munchen, Germany). ProBlott membranes
were incubated with peroxidase-labeled antibodies and revealed with the
ECL reagent of Amersham Pharmacia Biotech. Automated sequence analyses
were performed with an Applied Biosystems model 491 liquid phase
sequanator. The electrospray mass spectrum of the intact subunit i was
obtained on an Autospec mass spectrometer (Fisons VG-Analytical) fitted
with an electrospray source. Analysis conditions were described
previously (16). Proteolytic cleavage and peptide separation were as
described by Arselin et al. (32).
Cloning and Sequencing of the ATP18 Gene--
The yeast gene
ATP18 was amplified by polymerase chain reaction from yeast
genomic DNA. Two oligonucleotides, 5'-GAAGTCCTTTGAATTCTTCCG-3' (104,804-104,784, Crick strand, chromosome XIII) and
5'-CATCAGGATCCAACTACAGCA-3' (103,388-103,408, Watson strand,
chromosome XIII) were used. The 5' oligonucleotide contained an
EcoRI site. A BamHI site was created by the 3'
oligonucleotide. Polymerase chain reaction amplification resulted in a
1402-bp EcoRI-BamHI DNA fragment that was
inserted into an EcoRI-BamHI-cleaved pUC18
vector. After cloning, the 1402-bp EcoRI-BamHI
DNA fragment was inserted into an M13 mp9 vector for sequencing as
described by Sanger et al. (33). The oligonucleotide 5'-CATACGACGACAGATTAATTG-3' ( Disruption of the ATP18 Gene--
The pUC18 vector containing
the ATP18 gene was cleaved by EcoRV and
BglII. A 439-bp DNA fragment containing the ATP18
gene was replaced by an 1100-bp BglII URA3 fragment (34).
The resulting construct was cleaved by endonucleases EcoRI
and BamHI. This led to a 2465-bp DNA fragment, which was
used to transform the wild-type strain by using the lithium chloride
method (35). Transformants were selected on minimal medium,
supplemented with methionine and histidine and 2% glucose as a carbon
source. DNA of these clones was amplified by polymerase chain reaction
and checked for disruption.
Construction of the Complemented Strain--
The 1402-bp
EcoRI-BamHI DNA fragment bearing the wild-type
ATP18 gene was inserted into the polylinker of the low copy
shuttle vector pRS313. This construct was used to transform the deleted disrupted strain in the ATP18 gene. Transformants were
selected on minimal medium supplemented with methionine.
Identification of Supernumerary Subunits in the Yeast ATP Synthase
Complex--
The subunit e has been identified recently as a component
of the mitochondrial ATP synthase (14, 18). This protein was absent in
our previous preparations. The concentration of 1% (w/v) Triton X-100
that had been previously used to wash the yeast ATP synthase
immunoprecipitates was found to remove components migrating in SDS-PAGE
in the relative molecular mass range of 10 kDa. The same was true in
the case of our previous purification procedure for isolating large
amounts of enzyme. In the experimental procedure, there was an
ultrafiltration step, the aim of which was to concentrate the
solubilized ATP synthase (29). This step also increased the detergent
concentration and, as a consequence, removed loosely bound proteins.
Thus, to keep the Triton X-100 concentration at 0.1%, we modified the
purification procedure. To remove insoluble proteins, the crude 0.375%
Triton X-100 extract was centrifuged. The supernatant was then diluted
to obtain a detergent concentration of 0.1%, and centrifugation at
300,000 × g for 5 h made it possible to pellet
the complex. Finally, the pellet was dissolved in a buffer containing
0.1% Triton X-100, and the ATP synthase was purified by molecular
sieving and ion exchange chromatography (29). Purification of
supernumerary subunits was achieved by two chromatographic steps. Gel
permeation chromatography under denaturating conditions led to two well
separated peaks containing proteins of relative molecular masses of 5 and 10 kDa (Fig. 1A). Reverse
phase HPLC of the 10-kDa fraction made it possible to separate three
components (Fig. 1B), which were analyzed by the Tricine-SDS-PAGE of Schägger and Von Jagow (30); this procedure allows an efficient separation of proteins with molecular masses that
are in the range of 3-10 kDa. By contrast with what was observed with
the Laemmli method (36), subunits 4 and 6 were not resolved, and the
Reverse phase HPLC of the 5-kDa Superdex fraction gave two main peaks
(Fig. 1C). The first peak was identified as the
From these data, a 1402-bp DNA fragment was amplified by polymerase
chain reaction from our wild-type yeast strain D273-10B/A/H/U, cloned
in a pUC18 vector, and sequenced. A 177-bp DNA sequence encoding for a
protein containing 59 amino acid residues was identified and named
ATP18 (Fig. 3). The molecular mass of the predicted protein is 6687.5 Da. This value is in agreement with the measured mass of 6687.1 ± 2 Da (data not shown) obtained from the electrospray mass spectrum of
subunit i. Therefore, subunit i is devoid of a cleavable leader
sequence. Sequencing showed that the first amino acid residue of
subunit i was a methionine. Because there is no other initiating codon
upstream from the gene, and because a stop codon is present at Phenotypic Analysis of the Null Mutant
Wild-type and null mutant mitochondria were prepared by the protoplast
method. The uncoupled respiration rate of null mutant mitochondria with
NADH as substrate was 85% of that of the wild type (Table
II), which correlates with the decrease
in cytochrome oxidase amount measured in the cells. Although a similar
state 4 was obtained for both kinds of mitochondria, isolated null
mutant mitochondria displayed a low respiratory rate in the presence of
ADP (state 3), leading to a significant difference in respiratory control (Table II). The ADP/O ratio of ~1 calculated for the null mutant mitochondria indicates a lower efficiency of oxidative phosphorylations compared with the wild type.
Proton-pumping activities of the mitochondrial preparations were
measured in the same conditions as those used for the respiration rate
measurements (Fig. 4). In the presence of
ethanol, wild-type mitochondria displayed a fluorescent quenching of
rhodamine 123, which was transiently decreased with addition of 50 µM ADP, thus reflecting a decrease in the transmembranous
The oligomycin-sensitive ATPase activity of both of these two strains
was measured at pH 8.4 (25) either from freshly isolated mitochondria
or from frozen and thawed mitochondria (Table II). After freezing and
thawing of mitochondria, we have previously shown that there is a
2-fold increase in the wild-type mitochondrial ATPase activity (20). In
these experimental conditions, this ATPase activity increase
corresponds to the release of the natural inhibitory peptide of the
mitochondrial ATPase.3 Null
mutant mitochondria displayed a 30-37% oligomycin-insensitive ATPase
activity compared with a 9-13% for the wild type. It is clear that
under these experimental conditions, null mutant mitochondria showed an
uncoupling between F1 and F0. It is to be
pointed out that the mitochondrial ATPase activity was measured at pH
8.4, optimal conditions for the hydrolytic activity, but conditions that may render more deleterious the defect resulting from the absence
of subunit i. Whatever the experimental conditions used, the null
mutant mitochondrial ATPase activity was always 2-fold lesser than that
of the wild type. This lesser activity may represent a decrease in the
relative amount of the complex, a decrease in the turnover of the
enzyme, or both. As a consequence, a comparative analysis of enzyme
amounts was investigated by Western blot analysis of SDS-dissociated
mitochondria. Subunit 4, which is a component of the stalk, was
quantified. Comparison of the band intensity of subunit 4 as a function
of increasing mitochondrial membrane amounts showed that the amount of
enzyme was the same in each kind of mitochondria (Fig.
5). Thus, it is more likely that the turnover of the mutant enzyme is lowered, a finding corroborated by the
low ADP/O ratio and low ADP-stimulated respiration rate.
We report here that, as shown previously (18), subunit e is
associated with the yeast ATP synthase and that, in addition, this
subunit is easily removed from the complex by Triton X-100. Therefore,
this protein is not essential to the structure of the enzyme. Another
component of the ATP synthase, named subunit i, is also described. So
far, such an additional protein has not been identified in the beef
enzyme. The absence of subunit i in the null mutant does not alter the
structure or the assembly of the yeast ATP synthase. We have observed
that polyclonal antibodies raised against the Disruption of the ATP18 gene showed that this subunit is not essential
for oxidative phosphorylations. However, the efficiency of the
phosphorylating system is decreased with inactivation of the
ATP18 gene, and this probably originates from a reduced
turnover of the enzyme. It has been described that mutants in
F0 often display pleiotropic effects such as low oxygen
consumption and low cytochrome oxidase activity (40-44). The
experimental data give insight into a mechanistic defect that is
expressed during ATP synthase function rather than a deficiency caused
by a decrease in the cytochrome oxidase amount. On the basis of all the
data presented in this paper, we conclude that the null mutant ATP synthase, devoid of the supernumerary subunit i, shows in
vitro a defect in proton translocation through F0
during ATP synthesis and ATP hydrolysis. In vivo, this
alteration leads to a slow growth of mutant cells and the production of
a low amount of matter on nonfermentable carbon sources. Experiments
are in progress to elucidate the involvement of subunit i in the
activity of the ATP synthase, especially at the proton translocation
level, and to investigate the relationships of subunit i with other
known components of the yeast complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
,
,
,
, and
, which retains the ability to hydrolyze ATP
when in a soluble form; the second part is a detergent-soluble entity,
called F0, which is embedded in the membrane and is
composed of hydrophobic subunits forming a specific proton pathway. The connecting stalk is composed of elements of both F1 and
F0. When the two sectors are coupled, the enzyme functions
as a reversible H+-transporting ATPase or ATP synthase (1,
2). The model for energy coupling by F1F0 ATP
synthase that has gained the most general support is the binding change
mechanism (3). This concept has been strengthened by the crystal
structure of the major part of the bovine F1 (4). The
affinity changes for the substrates and products at the catalytic sites
are coupled to proton translocation via the rotation of subunits that
belong to F1 and F0 (for review, see Refs.
5-8). As a result, the Escherichia coli ATP synthase could
be a rotary motor with a rotor that is composed of subunits
and
and the dicyclohexylcarbodiimide binding protein oligomer, all other
subunits being parts of the stator.
EXPERIMENTAL PROCEDURES
, met6, ura3, his3; Ref.
20) was the wild-type strain. Cells were grown aerobically at 28 °C
in a complete liquid medium containing 2% lactate as a carbon source
(21) and harvested in logarithmic growth phase. Mitochondria were
prepared according to the methods of Lang et al., (22) and
Guérin et al., (23). Protein amounts were determined according to the method of Lowry et al., (24) in the
presence of 5% SDS using bovine serum albumin as standard. The
specific ATPase activity was measured at pH 8.4 according to the method of Somlo (25). Oxygen consumption rates were measured as described by
Rigoulet and Guérin (26). Variations of transmembrane potential (
) were evaluated by measurement of fluorescence quenching of rhodamine 123 with an SFM25 Kontron fluorescence spectrophotometer (27).
-subunit were added to the 100,000 × g
supernatant of the 0.375% Triton X-100 extract. The immunoprecipitated
materials were washed with increasing Triton X-100 concentrations. The
final pellet was dried under vacuum and then dissolved in 20 µl of
dissociation buffer devoid of reducing agent. A 10-µl aliquot of this
sample was analyzed by
SDS-PAGE.1 Large amounts of
ATP synthase were prepared according to a modification of a procedure
by Arselin et al. (29). To a known volume of a mitochondrial
suspension at 10 mg of protein/ml in 0.1 M Tris acetate, 1 mM ATP, 1 mM EDTA, pH 8.0, was added an equal
volume of 0.75% (w/v) Triton X-100. After a 20-min incubation time at 4 °C, the sample was centrifuged at 100,000 × g for
15 min at 4 °C, and the obtained supernatant was diluted with an
equal volume of the previous buffer and centrifuged at 300,000 × g for 5 h. The pellet dissolved in the minimal volume
of 20 mM Tris acetate, 65 mM sucrose, 1 mM EDTA, 0.1% Triton X-100, pH 7.5, was submitted to gel
permeation chromatography followed by an ion exchange chromatography (29).
20 °C.
37 to
17 upstream from the initiating codon) was used as primer.
RESULTS
-subunit migrated more slowly than subunit h (Fig. 1D). The first two peaks (Fig. 1B) were identified as subunits h
and f by their retention times (28 and 36 min, respectively), by
SDS-PAGE analysis (Fig. 1D, lanes 2 and 3,
respectively) and by Western blotting (not shown). The third component,
showing a retention time of 39 min, migrated at the same relative
molecular mass as subunit f on SDS-PAGE (Fig. 1D, lane 4).
From amino acid sequencing of the whole protein, we assume that the
protein is blocked, because sequencing gave very low yields of amino
acid. However, the following VNVLR amino acid residues were detected in
cycles 3-7. Trypsin cleavage of the protein resulted in a few peptides
that were purified by reverse phase HPLC (not shown). One of these
peptides had the sequence NLEDPNIDFER, a sequence that is found in
subunit e at the positions 72-82. Confirmation of the identification
of this subunit was brought by Western blot analysis. Subunit e
migrated as a broad band on SDS-PAGE at the same level as subunit f
(Figs. 1 and 2). When using low
concentrations of Triton X-100 (0.1-0.375%), this protein remained
associated with the yeast ATP synthase, but it was released in the
presence of 1% Triton X-100 (Fig. 2, B and C).
Subunit e is not essential to the structure of the enzyme, because it
has been reported that inactivation of the gene encoding for subunit e
results in slow growth and a decrease in mitochondrial respiration
(17). This behavior is in agreement with the weak association of the
subunit with the complex we have shown.
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Fig. 1.
Purification of subunits e and i.
A, gel permeation chromatography on a Superdex 75 column of
yeast ATP synthase subunits solubilized by 6 M guanidinium
chloride (see "Experimental Procedures"). B, reverse
phase chromatography of the 10-kDa peak from A. C, reverse
phase chromatography of the 5-kDa peak from A. D,
SDS-PAGE of 32 µg of ATP synthase (lane 1), subunit h
(lane 2), subunit f (lane 3), subunit e
(lane 4), and subunit i (lane 5). The slab gel
was silver-stained. su., subunit; oscp,
oligomycin sensitivity-conferring protein.
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Fig. 2.
Subunit e is loosely associated to the yeast
ATP synthase. A, SDS-PAGE of ATP synthase. The enzyme was
purified in the presence of 0.1% Triton X-100. Lane 1, 16 µg; lane 2, 32 µg. B, SDS-PAGE of ATP
synthase immunoprecipitates washed with 90 mM NaCl, 20 mM Tris acetate, pH 7.5, and increasing Triton X-100
concentrations. Only a part of the silver-stained slab gel is shown.
Lane 1, subunit h; lane 2, 0.1% Triton X-100;
lane 3, 0.375% Triton X-100; lane 4, 1% Triton
X-100. C, wild-type mitochondria (2 mg of mitochondrial
protein) were solubilized with 0.375% Triton X-100 in a final volume
of 0.4 ml. After centrifugation at 100,000 × g for 20 min at 4 °C, 0.1-ml aliquots of the supernatant were incubated with
0.1% Triton X-100 (lanes 1 and 4), 0.375%
Triton X-100 (lanes 2 and 5), and 1% Triton
X-100 (lanes 3 and 6). The 0.1% Triton X-100
sample was obtained by dilution with the extraction buffer devoid of
detergent. The samples were incubated for 30 min at 4 °C and then
centrifuged at 300,000 × g for 1 h to pellet the
enzyme. The 300,000 × g supernatants (lanes
4-6) were precipitated by 0.3 M trichloroacetic acid
and centrifuged at 10,000 × g for 10 min, and the
pellets were washed twice with cold acetone before solubilization with
50 µl of dissociation buffer. The 300,000 × g
pellets (lanes 1-3) were dissolved in the same volume of
dissociation buffer. A volume of 10-µl aliquots of each dissociated
sample was submitted to a Western blot analysis. Polyclonal antibodies
raised against subunit e (dilution, 1:10,000) were used to probe the
membrane. su., subunit; oscp, oligomycin
sensitivity-conferring protein.
-subunit because of its retention time (32 min) and by Western blot analysis (not shown). The second peak, named subunit i and showing a retention time of 37 min, was analyzed. Like the
-subunit, its relative molecular mass on SDS-PAGE was in the range of 5 kDa. It was poorly stained by the silver-staining technique, which explains its lack of
detection in purified ATP synthase (Fig. 1D, lane 1). The
N-terminal part of the protein was sequenced up to the 15th amino acid
(Fig. 3). Computer analysis against DNA
data banks fitted with a DNA fragment of S. cerevisiae
chromosome XIII (104,162-103,983, complementary strand) encoding for a
putative protein of 59 amino acid residues (37).
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Fig. 3.
DNA sequence of the ATP18 gene
encoding subunit i. The DNA sequence encoding the open reading
frame of the ATP18 gene is presented. The numbering of nucleotides
begins at the A of the initiating codon ATG. *, stop codon; ,
sequence determined by Edman degradation.
18 bp
upstream from the ATG, the first amino acid residue is the initiating
methionine. Subunit i is a basic protein with a calculated pI of 9.73 and is amphiphilic. It contains a hydrophobic N-terminal domain
(residues 5-27), thus suggesting a possible
-helix transmembranous
domain. The C-terminal part of the molecule (residues 30-50) is
hydrophilic. A similar DNA sequence has been reported for
Schizosaccharomyces pombe chromosome I.2 When comparing the
hypothetical protein of S. pombe and subunit i of S. cerevisiae, a 51% similarity was found.
ATP18--
The
ATP18 gene was disrupted. The resulting strain (
ATP18)
displayed the same doubling time as that of the wild-type strain with a
fermentable carbon source, whereas it showed a slow growth with an
oxidative carbon source (Table I). With
lactate as carbon source, a recovery of the doubling time was measured
with the complemented strain, thus showing that the ATP18
gene is likely involved in the oxidative phosphorylations. In addition,
at stationary phase, with lactate as a carbon source, a 2-fold lesser
amount of matter was formed by the mutant strain than by the wild-type strain. This result also points to a defect in the oxidative
phosphorylations. The cytochrome content of cells grown with lactate as
a carbon source were measured. The cytochrome oxidase amount of null
mutant cells was 60% of that of the wild-type cells, whereas
cytochrome b and c + c1 amounts were
not significantly modified.
Phenotypic analysis of wild-type, disrupted, and complemented cells
ATP18), and complemented
strain (
ATP18 + pRS313ATP18) cells were grown in complete
medium containing either 2% lactate or 2% glucose. Turbidimetry was
measured at 550 nm for the estimation of cell concentration after
appropriate dilution. The ratio dry mass/A550 was
the same for each of the strains. Cytochrome content was determined
from difference spectra of oxidized versus
Na2S2O4-reduced cells. ND, not determined.
Oxidative phosphorylation measurements and ATPase activities of
wild-type and null mutant mitochondria
ATP18) mitochondria
were prepared from yeast cells grown with 2% lactate as a carbon
source. Five and two different mitochondrial preparations were made
from wild-type and null mutant cells, respectively. Results are the
average of four different experiments and are presented with the S.D.
Oligomycin (6 µg/ml) and CCCP (3 µM) were added where
indicated. Respiration rates were obtained with NADH as substrate.
attributable to a proton influx through F0 during
ADP phosphorylation. After ADP consumption, the
increased, and
finally carbonyl CCCP addition produced a reversal of the fluorescent
quenching corresponding to the collapse of the
µH+. In
the case of null mutant mitochondria, ADP addition induced a lower
decrease in fluorescent quenching of the dye. In addition, the time
during which ADP was consumed was significantly increased. These two
points have to be correlated with the observed low ADP/O ratio of null
mutant mitochondria. Modifications of the transmembrane
mediated
by the ATPase proton-pumping activity were analyzed after energizing
mitochondria by ethanol, an activation step that is necessary to remove
the natural inhibitory peptide of the mitochondrial ATPase, which
otherwise would inhibit the ATPase activity (for review, see Ref. 38).
As expected, KCN addition by inhibiting proton pumping mediated by the
respiratory chain collapsed the
, and subsequent ATP addition
promoted a fluorescent quenching of the dye that was
dicyclohexylcarbodiimide- (Fig. 4) and oligomycin-sensitive (not
shown), thus reflecting the proton pumping mediated by the ATPase. In
the case of null mutant mitochondria, there was also an
ATP-dependent fluorescent quenching of the dye, but it
showed a slow and significant decrease with time. This unexpected
result could correspond to a loss of activity of the mutant enzyme. The ATP-dependent fluorescent quenching of null mutant
mitochondria was also oligomycin-sensitive (not shown). We verified
that ATP addition did not induce a proton leak with the null mutant
mitochondria (data not shown), as in the case of a strain having a
mutation in the hydrophobic part of subunit 4 (39).
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Fig. 4.
Mitochondrial energizing monitored by
fluorescent quenching of rhodamine 123. D273-10B/A/H/U
mitochondria (wild type) and null mutant mitochondria
( ATP18) were incubated in 2 ml of respiration medium.
Additions were 0.32 of mg mitochondrial proteins (m), 10 µl of ethanol (e), 50 µM ADP, 3 µM CCCP (c), 6 µg of
dicyclohexylcarbodiimide (d), and 200 µM KCN
(k); 1 mM ATP was added 1 min after KCN.
· · ·, additions were mitochondria, ethanol, KCN, ATP,
dicyclohexylcarbodiimide, and CCCP; - - -, additions were
mitochondria and ethanol.
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Fig. 5.
ATPase content in wild-type and null mutant
mitochondria. Mitochondria were dissociated in the presence of
SDS, and aliquots (3-30 µg of protein) were submitted to a Western
blot analysis. The blot was incubated with polyclonal antibodies raised
against subunit 4 and revealed by the ECL procedure of Amersham
Pharmacia Biotech. The film was scanned. The plot represents the
intensity measured as a function of the mitochondrial protein amount.
-
, wild-type mitochondria;
-
, null mutant
mitochondria.
DISCUSSION
-subunit made it
possible to precipitate the wild-type and the null mutant ATP synthase
from mitochondrial 0.375% Triton X-100 extracts, and that the obtained
immunoprecipitates showed similar patterns of subunits on SDS-PAGE (not shown).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. D. Promé for mass spectrometry analyses of subunit i and Drs. M. Rigoulet and C. Napias for stimulating discussions. We thank Dr. R. Cooke for his contribution to the editing of the manuscript.
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FOOTNOTES |
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* This work was supported by the CNRS, the Ministère de la Recherche et de l'Enseignement Supérieur, the Université Victor Segalen, Bordeaux 2, and the Etablissement Public Régional d'Aquitaine.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF073791.
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.: 33-5-5699-9048; Fax: 33-5-5699-9059;
E-mail: jean.velours{at}ibgc.u-bordeaux2.fr.
2 D. Harris and R. Squares, unpublished results, EMBL accession number Z99753.
3 J. Vaillier, G. Arselin, P.-V. Graves, N. Camougrand, and J. Velours, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; bp, base pair; HPLC, high performance liquid chromatography; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine..
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