From the Institut de Biochimie et
Génétique Cellulaires du CNRS, Université Victor
Ségalen, Bordeaux 2, 1 rue Camille Saint Saëns, 33077 Bordeaux, cedex France and
Department of Biochemistry and
Molecular Biology, Chicago Medical School,
North Chicago, Illinois 60064
Received for publication, September 6, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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The mammalian mitochondrial ATP synthase is
composed of at least 16 polypeptides. With the exception of coupling
factor F6, there are likely yeast homologs for each
of these polypeptides. There are no obvious yeast homologs of
F6, as predicted from primary sequence comparison of the
putative peptides encoded by the open reading frames in the yeast
genome. In this manuscript, we demonstrate that expression of bovine
F6 complements a null mutant in ATP14 gene in
yeast Saccharomyces cerevisiae. Subunit h of
the yeast ATP synthase is encoded by ATP14 and is just
14.5% identical to bovine F6. Expression of bovine
F6 in an atp14 null mutant strain recovers oxidative
phosphorylation, and the ATP synthase is active, although functioning
with a lower efficiency than the wild type enzyme. Like subunit
h, bovine F6 is shown to interact mainly with
subunit 4 (subunit b), a component of the
second stalk of the enzyme. These data indicated the subunit
h is the yeast homolog of mammalian coupling factor
F6.
The F0F1-ATP synthase is the major enzyme
responsible for the aerobic synthesis of ATP. It exhibits a tripartite
structure consisting of a head piece (F1 catalytic sector),
base piece (F0, membrane sector), and two connecting
stalks. F1 is a water-soluble unit retaining the ability to
hydrolyze ATP. F0 is embedded in the membrane and is mainly
composed of hydrophobic subunits forming a specific proton conducting
pathway. The connecting stalks are composed of components from both
F1 and F0. When F1 and
F0 are coupled, the enzyme functions as a reversible
H+-transporting ATPase or ATP synthase (1, 2).
The establishment of the crystal structure of the major part of the
bovine F1 (3) allowed experiments that demonstrated that
the enzyme is a molecular rotary motor, as shown by the
ATP-dependent rotation of the The E. coli ATP synthase and the bovine enzyme are composed
of 8 and 16 different subunits, respectively (11). In the case of
Saccharomyces cerevisiae, the
F0F1-ATP synthase is composed of at least 13 different subunits involved in the structure of the enzyme; the
disruption of any of the corresponding structural genes leads to a lack
of assembly of the holo-complex (12). Recently, the establishment of
the structure of the yeast enzyme at 3.9 Å resolution revealed the
structure of the F1 and the subunit c oligomer
of F0 (13).
Among the supernumerary subunits of the yeast ATP synthase
F0, subunit h has been described as an essential
component because inactivation of the ATP14 gene led to a
lack of oxidative phosphorylations (14). Recently cross-linking
experiments (15) have positioned this hydrophilic and acidic component
of 10,408 Da close to subunit 4 (subunit b), a
component of the second stalk of the ATP synthase. In this paper we
report the complementation of a yeast strain devoid of the yeast ATP
synthase subunit h by a single copy vector bearing a DNA
sequence encoding the bovine coupling factor 6. This is a rather
remarkable result because subunit h and bovine F6 share only 14.5% sequence identity.
Yeast Strains and Nucleic Acid Techniques--
The S. cerevisiae strain D273-10B/A/H/U (MAT
The expression plasmid for expression of bF6,
pbF6, was made essentially as described (17). In this
scheme, the coding region of mature bF6 replaces the coding
region of the gene encoding the
Recombination is effected in vivo in yeast and occurs across
a linearized plasmid (pRS314) (18) that contains the ATP2
gene that is cut in the center (gap repair) (for a diagram of this method, see Ref. 17). The recombination event occurs at the region
encoding the leader peptide, at one end, and at the stop codon of the
coding frame of bF6 (a clone containing the cDNA for
bovine F6 was kindly provided by Dr. John E. Walker,
Cambridge, UK). In this manner, the coding region of mature
bF6 precisely replaces the coding region of the
ATP2 gene.
The DNA sequence of the chimeric gene was sequenced to ensure the
correct recombination event and to ensure the absence of any mutations.
DNA sequence analysis was performed at the Iowa State University
sequencing facility (Ames, Iowa). The chimeric gene was removed from
the plasmid by digestion with XbaI and SalI and
subcloned into the low copy vector pRS313 at the same restriction sites
(18).
The forward and reverse primers used in the PCR reaction were:
bx_F6·pri: CTT CTA TCC ACT TCG TGG AAA AGA TGT ATG
GCC TCA aat aag gag ctt gat cct gtg and
bx_F6·rev·pri: CTT CCC TTG GTT TAA GCT TTA TTT CTT CTA
gga ttg tgg ttt ctc gac, respectively. The lowercase letters correspond
to the region that primes DNA synthesis in the PCR reaction using the
cDNA of bF6 as the template. The capital letters
correspond to the target site for homologous recombination in the
plasmid containing the gene encoding the Biochemical Procedures--
Cells were grown aerobically at
28 °C in a complete liquid medium containing 2% lactate as carbon
source (19) and harvested in logarithmic growth phase. Mitochondria
were prepared according to (20), frozen as droplets in liquid nitrogen,
and stored at
Mitochondrial Triton X-100 extracts were sedimented on sucrose
gradients as follows. Mitochondrial protein (3 mg at 10 mg/ml) was
incubated with an equal volume of 0.75% (w/v) Triton X-100. The
extract was spun at 100,000 × g for 10 min at 4 °C.
The supernatant was loaded on the top of a 10-30% linear sucrose
gradient containing 0.1% Triton X-100, 1 mM ATP, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0. The
gradient was centrifuged at 4 °C for 17 h, 46 min in a SW41
rotor (Beckman) at 41,000 rpm, and fractions (1 ml) were collected and
used for analysis.
Cross-linking Experiments and Analyses--
Frozen mitochondria
isolated from wild type and mutant cells were thawed, washed twice with
0.6 M mannitol, 2 mM EGTA, 50 mM
triethanolamine-HCl, pH 8.0, and suspended in the same buffer at a
protein concentration of 10 mg/ml. Samples were incubated for 30 min at
30 °C in the presence or absence of DSP dissolved in
dimethylformamide, and the reaction was quenched by the addition of 10 mM Tris, pH 8.0. The ATP synthase was extracted with
0.375% (w/v) Triton X-100, and after incubation for 15 min at 4 °C,
the extract was spun at 100,000 × g for 15 min at
4 °C, and the supernatant was used for analysis.
Bovine F6 was released from the ATP synthase (free bF6) by
sonication as follows. Washed mitochondria from yeast,
SDS-polyacrylamide gel electrophoresis was according to Schägger
and Von Jagow (25). Nitrocellulose membranes (Membrane Protan BA83, 0.2 µm from Schleicher & Schuell) were used for Western blot analyses.
The polyclonal antibodies, anti-bF6, were kindly provided
by Dr. Youssef Hatefi (Scripps Research Institute, La Jolla, CA) and
used with a dilution of 1:7,500. Antibodies against subunit
4 and the The Coding Sequence of the Bovine Coupling Factor 6 Complements a
Null Mutant Strain Devoid of Subunit h--
For expression in yeast,
the cDNA encoding mature bF6 was fused to the leader
sequence of the yeast
The strain containing the null mutation in the ATP14 gene
(14), which encodes subunit h, was transformed by a single
copy vector (pRS313) bearing the selection marker HIS3 and
the DNA encoding the bovine F6, pbF6. The
transformants were tested for growth on a complete medium containing
glycerol as the sole carbon source, YPG (3% glycerol, 2% peptone, 1%
yeast extract). Growth on medium containing glycerol or lactate
indicates that the cells are able to make ATP via oxidative
phosphorylation and thus have a functional ATP synthase. All of the
transformants were able to grow on YPG at 28 °C but not at 37 °C
(Fig. 1). One of the transformants was
selected, named Phenotypic Analyses of the Complemented Yeast
Strain--
Mitochondria were prepared from
At pH 8.4, the ATPase activity of
Potential ( Bovine F6 Associates with the Yeast ATP
Synthase--
The prior genetic and biochemical studies indicate that
bF6 can correct for the loss of subunit h,
albeit not to wild type levels. If bF6 is acting directly
by substituting for subunit h in the ATP synthase,
bF6 should be associated with the ATP synthase devoid of
subunit h. A number of biochemical studies were performed to
test this hypothesis. First Western blot analysis using
anti-bF6 antiserum detected a band with a relative
molecular mass of 10.6 kDa that was present in the SDS-dissociated
Association of bF6 to the yeast ATP synthase missing
subunit h was determined by immunoprecipitation of the
detergent (Triton X-100)-solubilized ATP synthase complex using
antibodies directed against the
To determine whether bF6 was cross-linked to the ATP
synthase, mitochondria treated with DSP were extracted with Triton
X-100, and the solubilized proteins were separated by sucrose-gradient centrifugation. Eleven fractions were collected and analyzed by Western
blot. The samples were reduced before SDS-polyacrylamide gel
electrophoresis to allow the clear identification of bF6. The blots were probed with antibodies raised against the DSP Primarily Cross-links Bovine F6 to Subunit
4--
If bF6 was acting directly by replacing subunit
h, then not only should it associate with the ATP synthase,
but it should also interact with the same peptides of the ATP synthase
as subunit h. To test this hypothesis, the protein partners
of bF6 were determined by Western blot analysis after
cross-linking the peptides with DSP. The results from Fig. 3 indicate
that the major cross-linked species with bF6 had a
molecular mass of about 36 kDa, suggesting that bF6 was
cross-linked to a peptide 20-30 kDa in size. As a consequence,
antibodies raised against subunits of the ATP synthase whose molecular
mass were within this range, OSCP and subunits 6,
d, and 4, were used in the analysis. In addition,
long polyacrylamide slab gels were used to clearly identify the
cross-linked products (Fig. 5).
In a wild type context, numerous cross-linked products involving
subunit 4 have been reported, and the most intense band that was found in the 36-kDa region has been identified as
4+g (15). This same cross-linked product was
present for both the ATP synthase from the wild type and
Two other bands of low intensity and showing relative molecular masses
of 42 and 56 kDa could be heterooligomers containing at least
bF6 and subunit d because the latter
cross-linked products were absent from the wild type sample (Fig.
5C). Antibodies raised against subunit 6 and OSCP
did not identify any bands that were also detected by antibodies
against bF6 (not shown). The increased intensity of a
26-kDa band upon cross-linking of the Subunit h is an essential component of the yeast ATP
synthase. It has been described as a supernumerary protein that, until this study, was not apparently related to any subunit described in
other ATP synthases (14). Primary structural analysis of nucleotide
data banks have identified the existence of an open reading frame in
Schizosaccharomyces pombe (TrEMBL accession number 059673)
that encodes an hypothetical protein 27% identical to subunit
h and another in Botryotinia fuckeliana
(GenBankTM accession number AL115386) that also encodes a
hypothetical protein 38% identical to subunit h. Yeast
subunit h is essential for yeast to grow on a nonfermentable
carbon sources, and mitochondria isolated from a yeast strain with a
null mutation in subunit h have an ATPase activity that is
oligomycin-insensitive and the catalytic sector dissociated from the
membrane components (14).
Mammalian coupling factor F6 is an essential component of
the mammalian mitochondrial ATP synthase. F6 is known to be
required for restoration of ATP-Pi exchange and
oligomycin-sensitive ATPase activity to factor 6-depleted ATP synthase
(27, 28). It is also involved in the binding of F1 to
F0 (29) and shields F1 from limited proteolysis
(30). As such, F6 is thought to be required for the
coupling of proton translocation to the synthesis of ATP.
The results in this manuscript are quite surprising and have important
implications. Both genetic and biochemical data indicate that subunit
h of the yeast ATP synthase is the homolog of mammalian coupling factor F6. This is a rather surprising because
both of these subunits are essential components of their respective
multimeric peptide complexes. Despite the apparent functional homology
of subunit h with F6, primary sequence alignment
of both subunits shows a very low sequence identity of just 14.5% and,
when allowing for amino acid replacements, a 54% similarity (Fig.
6). This low level of sequence identity
and homology is at the level seen between two random peptides. In
contrast, most of the remaining subunits of the ATP synthase
demonstrate a high degree of identity (31). For instance, the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-subunit (4, 5) and
consistent with the binding site hypothesis of Boyer (6). In
Escherichia coli, F1 and F0 are
linked by two stalks, one of which is made of subunits
and
, and
these also constitute a part of the rotor (7). Three other subunits,
of F1 and the two b-subunits of
F0, are also involved in the binding of F1 to
F0 and are thought to form the second stalk of the stator.
The stator is thought to fix the
3
3
oligomer to the a-subunit, thus allowing rotation of the c-subunit oligomer together with the
- and
-subunits.
(8-10) while holding the head piece in place.
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DISCUSSION
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, met6,
ura3, his3) (16) was the wild type strain. The strain with the
null mutation in ATP14 (MAT
, met6,
ura3, his3, ATP14::URA3) has
been described (14). The
ATP14 strain
containing the plasmid pbF6, was obtained by transformation
of the null mutant in ATP14 gene by the nonintegrative
single copy vector, pRS313, which contains the coding region of mature
bF6,1 the leader
peptide of the
-subunit of the yeast ATP synthase, and the upstream
and downstream transcriptional controlling elements of the
ATP2 gene.
-subunit of the ATP synthase,
ATP2. This allows the expression of bF6 to be
under the same controls as that of the ATP2 gene. The coding
region of bF6 is amplified by PCR, and this PCR fragment is
used to directly replace the coding region of the ATP2 gene. The replacement occurs by site-specific homologous recombination effected in yeast. In addition to the bases required to direct the
synthesis of the coding region of bF6, the PCR primers also contain a 30-base sequence that targets the PCR product to the desired site of recombination on the ATP2 gene, in this case
at the end of the region coding for the leader peptide and at the 3'-untranslated part of the gene.
-subunit of the
F1-ATPase (ATP2). The underlined sequence
corresponds to the additional Ala-Ser codons. These were added because
it is frequently the case that Ala-Ser are the first two amino acids after the processing site of the leader peptide. The Ala-Ser are thus
positioned after and adjacent to the leader sequence of what corresponds to the leader peptide of the
-subunit of the ATPase.
70 °C. Protein amounts were determined according to
Lowry et al. (21) in the presence of 5% SDS using bovine
serum albumin as a standard. Oxygen consumption rates were measured
with NADH as substrate (22). Variations of transmembrane potential
(
) were evaluated by measurement of fluorescence quenching of
rhodamine 123 with an SFM Kontron fluorescence spectrophotometer (23). The specific ATPase activity was measured at pH 8.4 according to Somlo
(24) and modified as follows. Freshly prepared mitochondria were
diluted with the same volume of either the isolation buffer (0.6 M mannitol, 2 mM EGTA, 10 mM
Tris-maleate, pH 6.8) or 0.75% Triton X-100 (w/v). Aliquots were taken
for protein concentration measurement, and determination of ATPase
activity was done as follows: mitochondrial protein (50 µg) was
incubated for 2 min in reaction medium (0.9 ml, 0.2 M KCl,
3 mM MgCl2, 10 mM Tris-HCl, pH 8.4)
in the presence or absence of F0 inhibitors. The reaction was started with the addition of 5 mM ATP and stopped after
2 min by the addition of 0.3 M trichloroacetic acid.
ATP14 + bF6,, were suspended in
100 mM NaCl, 22 mM triethanolamine, pH 8.0, at
a final concentration of 10 mg protein/ml and sonicated four times for
30 s at 120 V (Annemasse sonicator). The sample was centrifuged at
435,000 × g for 1 h at 4 °C. Aliquots of the supernatant containing free bF6 were incubated with DSP and
analyzed either by Western blot or loaded on a 10-30% linear sucrose
gradient, as described above.
-subunit were used with dilutions of 1:10,000 and 1:100,000, respectively. Membranes were incubated with
peroxidase-labeled antibodies and visualized with the ECL reagent of
Amersham Pharmacia Biotech. Molecular mass markers (Benchmark
Prestained Protein Ladder) were from Life Technologies, Inc. Sequence
alignments were performed as in (26). Secondary structure predictions
were made with the program at the PredictProtein Server.
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DISCUSSION
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-subunit. In addition, the codons for Ala-Ser
were added to the front end of the codons encoding the mature
bF6, as described (17). The expectation is that leader
sequence will be cleaved in front of the Ala-Ser residue after import
into the mitochondrion providing an N-terminal sequence of:
ASNKELD ...
ATP14 + bF6, and
this strain had a generation time of 300 min as compared with 164 min
for the wild type strain, at 28 °C in liquid medium containing
lactate as the carbon source. Loss of the plasmid, by growing the cells on a complete medium with glucose as carbon source, resulted in a
concomitant loss in the ability to grow on glucose minimal medium devoid of histidine and on YPG medium (not shown). Thus, these results
indicate a functional homology between yeast subunit h and
bF6.
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Fig. 1.
Complementation of the null mutant
atp14 by a single copy vector bearing the bovine
F6 gene. Wild type (wt) strain
(D273-10B/A/H/U), ATP14 null mutant strain,
and the complemented strain
ATP14 + bF6 were serially diluted, and 3 µl of each dilution
corresponding to the same cell number were spotted on solid complete
medium containing glycerol as the carbon source (YPG). The cells were
grown for 120 h at either 28 or 37 °C, as indicated.
ATP14 + bF6 strain to examine the effectiveness of
bF6 in replacing subunit h in the structure and
function of the ATP synthase. Respiration rates were measured with NADH
as substrate (Table I). In the presence
of CCCP, the uncoupled respiration rates of mitochondria
isolated from the wild type and
ATP14 + bF6 strains were similar. The main difference between
the two preparations is the respiration rate associated with the
phosphorylation of ADP (State 3) where the respiration rate of
ATP14 + bF6 mitochondria is only
51% of the uncoupled respiration rate, as compared with 63% for the
wild type. As a result, the respiratory control and the ATP/O ratio of
ATP14 + bF6 mitochondria are lower
than those of wild type mitochondria, thus reflecting a lower ATP
synthase activity and a lower efficiency of oxidative
phosphorylation.
Oxidative phosphorylation measurements of isolated mitochondria
ATP14 + bF6 mitochondria were prepared from cells grown in
complete liquid medium containing 2% lactate as carbon source. Data
are from typical experiments. Measurements were performed four times.
Respiration rates were obtained with NADH as substrate. CCCP
concentration was 3 µM.
ATP14 + bF6 mitochondria is partially inhibited by DCCD but
not by oligomycin, both of which are inhibitors of F0
(Table II). Addition of Triton X-100 (0.375%) solubilizes the ATP synthase and increases the ATPase activity of wild type mitochondria by removing IF1, the
F1 inhibitor protein. This stimulation also occurred for
the
ATP14 + bF6 mitochondrial extract, but the sensitivity to DCCD was nearly abolished. These data
suggest that the ATP synthase isolated from
ATP14 + bF6 mitochondria was highly unstable, consistent with
the temperature-sensitive phenotype of the cells on YPG medium (Fig.
1).
ATPase activities of isolated mitochondria
ATP14 + bF6 mitochondria were prepared from yeast cells
grown with 2% lactate as carbon source. Two different mitochondrial
preparations were made for each strain, and measurements were performed
in triplicate with 50 µg of mitochondrial protein. Samples treated in
the presence or absence of 0.375% Triton X-100 were prepared as
described under "Experimental Procedures." Oligomycin (6 µg/ml)
and DCCD (6 µg/ml) were added where indicated.
) measurements were performed in the respiration
medium (Fig. 2). With ethanol as a
substrate, the addition of ADP promoted a transient decrease in the
fluorescent quenching of rhodamine 123 because of proton uptake through
F0 during ATP synthesis. This effect was less pronounced
with mitochondria isolated from
ATP14 + bF6 consistent with the low rate of state 3 respiration. The reversibility of the ATP synthase was also examined.
Ethanol addition promoted a strong fluorescence quenching of rhodamine 123 because of the respiratory chain activity, and this was reversed by
potassium cyanide. Finally, addition of ATP caused a fluorescent quenching because of its hydrolysis and the coupled pumping of protons
out of the mitochondrion. Clearly, the
generated by ATP
hydrolysis in mitochondria isolated from
ATP14 + bF6 is sensitive to either oligomycin or DCCD at
concentrations used to inhibit the wild type mitochondria, but the
intensity of the fluorescent quenching was not as large, and it was not
as stable as compared with the wild type sample. These results suggest
that the ATP synthase from
ATP14 + bF6 is uncoupling during the course of the assay.
However, the addition of oligomycin or DCCD did not affect the
transmembrane potential generated by the respiratory chain, suggesting
that passive proton conduction through F0 is not occurring
in the absence of either ADP or ATP.
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Fig. 2.
Mitochondrial energization monitored by
fluorescent quenching of rhodamine 123. D273-10B/A/H/U
mitochondria (wild type) and ATP14 + bF6 mitochondria were incubated in 2 ml of respiration
medium. Additions were 0.6 mg of mitochondrial protein (m),
10 µl of ethanol (e), 37 µM ADP, 3 µM CCCP (c), 200 µM KCN
(k), 1 mM ATP, 6 µg of DCCD (d),
and 6 µg of oligomycin (o). Dotted line,
additions were mitochondria, ethanol, KCN, ATP, and oligomycin.
Dashed line, additions were mitochondria, ethanol,
oligomycin, and DCCD.
ATP14 + bF6, but not wild type,
mitochondrial sample (Fig.
3A). The mature
bF6 has a calculated molecular mass of 9,116 Da assuming
that processing occurs just prior to the Ala-Ser residues. If the
protein is not processed, other than removing the initiating Met, then
it would have a mass of 12,653 Da. These results suggest the 10.6-kDa
peptide represents bF6 and is likely processed at the
predicted point. However, N-terminal sequence analysis has not been
done on the yeast-expressed bF6 to verify the site of
processing.
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Fig. 3.
Cross-linking bF6 to putative
components of the ATP synthase. A, wild type
(wt) and ATP14 + bF6
mitochondria were dissociated and analyzed by Western blot. Blots were
probed with polyclonal antibodies raised against subunits 4 and bF6. B,
ATP14 + bF6 mitochondria (30 µg of protein) was cross-linked
with 0.2 and 0.5 mM DSP and treated with 2-mercaptoethanol,
as indicated, and the blot was probed with antibodies raised against
bF6. (C) bF6 was released from
F1F0 by sonication (see "Experimental
Procedures") and treated with DSP and 2-mercaptoethanol, as
indicated, and the blot was probed with polyclonal antibodies raised
against bF6.
-subunit. However, unlike ATP
synthase from wild type yeast, the antibodies precipitated only the
F1 sector (not shown). This suggested that the association
of F1 and F0 sectors was not stable during
immunoprecipitation. This is consistent with the prior studies that
indicated that the ATP synthase with bF6 was not as stable
as the wild type enzyme. To capture the interactions between
bF6 and components of the ATP synthase, the mitochondrial
proteins were treated with the thiol-cleavable homobifunctional cross-linking reagent, DSP. Fig. 3B shows that the
concentration of bF6 was greatly decreased upon incubation
of mitochondria with 0.2 and 0.5 mM DSP, concurrent with
the presence of bands of high molecular masses, as revealed by Western
blot analysis. The most intense band displayed a relative molecular
mass of 36 kDa, suggesting that bF6 (9.1 kDa) was
associated with a peptide of about 27 kDa. Incubation of DSP-treated
ATP14 + bF6 mitochondria with
2-mercaptoethanol to reverse the cross-linking, eliminated the major
36-kDa product, and provided a relative increase in the amount of
uncross-linked bF6. In the presence of 2-mercaptoethanol,
nonspecific bands are observed in the 45-kDa region. These bands
probably originate from aggregates that did not enter the gel in the
absence of the reducing agent but did enter the gel in the presence of
the reducing agent. Free bF6 was also prepared from
mitochondria isolated from yeast
ATP14 + bF6 to examine the behavior of bF6 upon
incubation with DSP in the absence of ATP synthase (Fig.
3C). The amount of free bF6 decreased upon
incubation with DSP, and this was reversed with 2-mercaptoethanol.
Importantly, DSP cross-linking of free bF6 did not provide
any specific bands of higher molecular masses. This suggests that
nonspecific cross-linking of free bF6 occurred with other
peptides, and these were reversed by reduction with 2-mercaptoethanol.
-subunit (an F1 subunit), subunit 4 (an F0
subunit), and bF6. The wild type ATP synthase sediments to
fractions 4-8 as shown by the cosedimentation of the
-subunit and
subunit 4 in the sucrose gradient (Fig.
4A). For the ATP synthase from
mitochondria isolated from yeast
ATP14 + bF6, the
-subunit and subunit 4 were
distributed in a much broader range of fractions, suggesting both a
more complex mixture of species and a less stable assembly of the ATP
synthase. This occurred in the absence of reaction with DSP (not shown)
or in the presence of 0.2 mM DSP (Fig. 4B).
However, when the concentration of DSP was increased to 0.5 mM, then a significant fraction of bF6 was seen
to sediment with the other subunits of the ATP synthase (Fig.
4C). This new location of bF6 in the sucrose
gradient was not the result of nonspecific cross-linkings of
bF6 to other proteins, because reaction of DSP with
bF6, which has been freed and separated from
F1F0 stayed at the top of the gradient, the
same position of bF6 without reaction with DSP (Fig.
4, D and E). In conclusion, the results support the
hypothesis that bF6 associates to the yeast ATP
synthase.
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Fig. 4.
Sedimentation analysis of bF6 in
a sucrose gradient. Mitochondrial Triton X-100 ) extracts
(Ext.) were separated on a 10-30% sucrose gradient. Protein from
fractions (30 µl) was separated by SDS-polyacrylamide gel
electrophoresis, electroblotted, and probed with antibodies raised
against the -subunit, bF6 and subunit 4, as
indicated. A, wild type mitochondrial Triton X-100 extract.
B and C, Triton X-100 extracts of
ATP14 + bF6 mitochondria were
treated with 0.2 mM DSP (B) and 0.5 mM DSP (C). D and E,
bF6 was released from F1F0 by
sonication and incubated without (D) or with 0.5 mM DSP (E). Note that
* is a degradation
product of the
-subunit.
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Fig. 5.
Cross-linking of bF6 with
components of the second stalk of the yeast ATP synthase.
Mitochondria were incubated in the presence or absence of DSP, and
samples (30 µg of protein) were analyzed by Western blot. The blots
were probed with polyclonal antibodies directed against subunits of the
ATP synthase, as indicated. A, ATP14 + bF6 mitochondria. B and C, wild
type (wt) and
ATP14 + bF6 mitochondria were incubated in the presence or
absence of 0.2 mM DSP, as indicated. The
asterisks indicate the position of two cross-linked
products with corresponding masses of 42 and 56 kDa.
ATP14 + bF6 strains (Fig. 5,
A and B). However, there was an additional band
at about 36 kDa that was obtained upon incubation with 0.2 and 0.5 mM DSP and that was seen with antibodies against either
subunit 4 or bF6. This band ran slightly ahead
of the 4+g cross-linked product, and it was absent from the wild type sample (Fig. 5B). This band is
thus concluded to be a cross-linked product of bF6 and
subunit 4.
ATP14 + bF6 mitochondria represents a cross-link involving
subunit d and a small component of the yeast ATP synthase
(Fig. 5C). The latter band was less apparent in the wild
type sample. Thus, these results indicated that bF6
associated with subunit 4 of the ATP synthase consistent
with the interactions of subunit h in the ATP synthase.
DISCUSSION
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DISCUSSION
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-and
-subunits of the yeast ATP synthase are highly conserved with
percent identities of about 72 and 75%, respectively. Thus, it is
surprising that bF6 is able to replace subunit h
and form a functional enzyme, even now, knowing that they are
functional homologs.
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Fig. 6.
Primary structure alignment of S. cerevisiae subunit h and bovine
F6. Sequence alignment of mature subunit h
and bF6 (Swiss-Prot accession numbers Q12349 and P02721,
respectively) was performed according to Risler et al. (26).
The amino acids conserved are indicated by an asterisk,
whereas double and single dots indicate
conservative and semi-conservative substitutions, respectively.
The biochemical studies here demonstrate that the complementation by
bF6 is due to the direct replacement of subunit
h with bF6 and not due to a secondary mechanism.
The biochemical studies indicate that bF6 occupies that
same spatial relationship in the yeast enzyme as subunit h.
Cross-linking products involving subunit h and subunit
4, a component of the second stalk, were obtained from
positions K98C (15) and Q203C of the latter
subunit,2 which are two
positions located in the hydrophilic part of subunit 4, thus
suggesting that subunit h also participates to form the second stalk or stator. Bovine F6 is a component of
F0 (11, 32), and it is associated with the stalk as shown
by reconstitution experiments (33). Nearest neighbors relationships
have been demonstrated, by cross-linking experiments, between
F6 and the b-subunit (subunit 4) (34)
and bF6 with the - and
-subunits (35). The data in
this study indicate that subunit 4 is the major peptide
cross-linked with bF6 using DSP within the yeast enzyme
missing subunit h. Thus, bF6 and subunit
h appear to occupy the same spatial arrangement in the
enzyme, and bF6 can directly replace subunit h
in the yeast enzyme.
Despite the lack of primary structural similarity, subunit h
and bF6 must share some structural features that define
them as functional homologs, and indeed, there are some features that are conserved. First, they are both relatively small peptides with
calculated molecular masses of 10,408 and 8,958 Da for subunit h and bF6, respectively. Second, both subunit
h and bF6 are acidic proteins showing pI of 4.06 and 5.23, respectively. Third, secondary structural computer analyses
predict two -helix regions for both subunits: the first
-helix
formed by residues 6-23 for bF6 and by residues 2-13 for
subunit h and the second
-helix formed by residues 34-50
for bF6 and residues 47-64 for subunit h.
Fourth, they both have an acidic tail, although it is slightly longer in subunit h. Thus, these conserved features may form some
of the basis required for the functional replacement of bF6
for subunit h. Of course, beyond these features, it is
possible that despite low primary sequence identity, that these
peptides fold into similar three-dimensional structures.
The partial complementation of the null mutant atp14 by the bovine coupling factor 6 is more consistent with the fact that F6 or h are important for stabilizing the ATP synthase than for a mechanistic role. Of all the F0 components, bF6 and subunit h are the only acidic proteins. One possible hypothesis is that these highly negative charged proteins help in the association of other positively charged components of the second stalk, such as subunits 4, d, and OSCP, three proteins with calculated pIs of 7.83, 8.92, and 9.3, respectively.
Expressions of other subunits of the mammalian ATP synthase have been
demonstrated to complement the corresponding null mutations in yeast.
Expression of bovine -,
-,
-, and
-subunits (17) and rat
OSCP (36) have all complemented the corresponding null mutant strains
in yeast. However, although some of these homologous peptides did not
show a large amount of identity, it was always high enough to suggest
them as homologs by simple primary structural analysis. Subunit
h and bF6 are so divergent that even a one on one comparison of their primary structure provided no clue that they
were indeed homologs. The results of this study are even more startling
because these peptides are not the sole peptide in an enzyme complex,
but must interact within a heterosubunit multimeric enzyme complex. The
implications of this are significant because they indicate that primary
structural analysis cannot be used as the sole evidence that functional
peptide homologs do not exist between two species. This is true even
when the peptide is within a multimeric peptide complex that otherwise
might be highly conserved.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Brèthes and Dr. P. V. Graves for helpful discussions and for critical reading this manuscript.
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FOOTNOTES |
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* This work was supported by the Center National de la Recherche Scientifique, the Ministère de la Recherche et de l'Enseignement Supérieur, the Université Victor Ségalen, Bordeaux 2, and the Etablissement Public Régional d'Aquitaine and by National Institutes of Health Grant GM44412 (to D. M. M.).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.
§ 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.: 33-5-56-99-90-48; Fax: 33-5-56-99-90-51; E-mail: jean. velours@ibgc.u-bordeaux2.fr.
¶ Recipients of a research grant from the Ministère de la Recherche et de la Technologie.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008123200
2 V. Soubannier and J. Velours, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: bF6, bovine coupling factor 6; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, dicyclohexylcarbodiimide; DSP, dithiobis[succinimidylpropionate]; OSCP, oligomycin-sensitivity-conferring protein; PCR, polymerase chain reaction.
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