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INTRODUCTION |
ATP synthase (F1F0-ATPase) uses energy
produced from the electrochemical gradient to produce ATP from ADP and
Pi. Production of ATP is thought to occur by the binding
change mechanism (1). The F1 sector has three sites that
catalyze the production of ATP and that are repetitively and
sequentially driven through defined conformational states. Recent
studies have demonstrated that one element of the mechanism driving
these sequential events is the rotation of the
subunit in the
F1 sector of the complex (2). Moreover, the rotary process
by which ATP is synthesized is thought to involve a stator that
prevents the futile rotation of the
3
3
hexamer. There is evidence for such a stator consisting of subunits b
and
in the bacterial enzyme (3, 4). Conservation of the general
mechanism between the F1F0-ATPases might
suggest that a stator of similar structure exists in mitochondrial
enzymes. In eukaryotes, subunits b and
OSCP,1 which are homologues
of the bacterial subunits b and
, respectively, would be prime
candidates for components of a stator in the mitochondrial ATP synthase
(mtATPase). However, mtATPase contains additional subunits that do not
have a bacterial homologue, which means that the composition of the
stator in higher organisms may be more complex.
For an understanding of the structure and function of this stator in
the eukaryotic mtATPase, it is important to know the identity and
number of each subunit within the enzyme complex. However, the
stoichiometry and composition of several subunits in mtATPase enzymes
remain ill-defined. It is generally agreed that bacterial ATP synthase
contains two identical copies of subunit b per complex and that the
corresponding chloroplast enzyme contains one copy each of two
non-identical but homologous subunits, b and b' (I and II). By
contrast, in the mammalian system, the stoichiometry of subunit b and
other possible stator stalk components such as subunits OSCP and d and
coupling factor 6 varies according to the report. Collinson et
al. (5) determined the molar ratio of b:OSCP:d:F6
(where F6 is coupling factor 6) to be 1:1:1:1 using three
independent methods. Using a different approach, Hekman et
al. (6) proposed a stoichiometry of 2:1:1:2 for the same group of
subunits (b:OSCP:d:F6). Some support for a stoichiometry of
2 for subunit b comes from the earlier studies of Lippe et al. (7). The stoichiometry of 1 for OSCP reported by Collinson et al. (5) and Hekman et al. (6) contradicts the
earlier report of Penin et al. (8), who proposed a
stoichiometry of 2 for OSCP in the porcine enzyme.
Remarkably, the stoichiometry of subunits b, OSCP, and d in mtATPase
from the yeast Saccharomyces cerevisiae has received little
attention. In light of the discrepancies between reports on the
stoichiometry of subunits in mammalian mtATPase complexes and
differences in subunit composition of bacterial and eukaryotic enzymes,
a resolution of these issues is required. The stoichiometry of subunits
b, OSCP, and d in yeast cannot be merely predicted using models of the
bacterial complex. Therefore, we have set out to establish the
stoichiometry of subunits b, OSCP, and d in yeast. To achieve this, we
exploited a technique initially used to isolate, by immobilized metal
ion affinity (Ni2+-NTA) chromatography, mtATPase complexes
containing individual subunits tagged with hexahistidine
(Hex6), namely, subunit d, OSCP (9), or subunit b. Using
such hexahistidine tagging technology, we demonstrate that the
stoichiometry of subunits d, OSCP, and b in yeast mtATPase is 1 in each case.
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EXPERIMENTAL PROCEDURES |
Materials--
Ni2+-NTA resin was purchased from
QIAGEN Pty. Ltd. (Melbourne, Australia). Dodecyl
-maltoside,
complete protease inhibitors, and anti-hexahistidine monoclonal
antibodies were purchased from Boehringer Mannheim (Sydney, Australia).
N-Octyl glucoside was purchased from Sigma. Vistra ECF
substrate was purchased from Amersham Pharmacia Biotech (Sidney, Australia).
Construction of Expression Vectors--
The ATP4 gene
cassette, encoding subunit b, was modified by polymerase chain reaction
to encode a C-terminal addition of hexahistidine and cloned into the
BamHI expression site of the vector pAS2 to form
pAS2-SUB-Hex6. Plasmid pAS2 is a derivative of pAS1 (10) and differs in that the LEU2 selectable marker is replaced
by HIS3. Genes encoding proteins containing hexahistidine
are denoted by the suffix -Hex6. The vector pAS2-SUB,
carrying the expression cassette encoding subunit b without a
hexahistidine addition, was constructed in a similar fashion.
Construction of the plasmids pAS1-OSCP-Hex6,
pAS3-SUD-Hex6, pAS1-OSCP, and pAS3-SUD has been described
previously (9). Expression cassettes carried by the pAS series of
vectors are under the transcriptional control of the PGK1
promoter. The plasmids pRJ21-SUD, pRJ21-OSCP, and pRJ21-SUB, expressing
subunits d, OSCP, and b, respectively, under the transcriptional control of the GAL1 promoter, have been described (11). All plasmids were introduced into a yeast strain null for expression of the
corresponding chromosomal gene. YRD15 (MAT
his3-11, 2-15 leu2-3,
2-112 ura3-251, 3-373,
[rho+])is the parent strain for these null
mutants (11). Generation times of strains were determined at 28 °C
in SaccE medium, which is a rich medium containing 2% ethanol as a
carbon source (12).
Isolation of ATP Synthase from Mitochondrial
Lysates--
Mitochondria were prepared from yeast following zymolase
digestion of the cell wall (13). Lysates were prepared from isolated mitochondria, and Ni2+-NTA chromatography was performed as
described by Bateson et al. (9). Assembled ATP synthase
complexes were immunoprecipitated from lysates of mitochondria using an
immobilized monoclonal antibody directed against the
-subunit of the
F1 sector (14).
ATPase Assay--
Mitochondria were isolated (13), and ATPase
activity was monitored spectrophotometrically by the oxidation of NADH
in an enzyme-linked assay containing pyruvate dehydrogenase and lactate dehydrogenase (15). Assays were performed at 28 °C and contained 30 µg of mitochondrial protein. Oligomycin sensitivity of the ATPase was
determined by the addition of oligomycin (100 µg/mg mitochondrial protein)
SDS-PAGE--
SDS-PAGE was carried out according to standard
protocols (16) using a Bio-Rad minigel apparatus. Gels contained 15%
acrylamide and were stained for protein with silver (17).
Western Blotting and Image Analysis--
Proteins were
transferred to nitrocellulose membrane after SDS-PAGE by standard
procedures (18). Membranes were probed with rabbit polyclonal antisera
against subunit d or OSCP (diluted 1:1000), mouse monoclonal antibodies
against subunit b (diluted 1:7000), and monoclonal antibodies directed
against the hexahistidine tag (diluted 1:500). Secondary antibodies
were alkaline phosphatase-conjugated anti-rabbit and anti-mouse IgG.
Signals were generated by covering the nitrocellulose filter with a 50 µl/cm2 concentration of the chemifluorescent Vistra
substrate (diluted 1:4 with water) and incubating for 5 min at room
temperature. Chemifluorescence at 540-560 nm was detected using a
Storm 820 PhosphorImages (Molecular Dynamics Australia Ltd., Pty.,
Melbourne, Australia) using excitation at 450 nm. Signals were
quantified using ImageQuant software (Molecular Dynamics Australia
Ltd., Pty.). In separate experiments, a linear response of the
detection technique was established for each polypeptide/antibody
combination by analyzing a serial dilution of each sample.
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RESULTS |
Principle of the Method for Determining Subunit
Stoichiometry--
We have previously demonstrated that when
OSCP-Hex6 or subunit d-Hex6 is expressed in a
strain lacking the corresponding endogenous subunit, assembled mtATPase
complexes can be adsorbed from a mitochondrial lysate to
Ni2+-NTA resin by binding of the Hex6-tagged
subunit to the resin (9). We have also recovered assembled mtATPase
complexes containing a tagged subunit
b.2 Recovery of assembled
mtATPase complexes via a Hex6-tagged subunit has now been
exploited in this study to determine the stoichiometry of the three
subunits, d, OSCP, and b.
Strains YMB4, YMB5, and YMB6 are capable of coexpressing wild-type
subunits d, OSCP, and subunit b, respectively, with the Hex6-tagged version of the same subunit. In these strains,
expression of the wild-type subunit, under the tight transcriptional
control of the GAL1 promoter, can be strongly induced by the
addition of galactose to the growth medium. Under the conditions used
in these experiments, expression of the Hex6-tagged
subunit, under the transcriptional control of the PGK1
promoter, was essentially constitutive. The addition of galactose to
cultures of these cells in SaccE growth medium would result in the
induction of expression of the wild-type subunit, while, at the same
time, expression of the Hex6-tagged version would continue.
Cells expressing both the untagged wild-type and
Hex6-tagged subunits would be expected to contain a mixed
population of mtATPase complexes assembled from both forms of each
subunit. We reasoned that the stoichiometry of each subunit could then
be determined by assessing the recovery of untagged and
Hex6-tagged subunits from such mixed mtATPase populations
present in mitochondrial lysates using Ni2+-NTA
chromatography. The prediction was that if the stoichiometry of the
relevant subunit were 1, there would be only two species of mtATPase
complexes present, those containing a single copy of either an untagged
wild-type subunit or the corresponding Hex6-tagged subunit.
Only complexes containing the Hex6-tagged subunit would be
adsorbed to the Ni2+-NTA resin from mitochondrial lysates;
therefore, only tagged subunit would be detected in the mtATPase
complexes recovered. If, however, the stoichiometry for a subunit was
>1, then more than two species would be present in the population of
mtATPase complexes. A subpopulation of complexes would exist that
contains both a wild-type and a Hex6-tagged subunit.
Subsequent Ni2+-NTA chromatography of lysates containing
such a subpopulation would result in the recovery of untagged wild-type
subunit as an integral component of complexes recovered by virtue of
the presence of the Hex6-tagged subunit. The presence of
the untagged wild-type polypeptide in Ni2+-NTA eluates
would therefore indicate that the stoichiometry of a particular subunit
was >1.
mtATPase Containing Hex6-tagged Subunits Is
Functional--
The ability of the each of the Hex6-tagged
subunits to act as a functional replacement for the corresponding
endogenous subunit was tested. Yeast cells lacking expression of genes
encoding subunit d, OSCP, or b are unable to grow on nonfermentable
substrates owing to the absence of a functional ATP synthase. Strains
YMB1, YMB2, and YMB3, expressing subunit d-Hex6,
OSCP-Hex6, and subunit b-Hex6, respectively, in
the absence of the corresponding endogenous subunit, were assessed for
growth in liquid SaccE medium containing ethanol as a nonfermentable
carbon source. The growth rate of these strains at 28 °C was
compared with that of strains A7NP, A5NP, and A4NP, expressing subunits
d, OSCP, and b, respectively, each lacking a Hex6 tag at
the C terminus. Equivalent generation times were observed for all
strains (data not shown).
The function of each of the Hex6-tagged subunits was
investigated in more detail. ATPase activity was measured in lysates of
isolated mitochondria in the presence and absence of oligomycin, an
inhibitor of the F0 proton channel. Such a measurement
gives an indication of the degree of functional coupling between the F1 catalytic sector and the F0 membrane sector
of the enzyme complex. The ATPase activity of mitochondrial lysates
prepared from strains YMB1, YMB2, and YMB3 was found to be closely
comparable to the ATPase activity observed in mitochondrial lysates of
the corresponding control strains A7NP, A5NP, and A4NP (data not
shown). Inhibition by oligomycin of ATPase activity in each of the
mitochondrial preparations was found to be in the range of 82-88% of
the uninhibited activity. Therefore, the modification of subunit d,
OSCP, or b to contain the C-terminal addition of hexahistidine
compromised neither the ability of each of these subunits to assemble
into mtATPase complexes nor the capacity of the resultant complex to generate adequate ATP synthesis for cellular growth.
Recovery of Assembled mtATPase Complexes from Mitochondrial Lysates
by Ni2+-NTA Chromatography--
In preliminary
experiments, the relative levels of tagged and untagged forms of each
of the subunits d, OSCP, and b, expressed in YMB4, YMB5, and YMB6
cells, respectively, were monitored during growth. Cell lysates were
prepared from cells of each strain cultured in growth medium containing
ethanol and galactose and subjected to SDS-PAGE. Following transfer of
proteins to nitrocellulose membrane, blots were probed with appropriate
subunit-specific antisera (data not shown). Mitochondria were prepared
from cells with a high content of the untagged subunit and a low
content of the Hex6-tagged subunit. The ATPase activity in
isolated mitochondria was determined and found to be closely comparable
to that in control mitochondria. Inhibition by oligomycin was similar
and in the range of 84-92% of the uninhibited activity (data not
shown). These results indicate that cells expressing subunit
d-Hex6, OSCP-Hex6, or subunit
b-Hex6 in the presence of the corresponding untagged subunit were not compromised in their ability to assemble functionally coupled complexes.
Mitochondria were lysed, and assembled mtATPase complexes were isolated
by immunoprecipitation with an immobilized F1-
antibody or by adsorption to Ni2+-NTA resin. Proteins purified by
these methods were subjected to SDS-PAGE and visualized by silver
staining (Fig. 1; only data for the
Ni2+-NTA affinity chromatography are shown). The expected
polypeptide profile for a fully assembled ATP synthase (lane
1) was generated using a preparation of the purified complex
isolated from control cells (19). Polypeptides corresponding to each of
the subunits could be identified in eluates from Ni2+-NTA
incubated with lysates of mitochondria containing modified subunits d,
OSCP, and b (lanes 2-4). It was concluded that, in each
case, assembled mtATPase was efficiently recovered by both Ni2+-NTA chromatography and immunoprecipitation (data not
shown). In lane 2 (subunit d-Hex6), a
polypeptide corresponding to subunit d was absent and replaced by a
polypeptide migrating just below the position for OSCP. In lane
3 (OSCP-Hex6), a polypeptide corresponding to OSCP was
absent and replaced by a polypeptide migrating just below subunit b. In
lane 4 (subunit b-Hex6), a polypeptide
corresponding to subunit b was absent and replaced by a polypeptide of
decreased mobility. The change in relative mobility in each case was
consistent with the additional mass (823 Da) of six histidine
residues.

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Fig. 1.
Isolation of mtATPase complexes containing
Hex6-tagged subunits from cells grown in the presence of
galactose. ATP synthase isolated from the parental strain YRD15
(lane 1) by the method of Rott and Nelson (19) was included
as a standard and is representative of a fully assembled complex.
Proteins from lysates of mitochondria isolated after growth in medium
containing galactose prepared from yeast strains YMB4 (lane
2), YMB5 (lane 3), and YMB6 (lane 4) were
recovered by Ni2+-NTA chromatography. Eluates were
subjected to SDS-PAGE analysis and stained with silver. The positions
of selected subunits of the standard ATP synthase preparation are
identified on the left. Arrowheads (lanes 2-4)
indicate the positions of the Hex6-tagged subunits (subunit
d-Hex6, OSCP-Hex6, and subunit
b-Hex6, respectively).
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The Stoichiometry of Subunits d, OSCP, and b Is 1 in Each
Case--
Proteins recovered from lysates of YMB4, YMB5, or YMB6
mitochondria by immunoprecipitation with an immobilized
F1-
antibody or by Ni2+-NTA chromatography
were subjected to SDS-PAGE under conditions that could resolve both the
tagged and untagged forms of each subunit. Separate blots were probed
with monospecific antisera against subunit d, OSCP, or b or
hexahistidine (Fig. 2).

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Fig. 2.
Recovery and immunological detection of
mtATPase subunits. After growth of strains YMB4 (A),
YMB5 (B), and YMB6 (C) in medium containing
galactose, mitochondria were isolated. Proteins were recovered from
mitochondrial lysates by immunoprecipitation with an immobilized
F1- antibody (lane 1) or by
Ni2+-NTA chromatography (lanes 2 and
4). Samples of protein that had not been adsorbed to the
Ni2+-NTA resin during the chromatography procedure were
also analyzed (lane 3). Proteins were separated by SDS-PAGE
and transferred to nitrocellulose membranes. Membranes were probed with
antisera containing antibodies with the following specificities:
subunit d (Sud; A, lanes
1-3), OSCP (B, lanes
1-3), subunit b (Sub; C,
lanes 1-3), and hexahistidine (A-C,
lane 4 in each case). The blots were developed with Vistra
ECF substrate, and bands were visualized by scanning for
chemifluorescence using a PhosphorImager.
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Two polypeptides were detected in material immunoprecipitated from
lysates of each mitochondrial preparation when blots were probed with
antisera against untagged wild-type subunits (Fig. 2, A-C,
lane 1). In each case, a single polypeptide with similar mobility to the slow migrating polypeptide observed in lane
1 was detected when a portion of the blot was probed with a
monoclonal antibody against hexahistidine (Fig. 2, A-C,
lane 4). Therefore, the polypeptides of low mobility
corresponded to subunit d-Hex6 (panel
A), OSCP-Hex6 (panel B),
and subunit b-Hex6 (panel C)
respectively, whereas the polypeptide of high mobility corresponded to
the untagged form of each of the subunits. These results indicate that
both untagged and Hex6-tagged subunits were present in
assembled mtATPase complexes isolated from the mitochondrial
preparations. Quantitative analysis of the signal in lane 1 of the blots is presented in Table I.
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Table I
Quantification of subunits recovered by immunoprecipitation and
Ni2+-NTA chromatography
Chemifluorescent signals corresponding to untagged and
Hex6-tagged subunits on immunoblots of immunoprecipitations and
Ni2+-NTA purifications were quantified using ImageQuant
software on the PhosphorImager scan. The amounts of untagged and
Hex6-tagged subunits recovered are shown as a percentage of the
total population of untagged and Hex6-tagged subunits and
represent the mean ± S.D. of three loadings of the sample.
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A single polypeptide was detected in eluates of Ni2+-NTA
resin incubated with lysates of each mitochondrial preparation when blots were probed with antisera against untagged wild-type subunits (Fig. 2, A-C, lane 2). Quantitative analysis of
the signal in lane 2 indicated that, in assembled complexes
adsorbed to Ni2+-NTA, the Hex6-tagged subunit
represented some 80-95% of the total subunit recovered (Table I).
This result indicates that the stoichiometry of each subunit in
mtATPase is 1. No significant amounts of the Hex6-tagged
subunits were detected in the material not adsorbed to the
Ni2+-NTA resin (Fig. 2, A-C, lane
3), indicating that adsorption to the resin of complexes
containing the Hex6-tagged subunit was complete in each
case. This is an important consideration as it may be expected that
complexes having two or more copies of a Hex6-tagged
subunit would have a significantly higher affinity for the
Ni2+-NTA resin and would, if the resin was present in
limiting amounts, displace complexes containing only one copy of a
Hex6-tagged subunit.
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DISCUSSION |
In this study, the stoichiometry of subunits d, OSCP, and b in the
yeast S. cerevisiae was defined using hexahistidine
technology. Ni2+-NTA chromatography was performed on a
population of mtATPase complexes that were isolated from cells
expressing a Hex6-tagged subunit along with the
corresponding wild-type subunit. The question was then asked, which
subunits, tagged or untagged, along with other subunits of the mtATPase
are adsorbed to Ni2+-NTA resin? The answer to this question
provided a definitive statement about the stoichiometry of these
subunits. On the basis that only the Hex6-tagged subunit
was recovered in eluates, it was concluded that there is no more than
one of each of subunits d, OSCP, and b per mtATPase complex.
Although these results are in accordance with a recent stoichiometric
determination for bovine ATP synthase (5), the implications of these
findings for ATP synthase structure across species become evident when
we look at the stoichiometry of these subunits in the bacterial system.
In Escherichia coli, the stoichiometry of subunits b and
(OSCP homologue) is well established as b2
(20, 21).
Although the bacterial enzyme is considered as the "prototype" of
all F1F0-ATPases, knowledge of its structure
and subunit composition may not be directly applicable to the
prediction of the structure and composition of eukaryotic enzymes.
In E. coli, subunit b is proposed to be part of a stator
that holds the F1
3
3
headpiece in place during rotation of the
subunit. The N-terminal
portion of subunit b is anchored in the membrane; however, the
remainder of the protein is considered to form an
-helical segment
that extends and interacts with the F1 sector (22).
Chemical cross-linking experiments suggest that the C-terminal portion
of subunit b interacts with subunit
, which in turn interacts with
subunits of the F1 sector (3, 23). Recently, subunit b has
been shown to cross-link with subunit
(4). Homologues of subunit b
have been found in all F1F0-ATPases studied so
far. Whereas in E. coli there are two copies of subunit b,
our results suggest there is only one copy of subunit b per complex in
yeast. This difference in stoichiometry means that either another
subunit fulfills the role of the second copy of subunit b or the stator
stalk has a different composition in the mitochondrial enzyme of
eukaryotes. It should be noted that considerable (~30%) homology has
been reported between bovine OSCP and the hydrophilic region of
E. coli subunit b (25). Thus, OSCP in the eukaryotic complex
may fulfill the role of both the second subunit b and subunit
.
Alternatively, a subunit for which there is no homologue in E. coli may fulfill the role of the second copy of subunit b. Such a
subunit may perform a role that would be superfluous in the bacterial
system or, alternatively, may be the functional equivalent, but not a
homologue, in primary amino acid sequence terms, of a bacterial subunit.
We hypothesize that, in yeast, subunit d may take the place of a second
subunit b. In yeast cells lacking expression of subunit d, a component
of the F0 sector, subunit 6, is not assembled into the
inner membrane of the mitochondrion (26). The results of chemical
cross-linking experiments in E. coli indicate that subunit b
is close to subunit a, the homologue of subunit 6 in the mitochondrial ATP synthase (27, 28). Subunit d of the bovine mtATPase can be
cross-linked to subunit A6L, the homologue of subunit 8 in yeast (29).
One may further speculate that the function of the small membrane
integral and hydrophobic subunit 8 replaces the role of the N-terminal
region of subunit b. The C-terminal portion of subunit 8 includes three
positively charged residues and extends into the matrix and would be
available to make contact with subunit d (24). Thus, in yeast, the
combination of the hydrophobic integral membrane protein, subunit 8, and subunit d may serve the function of the second copy of subunit b.
We are now engaged in probing the relationship of subunits 8 and d in yeast.
Although the study of the bacterial ATP synthase has provided us with a
prototype on which to develop other models, its different subunit
stoichiometry and more simple composition mean that some features
cannot be simply extended and expected to apply to mtATPase. The use of
a yeast model allows a wide range of molecular biological approaches to
be used for the study of mtATPase, while, at the same time, providing a
more evolutionarily related model on which to develop our detailed
understanding of the structure and function of the eukaryotic
mtATPase.