From the Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Victoria 3800, Australia
Received for publication, January 29, 2003, and in revised form, February 25, 2003
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ABSTRACT |
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The detailed membrane topography and
neighboring polypeptides of subunit 8 in yeast mitochondrial ATP
synthase have been determined using a combination of cysteine scanning
mutagenesis and chemical modification. 46 single cysteine substitution
mutants encompassing the length of the subunit 8 protein were
constructed by site-directed mutagenesis. Expression of each cysteine
variant in yeast lacking endogenous subunit 8 restored respiratory
phenotype to cells and had little measurable effect on ATP hydrolase
function. The exposure of each introduced cysteine residue to the
aqueous environment was assessed in isolated mitochondria using the
fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein
5-maleimide in osmotically lysed mitochondria and are thus extrinsic to
the lipid bilayer, indicating a 21-amino acid transmembrane span. The
C-terminal region of subunit 8 was partially occluded by other ATP
synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in
maintaining the stability of the F1-F0
interaction. Cross-linking using heterobifunctional reagents revealed
the proximity of subunit 8 to subunits b, d, and f in the matrix and to
subunits b, f, and 6 in the intermembrane space. A disulfide bridge was
also formed between subunit 8(F7C) or (M10C) and residue Cys-23
of subunit 6, demonstrating a close interaction between these two
hydrophobic membrane subunits and confirming the location of the N
termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial
F1F0-ATP synthase.
Mitochondrial F1F0-ATP synthase
(mtATPase)1 consists of a
membrane-extrinsic sector (F1) linked to a
membrane-embedded proton channel (F0) by two protein stalks
(1). Synthesis/hydrolysis of ATP occurs on the structurally well
characterized F1 sector comprised of five subunits
By contrast to that of F1 no high-resolution structure of
the F0 sector is available. In yeast mitochondria
F0 is comprised of at least 12 different polypeptides:
OSCP, b, d, e, f, g, h, i/j, and k that are nuclearly encoded, and
subunits 6, 8, and 9 that are mitochondrially encoded (1).
F0 has two important functions. First, some F0
subunits form a "stator" stalk anchored in the membrane, which
during coupled ATP synthesis/hydrolysis prevents futile rotation of
mtATPase subunits relative to the rotor. This structure has been
visualized in ATP synthases from several organisms (14-16). In the
structurally less complex ATP synthase of Escherichia coli
the stator stalk is composed of subunit In yeast subunit 8 (Y8) is a 48-amino acid intrinsic membrane protein
essential for the assembly and function of mtATPase (1). Whereas
divergent in amino acid composition, Y8 and its other eukaryotic
homologs exhibit three highly conserved regions: an N-terminal MPQL
motif, a central hydrophobic domain (CHD), and a C-terminal positively
charged region (21-23). Allotopic expression (24), whereby a
mitochondrial gene is recoded for nuclear expression with subsequent
delivery of the protein back to mitochondria, has enabled our
laboratory to undertake a detailed molecular genetic approach to
investigate the structure and function of Y8. Detailed studies on
genetically modified Y8 variants, allotopically expressed in yeast
lacking endogenous Y8, have suggested that the N-terminal motif of Y8
plays a functional role in mtATPase (25-27) while the positively
charged amino acid region at the C terminus of Y8 is involved in both
the assembly and function of the F0 sector (27-29). A
surprising feature of Y8 is the functional accommodation of charged
amino acid residues within the CHD (30-33). Analysis by Roucou
et al. (33) has shown that variants bearing either positive or negatively charged amino acids in the CHD display structural destabilization of the F1-F0 interaction but
nevertheless retain function.
Cysteine scanning mutagenesis has been used extensively to analyze
membrane proteins in their native state and provides a powerful
alternative for examining the molecular neighborhood surrounding
membrane proteins (34-36). In a previous study, analysis of
allotopically expressed Y8 variants having a cysteine substitution at
the N- or C-terminal residue of Y8 demonstrated that the CHD spans the
inner mitochondrial membrane with Nout-Cin
orientation (37). Here we have combined cysteine-scanning mutagenesis
with allotopic expression to construct a series of single cysteine replacements encompassing the entire length of the Y8 protein (excluding three residues within the C-terminal positively charged region whose replacement would abrogate function (28)). Without exception each cysteine replacement was tolerated and all of the expressed Y8 variants restored mtATPase function in cells lacking endogenous Y8, demonstrating that none of the substituted amino acid
residues of Y8 are essential for either ATP synthesis/hydrolysis activity or proton flow. Specific labeling experiments using the thiol-modifying chemical reagent fluorescein 5-maleimide (FM) showed
that the membrane-spanning CHD of Y8 encompasses residues 15 to 35. By
assessing the accessibility of introduced cysteine residues to FM in
partially dissociated mtATPase complexes, the C terminus of Y8 was
found to be occluded by other mtATPase subunits, especially in a small
region close to Val-40 demonstrated to play an important role in
maintaining the structural interaction between the F1 and
F0 sectors. In addition, site-directed cross-linking experiments demonstrated extensive interactions between Y8 and subunits
d, f, b, and 6. These results lead us to propose a significant role for
Y8 as part of the stator stalk of mtATPase.
Construction of Y8 Cysteine Replacements--
The gene cassettes
N9L-D/Y8-1 and N9L-D/Y8-1-FLAG, yeast expression vector pPD72, and
yeast strains YM2 and FTC2 were as described previously (37). Briefly,
each gene cassette encodes a full-length wild-type Y8 protein bearing a
7-amino acid extension at the N terminus (YSSEISS, numbered from
Site-directed mutagenesis was carried out on the N9L-D/Y8-1 gene
cassette as described (37) to generate 46 single cysteine replacements
in the expressed Y8 proteins. The Y8(M1C) FLAG variant was constructed
by ligation of a BamHI/KpnI fragment containing the N9L-D sequence and the first 15 nucleotides of the synthetic Y8
gene bearing the M1C mutation into a similarly digested pUC9 vector
containing the FTC2 variant. The (M1C) FLAG cassette was then excised
by BamHI/NotI restriction digestion and ligated
into the pPD72 vector as described (37).
A DNA sequence encoding an HA epitope (YPYDVPDYA) was introduced into
the N9L-D/Y8-1 cassette using two rounds of PCR mutagenesis. The
expressed protein is designated NHAY8. First, the upstream and
downstream flanking primer pair, 5'-CAGGAAACAGCTATGACC-3' and
5'-GGAACGTCGTATGGGTAAGACGAGATCTCGGAAGAGTAGGC-3', respectively, were
used to introduce nucleotides into the N9L-D/Y8-1 cassette specifying
the amino acid sequence YPYDV inserted between the YSSEISS sequence
that was retained following import of allotopically expressed Y8
into mitochondria and the N terminus of the Y8 peptide. A second round
of PCR using the flanking primer pair 5'-CAGGAAACAGCTATGACC-3' and
5'-GGCTCGAGGAGGCGTAGTCAGGAACGTCGTATGGGTAAGACGAGATC-3' was then used to
incorporate adjacent nucleotides into the N9L-D/Y8-1 cassette
specifying amino acids PDYA, to form the complete HA epitope, along
with three additional serine residues between this epitope and the Y8
passenger protein. Thus, the N9L-D/NHAY8-1 cassette encodes a protein
that contains the N9L-D-YSSEISS sequence (as for YM2) followed by the
sequence YPYDVPDYA-SSS-subunit 8, with the original matrix protease
cleavage site maintained. A BamHI/XhoI
restriction fragment bearing the nucleotide sequence encoding N9L-D/NHA
was ligated into similarly digested pUC9 containing the gene cassette
encoding selected N9L-D/Y8-1 cysteine variants to create constructs
NHAY8(L4C), NHAY8(F7C), NHAY8(M10C), NHAY8(F44C), and NHAY8(L48C). The
fidelity of all constructs was checked by DNA sequencing.
Expression of Cysteine Variants in Yeast and Tests for
Restoration of Respiratory Function--
Yeast strain M31
(MAT Thiol-specific Labeling of Mitochondrial Proteins and Analysis of
Protein Fluorescence--
Thiol-specific labeling of mitochondrial
proteins by FM was carried out for 4 h on mitochondria osmotically
lysed by exposure to a hypotonic solution (denoted lysate) as described
(37). Isolated proteolipids were separated by Tricine SDS-PAGE (18% polyacrylamide) according to the method of Schagger and von Jagow (40).
Analysis of protein fluorescence was as described (37). For Y8 cysteine
variants that remained unmodified under these conditions, labeling by
FM was repeated as above in the presence of 1% SDS prior to
proteolipid extraction.
Cross-linking of Mitochondrial Proteins--
Cross-linking of
mitochondrial proteins using the heterobifunctional reagents
p-azidophenacyl bromide (APA-Br) and
N-(4-p-azidosalicylamidobutyl)-3'-(pyridyldithio)-propionamide (APDP) was as described, except that mitochondrial membranes equivalent to 0.5 mg of protein were used (37). Cross-linking using
CuCl2 was performed as follows. Mitochondrial membranes
(equivalent to 0.5 mg of protein) were pelleted at 131,440 × g for 15 min at 4 °C and resuspended in 200 µl of
buffer C (50 mM Tris-HCl, 2 mM
MgCl2, pH 7.5). CuCl2 was added to 1.5 mM (from a 25 mM stock solution, prepared
fresh) and the samples were incubated on ice for 2 h. EDTA was
then added to 10 mM to chelate the remaining Cu2+ and the samples were incubated for 5 min.
Mitochondrial membranes were then pelleted at 131,440 × g for 15 min at 4 °C and the pellet resuspended in 125 µl of 4% SDS (w/v) and 125 µl of dissociation buffer (125 mM Tris-HCl, 2% SDS (w/v), 50% glycerol (v/v), 0.02% bromphenol blue (w/v), pH 6.7). To reduce samples 1 µl of 14.3 mM 2-mercaptoethanol was added and the samples were
incubated on ice for 30 min, then at 65 °C for 10 min prior to
analysis by SDS-PAGE and immunoblotting.
Removal of the F1 Sector from Mitochondrial
Membranes--
Removal of the F1 sector from mitochondrial
membranes was achieved using the modified method of McEnery et
al. (41). Mitochondrial membranes (equivalent to 2 mg of protein)
were pelleted at 131,440 × g for 15 min at 4 °C,
and then resuspended in 0.8 ml of PAB buffer (0.15 M
K2HPO4, 25 mM EDTA, 100 mM 2-mercaptoethanol, pH 7.9). After 5 min incubation on
ice, the mitochondrial membranes were centrifuged as before and the
pellet resuspended in 1.0 ml of GPAB buffer (0.15 M
K2HPO4, 25 mM EDTA, 100 mM 2-mercaptoethanol, 3.0 M guanidine-HCl, pH
7.9). Following incubation on ice for 13 min, membranes were pelleted
as before and washed twice in PA buffer (150 mM
K2HPO4, 25 mM EDTA, pH 7.9) by
resuspension and centrifugation. For fluorescent labeling experiments
membrane samples were then resuspended in 0.2 ml of buffer A and
labeling performed as described above. Samples not intended for
labeling studies were prepared for denaturing glycine SDS-PAGE as
described (37).
Isolation of Monomeric MtATPase and in Situ ATP
Hydrolysis--
Monomeric mtATPase complexes were isolated using clear
native gel electrophoresis according to the method of Arnold et
al. (42), but with the omission of Serva Blue G dye from the
sample. Mitochondrial membranes (equivalent to 100 µg of protein)
were pelleted at 131,440 × g for 15 min at 4 °C and
resuspended in 20 µl of native gel sample buffer (50 mM
NaCl, 2 mM aminohexanoic acid, 1 mM EDTA, 50 mM imidazole, 5 mM phenylmethylsulfonyl
fluoride). Lauryl maltoside was added to a final concentration
of 2.86% (w/v) and the samples (total sample volume 26 µl) were
incubated on ice for 20 min. Insoluble material was pelleted at
131,440 × g for 15 min at 4 °C, and 20 µl of the
sample was applied to a 4-13% polyacrylamide gradient gel (dimensions
13 × 10 × 0.075 cm). Electrophoresis was carried out for
3 h with an initial current of 15 mA and voltage increasing to a
maximum of 500 V. On completion of electrophoresis, gels were
transferred to small plastic containers and in situ ATP
hydrolase activity was assessed using the method of Yoshida et
al. (43). Gels were photographed, then fixed in a solution of
methanol (50%) (v/v), acetic acid (10%) (w/v) for 30 min, and then
stained in acetic acid (10%) (v/v), Serva Blue G dye (0.025%) (w/v)
overnight. Gels were destained in acetic acid (10%) (v/v) for at least
2 h with several changes of solution until protein bands were
distinctly blue against a clear background. Coomassie-stained gels were
analyzed using a ProXPRESS multiwavelength fluorimager (PerkinElmer Life Sciences) and Phoretix 2D analysis software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom).
Second Dimension Protein Analysis by SDS-PAGE--
Denaturing
second dimension gel electrophoresis analysis of isolated mtATPase
complexes was performed as follows. Monomeric mtATPase complexes
isolated using native gel electrophoresis and stained with Serva Blue G
dye were carefully excised from the gel and each polyacrylamide
fragment rinsed in 1 ml of distilled water in a 1.5-ml snap-cap tube.
After a 60-s centrifugation at 20,798 × g, the water
was aspirated off and 50 µl of SDS (5%) (w/v) was added to the tube.
The gel fragment was then finely ground using a thin glass rod and
centrifuged at 20,798 × g for 1 min. Grinding and
centrifugation were repeated and then 50 µl of 2× dissociation
buffer (125 mM Tris-HCl, 2% SDS (w/v), 50% glycerol
(v/v), 0.02% bromphenol blue (w/v), 2% 2-mercaptoethanol (v/v), pH
6.7) was added to the SDS-gel slurry and the samples were heated to
65 °C for 5 min. Following incubation at 4 °C overnight, samples
were stored at Immunoblotting--
Membranes prepared as above were probed with
mouse monoclonal antibodies against either the FLAG (diluted 1:380) or
HA epitopes (diluted 1:2000) (Sigma), or yeast mtATPase subunits Characterization of Single Cysteine Mutants--
A total of 46 single cysteine replacements were made in the Y8 protein at positions
ranging from the amino acid directly preceding the N-terminal
methionine of allotopically expressed Y8 (position
Next, performance of the mtATPase was assessed by measuring ATPase
activity. In each case, the ATP hydrolysis rate was similar to that
measured for mitochondria isolated from strain YM2. In the presence of
the inhibitor oligomycin, however, mitochondria isolated from cells
expressing Y8 variants Y39C or V40C showed faster ATP hydrolysis rates
(p < 0.01 and p < 0.001, respectively) than mitochondria isolated from strain YM2 (Table I). The
decrease in sensitivity to oligomycin displayed by these two cysteine
variants compared with the wild type reflects less efficient coupling
of ATP hydrolysis on the F1 sector with proton pumping
across the membrane in the F0 sector. Collectively, these
results indicate expression of the individual Y8 cysteine substitutions
is generally well tolerated at the level of whole cell growth, with an
effect apparent only when cysteine substitutions are made in a small region of the C-terminal domain of Y8.
Physical Destabilization of the F1F0
Complex by Y39C and V40C Variants--
To investigate whether the Y39C
and V40C substitutions resulted in structural changes within mtATPase,
monomeric enzyme complexes were isolated from mitochondria by clear
native gel electrophoresis. The gels were first assayed for ATP
hydrolysis activity in both the presence and absence of oligomycin, and
then stained with Coomassie Blue to examine any difference in the
profile of the isolated complexes.
In each sample ATP hydrolase activity could be detected as a discrete
band (Fig. 1A, lanes
1-3) demonstrating that the enzyme was isolated in an intact and
functional state. In the presence of oligomycin ATP hydrolase activity
was completely inhibited in enzyme isolated from strain YM2, expressing
unmodified Y8 (Fig. 1A, lane 4). By contrast, a
very small amount of residual activity was seen for mtATPases isolated
from cells expressing either the Y39C or V40C variants (Fig.
1A, lanes 5 and 6). This clearly
demonstrates that while the isolated mtATPases are both intact and
coupled, in mtATPases assembled with either the Y39C or V40C variants
the structure of the complex has become sufficiently destabilized such
that it is less sensitive to oligomycin in the in situ
assay. Whereas not excluding the possibility of alteration to the
binding of oligomycin the results indicate that there exists a
heterogenous population of mtATPases in Y39C or V40C mitochondria, some
of which are insensitive to oligomycin inhibition.
Next, the gels were stained with Coomassie Blue to demonstrate the
presence of intact mtATPases. In each case, a single protein band was
stained at a position corresponding exactly to that of the
oligomycin-sensitive, ATPase-active band observed after in situ assay (Fig. 1B, lanes 1-6).
Immunoblotting analysis confirmed the identity of this band as intact
mtATPase because several subunits belonging to both the F1
(subunits Strategy for Determination of Solvent-exposed Regions of
Y8--
We next sought to determine the extent of the
membrane-embedded and extra membranous regions of Y8. Our approach was
to determine the local side chain environment using FM to specifically
label the cysteine residues introduced into Y8. FM is a small,
non-polar compound that is highly reactive with thiol groups in an
aqueous environment (45). It is also sufficiently hydrophobic to
partially penetrate into hydrophobic environments and even to cross the mitochondrial membrane at high concentrations (37, 46). Importantly, FM
is unable to react with a thiol group that is either: 1) very close to
and facing the boundary of the lipid bilayer; or 2) embedded within it,
because generation of a reactive thiolate anion at, or in, the membrane
is impeded by the low dielectric constant of that environment (47, 48).
Thus, FM should react readily with any available thiol group in an
aqueous environment, less readily in a hydrophobic environment, and not
at all within a lipid environment. Accordingly, our expectation was
that as the cysteine residue introduced into Y8 was located closer to
the aqueous-lipid interface, its reactivity with FM would steadily decrease until no reaction is observed because of the proximity of the
lipid bilayer. Such decreased reactivity would be reflected as a
decreased intensity of fluorescence that was detectable following labeling of membrane preparations containing Y8 and analysis of the
partially purified proteins separated by SDS-PAGE. Use of this strategy
potentially enables accurate delineation of the boundaries of the
lipid-aqueous interface for both the N- and C-terminal domains of
Y8.
Accessibility of Introduced Cysteine Residues in Y8--
To assess
the exposure of cysteine residues to the aqueous phase, isolated
osmotically lysed mitochondria were incubated in the presence of 1 mM FM for 4 h. After removal of excess FM,
proteolipids were extracted from the membranes and separated by Tricine
SDS-PAGE. The gel was scanned for FM fluorescence (upper
panel in Fig. 2, A and
B) and then silver-stained (lower panel in Fig.
2, A and B). The stained gel was then overlaid
onto the fluorescent image to correlate stained proteins with the
fluorescent signal obtained. Y8 variants S
Y8 variants bearing cysteine replacements at residue positions
At the N terminus of Y8 a gradient of accessibility to FM was evident
proceeding from the N terminus toward residue 14 (Fig. 3A).
Cysteines at positions
At the C terminus of Y8 a gradient of decreasing accessibility was
again evident from residue position 48 through 36 (Fig. 3B).
As for the N-terminal residues, increased concentrations of FM or
extended periods of incubation failed to increase the reactivity of any
residue at the C terminus of Y8. Moreover, shorter incubation times (1 h) again resulted in an overall decrease in detectable FM fluorescence
and a loss of fluorescent signal from variants L38C and L36C (data not
shown). Residues at positions 38 and 36 therefore are likely to lie in
close proximity to the lipid bilayer, whereas residue 35 is located
within, or faces the lipid bilayer and is unavailable for modification
because a substituted cysteine at this position is not reactive with
FM. It was also noted that, whereas cysteines at positions 48 through 36 are reactive with FM and thus exposed to the aqueous phase, they are
comparatively less reactive than are most residues at the N terminus of
Y8 (compare Fig. 3, A and B). Because the C terminus of Y8 is located in the mitochondrial matrix (37, 49) it seems
likely that the apparent reduction in reactivity may be because of
steric occlusion by other mtATPase subunits.
Effect of Partial Removal of the F1 Sector on
Accessibility of the C terminus of Y8--
If the comparatively
reduced reactivity of C-terminal versus N-terminal cysteine
substitutions observed is because of steric occlusion of Y8 by other
mtATPase subunits, the removal of the F1 sector from
membranes may increase their accessibility to FM. To demonstrate
removal of the F1 sector of mtATPase from mitochondrial membranes, mitochondria isolated from strain FTC2 (expressing wild type
Y8 with a C-terminal FLAG epitope) were treated with guanidine HCl and
the remaining protein complement assessed by SDS-PAGE and
immunoblotting. Membranes were probed with monoclonal antibodies
against mtATPase subunit
Mitochondria isolated from yeast expressing the Y8 cysteine variants
S46C, V40C, L36C, I35C, and YM2 were lysed and incubated with guanidine
HCl prior to incubation with FM. Accessibility of residue 46 was
assessed because it is adjacent to Leu-48 but displays only
~50% of the relative accessibility, and that of residue 40 as it
marks the boundary of a slight decrease in relative accessibility at
the C terminus that extends from residue positions 40 through 46 (Fig.
3B). Cysteine residues at positions 36 and 35 were chosen as
they are reactive or not with FM in the mitochondrial lysate,
respectively, and are presumed to denote the membrane boundary of Y8.
After guanidine HCl treatment the reactivity of cysteine residues at
positions 40 and 46 increased substantially compared with untreated
controls (Fig. 5, lanes
7-10). The reactivity of the Y8 variant L36C, previously
localized to the membrane boundary, was not visibly improved by prior
incubation of membranes with guanidine HCl (Fig. 5, lanes 5 and 6) suggesting that its low reactivity was because of the
proximity of the lipid bilayer and not steric occlusion of FM binding
by other mtATPase subunits. Residue 35, localized within the lipid
bilayer, remained unreactive to FM after guanidine HCl treatment (Fig.
5, lanes 3 and 4) indicating that the position of
Y8 within the membrane remained unchanged. As expected, no fluorescence
was ever detected for YM2 (Fig. 5, lanes 1 and
2).
These results demonstrate first that the reactivity of cysteine
substitutions V40C and S46C can be substantially increased by at least
partial removal of the F1 sector from the membrane, whereas
reactivity of the L36C substitution cannot be improved in this manner.
Second, the increased reactivity displayed by the V40C and S46C
substitutions is not because of the removal of phospholipids
surrounding the introduced cysteines nor any alteration to the position
of the membrane-embedded domain of Y8 because the reactivity of
cysteines at positions 35 and 36 remained unchanged. Thus, the
reactivity of cysteines at positions 40 and 46 is to some degree
determined by the partial occlusion of the C terminus of Y8 by other
subunits of mtATPase.
Cross-linking of Y8 to mtATPase Subunits b, d, f, and
6--
Previously, we have demonstrated cross-linking between the
matrix-located C terminus of Y8 and each of subunits d and f, and the N
terminus of Y8 to subunit f in the intermembrane space (37). Here we
have attempted to define more fully interactions involving Y8 with
other mtATPase subunits by using additional cysteine substitution mutants in cross-linking experiments. For these studies, an HA epitope
was fused to the N terminus of the Y8 protein to facilitate detection
of cross-linked products. Cells expressing the HA-tagged Y8 variants
demonstrated normal respiratory growth compared with strain YM2, and
the epitope-tagged Y8 was detected in isolated, functional mtATPase
complexes (data not shown). Mitochondria prepared from selected strains
expressing HA-tagged Y8 cysteine variants were incubated with APDP or
APA-Br and the cross-linked products analyzed by SDS-PAGE and
immunoblotting. APDP is capable of cross-linking over a distance of 21 Å, whereas APA-Br can cross-link proteins up to a distance of 9 Å (37), thus the presence or absence of cross-linked products reflects
both distance constraints between the cross-linked partners and
accessibility of the reagents to the introduced cysteine. At each of
the N and C termini of Y8, several cross-linked products were detected
using antibodies against the HA epitope in Y8 (Fig.
6, A and B). No
cross-linking was ever detected for wild-type Y8 (Fig. 6, A
and B) demonstrating that each of the cross-linked products
observed involved the unique introduced thiol in each of the Y8
variants.
Immunoblotting analysis using antibodies against other mtATPase
subunits was undertaken to identify partners involved in cross-links to
Y8. Each of the ~22- and ~24-kDa adducts formed using APDP was
cross-reactive with antibodies against mtATPase subunit f (Fig.
7A). The apparent anomalous
migration of the Y8-subunit f cross-linked product is similar to that
observed in experiments involving cross-linking of subunit b to d (50).
This effect is presumably caused by alternate locations of the
cross-linking between the two proteins, leading to differences in their
hydrodynamic volumes during SDS-PAGE and thus reflected as a difference
in apparent mobility in the gel (50). Identical results were obtained when cross-linking was performed using APA-Br (not shown), except that
the ~22-kDa Y8-subunit f cross-link was absent in the L4C sample but
present in the M10C sample; also, both of the ~22- and ~24-kDa
Y8-subunit f adducts were simultaneously present in the F7C, F44C, and
L48C samples when the shorter cross-linker was used (Fig. 6).
Presumably this reflects steric constraints on the ability of each
cross-linker to form a product when bound at different positions in
Y8.
The ~40-kDa adduct formed from Y8 F7C, M10C, and F44C variants using
APDP was cross-reactive with antibodies against mtATPase subunit b
(Fig. 7B). Identical results were obtained using APA-Br (not
shown), except that the ~40-kDa Y8-subunit b product was not formed
from the F7C or M10C replacements (Fig. 6). The ~30-kDa adduct formed
from each of the F44C and L48C variants using APDP was cross-reactive
with antibodies against mtATPase subunit d (Fig. 7C).
Identical results were obtained when cross-linking was performed using
APA-Br (not shown).
The ~35-kDa adduct formed from the Y8 F7C and M10C replacements was
cross-reactive with antibodies against mtATPase subunit 6 (Fig.
7D). Identical results were obtained when cross-linking was
performed using APA-Br (not shown), except that the ~35-kDa Y8-subunit 6 product was not formed from the F7C variant (Fig. 6).
Also, this Y8-subunit 6 adduct was formed in response to UV irradiation
alone in the M10C sample (Fig. 7D, lane 3)
suggesting it may represent oxidation of the unique thiol group on
Y8.
Disulfide Formation between Y8 F7C or M10C and Subunit 6 C23--
The UV-induced Y8-subunit 6 adduct could be reduced in the
presence of 2-mercaptoethanol (Fig. 6A, lane 13)
suggesting it may involve a disulfide bridge. Incubation of
mitochondria with CuCl2 strongly induced formation of the
~35-kDa product in each of the F7C and M10C variants but not in the
wild-type sample (Fig. 8A,
lanes 1-3). Subsequent incubation with 2-mercaptoethanol
completely reduced the disulfide bridge (Fig. 8A,
lanes 4-6). This CuCl2-induced product was
confirmed as a Y8-subunit 6 cross-link using antibodies against
mtATPase subunit 6 (Fig. 8B).
Membrane Topography of Subunit 8--
Y8 is an intrinsic membrane
protein whose N terminus is in the intermembrane space and C terminus
in the mitochondrial matrix (37). In this work, application of FM
affinity labeling enabled identification of the membrane-intrinsic, as
opposed to membrane-extrinsic, regions of Y8 in its assembled form
within the mtATPase complex. Thus, a 14-amino acid region at the N
terminus of Y8, and a 13-amino acid region at its C terminus, can be
modified by FM in a mitochondrial lysate and are thus located outside
the lipid bilayer. Accordingly, Y8 possesses a 21-amino acid
transmembrane span, in agreement with hydropathy profiles (21, 23). The
membrane-extrinsic regions of Y8 are expected to interact with other
mtATPase subunits. Indeed, the C-terminal region of Y8, exposed in the
matrix, was partially occluded from FM binding in the intact,
membrane-embedded mtATPase. Partial removal of the F1
subunits increased the exposure of this region of Y8 to the aqueous
phase, allowing more efficient reaction with FM. Therefore, the
C-terminal region is intimately involved with other matrix-located
components of mtATPase.
The N terminus of Y8 maintains a highly conserved MPQL motif (23, 51)
that has previously been assigned a role in function (23, 27). Cysteine
substitutions for any of these 4 conserved amino acids had no
demonstrable effect on cell growth or ATP hydrolase activity,
suggesting that in yeast none of these residues is individually required for mtATPase function. However, it was noted that
substitutions in this region caused aberrant mobility of the respective
Y8 cysteine variants, especially for the M1C replacement, during
SDS-PAGE. The reason for this phenomenon is not known, but may reflect
altered processing of the Y8 pre-protein or modification of Y8 because of the introduction of a cysteine at these positions.
Localization of Subunit 8 in the Complex with Respect to Other
mtATPase Subunits--
Y8 plays an essential role in maintenance of
the structural integrity of the interaction between F1 and
F0, and is thus essential for mtATPase function (1).
Previous work demonstrated interactions between Y8 and mtATPase
subunits d and f based on analysis of Y8 variants bearing cysteine
substitutions at either their extreme N or C termini (37). Here we have
employed site-directed cross-linking to demonstrate contacts between Y8
and subunits d, f, b, and 6. This leads to novel information about the
molecular neighborhood of Y8 in yeast mtATPase.
In a mitochondrial lysate, Y8 could be cross-linked to each of subunits
b and f in both the matrix and intermembrane space. In the matrix,
cross-linking to subunit b was from Y8(F44C) at a distance no greater
than 9 Å. Subunit b has two extra membranous regions that project into
the matrix between residues 1-26 and 72-209 (50), whereas N-terminal
residues 1-66 of subunit f are also extrinsic to the membrane (52).
Accordingly, these regions of each subunit are available to interact
with Y8. In the intermembrane space, cross-linking occurred from
Y8(F7C) and Y8(M10C) at a distance between 9 and 21 Å to subunit b,
whose polar loop is exposed from residues 46 to 55 (53, 54).
Cross-linking to subunit f, exposed between residues 85 and 94 (52),
was from a maximum of 9 Å to a maximum of 21 Å depending on the
location of the introduced cysteine (see Fig. 6). Overall, these
results demonstrate that Y8 is in close proximity to each of subunits b
and f across the membrane and in both compartments of the mitochondrion.
Y8 was also able to cross-link with subunit d in the matrix at no
greater than 9 Å. At present the orientation of subunit d is unknown,
but cross-linking of subunit d to each of subunits b (50) and i/j (55)
in the matrix has also been demonstrated.
A disulfide bridge was formed between Y8(F7C) and Y8(M10C) to subunit 6 in the intermembrane space, demonstrating a direct interaction between
these two subunits. Subunit 6 possesses a single endogenous cysteine
(Cys-23) in its first extra membranous region and located close
to the membrane (53, 56). In addition to the close interaction between
subunits 8 and 6, this result emphasizes two points. First, the N
termini of each of Y8 and subunit 6 are located in the intermembrane
space. Second, the following residues of these proteins all lie close
to the membrane boundary: Y8(F7C) and Y8(M10C), and subunit 6 Cys-23. Aspects of the detailed arrangement of other
F0 subunits, as understood on the basis of cross-linking
analyses here and elsewhere (37, 53-55, 57-59), are shown in Fig.
9.
Subunit 8 Is Part of the Stator Stalk of mtATPase--
Several
lines of evidence now support the view that Y8 is a part of the stator
stalk in yeast mtATPase. Our cysteine scan revealed no significant
effects on function for any of the Y8 variants constructed. This
suggests Y8 is a structural component of mtATPase, because no amino
acid of Y8 directly participates in either ATP synthesis/hydrolysis or
proton pumping. A structural role for Y8 is also consistent with the
notion that the overall shape of Y8 and broad chemical character of its
amino acids (rather than their specific identities) are important for
its role in mtATPase function (32, 60). The introduction of charged
residues within the CHD by Roucou et al. (33) showed that Y8
is required for maintenance of the interaction between F1
and F0 as well as correct F0 assembly. Here we
have defined two residues, Tyr-39 and Val-40, which are similarly
important for the integrity of the structural linkages between
F1 and F0. Furthermore, occlusion of the C
terminus of Y8 by other mtATPase proteins suggests that these
effects on stability of the enzyme may be mediated through other
mtATPase subunits.
The data presented here show that Y8 maintains close interactions with
subunits b, d, f, and 6. Each of these subunits has proposed roles as
part of the stator stalk in yeast mtATPase (18, 37, 55). This view is
reinforced by the interdependence of each subunit for the assembly of
the F0 sector. Each of subunits b, d, f, or 8 is required
for the stable integration of subunit 6 into the membrane; the loss of
any one of these subunits leads to a functionally uncoupled
F1 sector that becomes loosely bound to the membrane (18,
30, 59, 61, 62). Moreover, subunit f is also required for the presence
of subunits b, 8, and 9 (59). It has also been suggested that subunit
i/j is required for the incorporation of both of subunits 6 and f into
the complex (63). The direct interaction between Y8 and the N terminus
of subunit 6 implies that these subunits interact directly during their
assembly into mtATPases in the mitochondrial membrane.
The stator stalk of bacterial F1F0-ATPase is
comprised of two copies of subunit b, which dimerize along a large
portion of their length (17, 64). However, only a single copy of
subunit b is present in yeast mtATPase (18). If the stator stalks of bacterial, chloroplast, and mitochondrial
F1F0-ATPases share a similar overall design,
then other proteins should fulfill the role of the missing subunit b in
mtATPase (18). Cross-linking studies have demonstrated extensive
interactions involving mtATPase subunits b, d, f, and 6 (50,
53-55, 57-59). We now show interactions involving each of these
subunits with Y8, highlighting the location of Y8 deeply embedded
within, and located close to, each of the major structural components
of the stator stalk. Accordingly, we propose that subunits d, f, and Y8
fulfill the role of the missing subunit b in forming the connection
between F1 and F0 in yeast mtATPase. Further
elucidation of the structural interactions between these subunits will
reveal the detailed architecture of the stator stalk in mtATPase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-
3-
1-
1-
1
(2-4) and is coupled to proton flux through the F0 sector.
The crystal structure of an incomplete mtATPase from yeast determined
by Stock et al. (3) showed that the
-
-
subunits
form a central stalk that is closely associated with a ring of 10 copies of membrane-embedded subunit 9. Together these proteins
constitute the "rotor" of mtATPase, which has been conclusively
demonstrated to rotate during ATP synthesis/hydrolysis relative to the
other subunits of the complex (5-13).
and two copies of subunit b
(17). However, only a single copy of subunit b is present in the
mtATPase of the yeast Saccharomyces cerevisiae (18); thus,
in conjunction with OSCP (the mitochondrial homolog of subunit
),
other F0 subunits contribute to form the stator stalk.
Second, movement of protons through the membrane-embedded channel of
F0 allows conversion of the proton gradient generated by
respiratory chain activity into chemical energy, in the form of ATP
made by phosphorylation of ADP on F1. Based on molecular genetic and biochemical studies in yeast the assembly and stability of
the membrane-embedded proton channel of mtATPase depends on the
presence of the three mitochondrially encoded protein subunits 6, 8, and 9 (19). Subunits 6 and 9 are the homologs of bacterial ATP
synthase subunits a and c, respectively, and have well defined roles in
proton channel function (20). Subunit 8 is not found in bacteria but is
an additional subunit present in the mtATPase of mammals and fungi. The
absence of all but one of these F0 subunits from the yeast
F1-c10 crystal structure (3) highlights the need for additional biochemical approaches aimed at elucidating the
structure and function of F0 subunits.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 to
1), which is retained following import of Y8 into mitochondria and
processing of the N9L mitochondrial import sequence by matrix protease.
The N9L-D/Y8-1-FLAG construct expresses the same protein bearing a
C-terminal FLAG epitope (DYKDDDDK). Cells expressing the N9L-D/Y8-1
cassette are denoted YM2, those expressing N9L-D/Y8-1-FLAG are denoted FTC2.
ade1
his6)[aap1
] lacking endogenous Y8 has
been described previously (30). M31 cells were transformed by the
method of Klebe et al. (38) with the pPD72 expression vector
that carries a functional ADE1 gene (28). Transformants displaying the Ade+ phenotype were selected and restoration
of respiratory-competent phenotype by the expressed Y8 cysteine
variants was assessed by testing growth on medium containing ethanol as
the sole carbon source. Generation times, ATP hydrolysis activity in
isolated mitochondria, and its sensitivity to oligomycin were measured as described previously (39). Comparisons (Student's t
test) were made against strain YM2 (28), allotopically expressing unmodified Y8.
20 °C until use. Prior to electrophoresis, the
samples were heated to 65 °C for 5 min and then centrifuged at
20,798 × g for 5 min. Immediately following
centrifugation 20 µl of the sample was removed, taking care to avoid
any fragments of acrylamide gel present in the tube. Samples were
electrophoresed (glycine SDS-PAGE) in a 15% polyacrylamide denaturing
gel and transferred to PVDF membrane for immunoblotting.
(diluted 1:5000),
(diluted 1:7000), or b (diluted 1:5000) from our
library of mtATPase-specific antibodies (44). Rabbit polyclonal
antisera were also employed against yeast mtATPase subunits
, d, or
OSCP (diluted 1:1000) (44), f, 6, or h (diluted 1:10000, kindly
provided by Dr. J. Velours), and e, g, i/j, or k (diluted 1:500, kindly provided by Dr. R. A. Stuart). Secondary antibodies and detection were as described (37). Antibody binding was quantified using ImageQuant software (Amersham Biosciences).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) to the most
C-terminal residue (position 48). The lysine at position 37 and two
arginines at positions 42 and 47 were not substituted as each of these
residues contributes to the C-terminal positively charged region
required for assembly of Y8 into mtATPase (28). To test the
functionality in vivo of Y8 cysteine variants, M31(aap1
) cells expressing each single
cysteine variant were plated onto medium containing ethanol as the sole
carbon source. Without exception, expression of each variant was able
to restore respiratory function. However, cells expressing either the
Y39C or V40C Y8 variants displayed apparently slower growth on solid
medium than did control strain YM2 at 37 °C, yet displayed normal
growth at either 18 or 28 °C. To further assess functionality of the
Y8 variants at the whole cell level, the generation time of each Y8
variant-expressing strain was assessed in liquid medium containing
ethanol as the sole carbon source. Of the 46 cysteine variants tested,
only three had a significant effect (Student's t test) on
whole cell growth rate. Expression of the L38C variant resulted in a
significant decrease (p < 0.01) in generation time
compared with strain YM2 expressing unmodified Y8 (Table
I). The reason for this increased rate of
growth is unclear and has not been examined further. Because yeast
should have an excess capacity for ATP production, increased ATP
synthesis should not promote growth. By contrast, expression of Y8
variants Y39C or V40C, respectively, displayed a significant increase
in generation time compared with that of strain YM2 (p < 0.01 in each case) (Table I).
Growth properties and mitochondrial ATP hydrolase activities of cells
expressing Y8 variants
1 mitochondrial
protein. Whole cell generation times represent the mean ± S.D. of
triplicate assays.
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Fig. 1.
Structural perturbation of the
F1-F0 interaction by the Y8 substitutions Y39C
and V40C. Intact, monomeric mtATPase complexes were isolated by
native gel electrophoresis (4-13% polyacrylamide gradient). Prior to
staining, the ATPase activity of complexes (panel A) was
demonstrated in situ in the absence (lanes 1-3)
or presence (lanes 4-6) of oligomycin. The polyacrylamide
gels were then stained with Coomassie Blue (panel B), and
the identity of proteins comprising each band was established by second
dimension SDS-PAGE (15% acrylamide) and immunoblotting analysis (not
shown). The source of mitochondria is indicated at the top
of each panel.
,
and
) and F0 (subunits b, d, f, OSCP,
and 6) sectors were identified (data not shown). In each of the Y39C
and V40C variants a second prominent stained band of faster mobility
was detected (Fig. 1B, lanes 2-3 and
5-6), which was either absent or below the limit
of detection in the YM2 sample (Fig. 1B, lanes 1 and 4). Immunoblotting analysis demonstrated the presence of both the
and
subunits in each of these bands but not any F0
subunits tested (data not shown); thus, this band represents a
dissociated F1 portion of mtATPase.
1C and L48C have
previously been demonstrated to react strongly with FM in a
mitochondrial lysate (37) and effectively act as positive controls for
labeling of the N and C termini of Y8, respectively. Cysteine residues
introduced at positions
1 through 14 at the N terminus of Y8 were all
accessible and available for modification by FM (Fig. 2A,
lanes 2-16) as were cysteines introduced at positions 36 through 48 in the C terminus (Fig. 2B, lanes
18-27). As expected, no fluorescence was observed for unmodified
Y8 (Fig. 2A, lane 1). These cysteine residues are
therefore, a priori, exposed to the aqueous phase and
available for modification in a mitochondrial lysate. While decreasing
the period of FM incubation to 1 h resulted in a reduction of the
intensity of labeling for all of the reactive residues, neither
extended incubation times (up to 8 h) nor increased concentrations
of FM (2 or 4 mM) resulted in detectable modification of
introduced cysteine residues at positions 15 through 35, although Y8
could still be detected by silver staining (data not shown). These
non-reactive residues could, however, be modified in the presence of
SDS (data not shown) suggesting that they can indeed be modified but
are not accessible to FM in a mitochondrial lysate, as expected for
residues sequestered within the lipid bilayer. Thus, we conclude that
the first 14 and last 13 amino acids at the N and C termini of Y8,
respectively, are exposed to the aqueous phase, extrinsic to the lipid
bilayer, and that residues 15 through 35 are embedded in the inner
mitochondrial membrane.
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Fig. 2.
Reactivity of introduced cysteine residues in
Y8 with FM. Isolated mitochondria were osmotically lysed and
treated with the thiol-specific reagent FM. Proteolipids were extracted
and separated by Tricine SDS-PAGE (18% polyacrylamide) and analyzed by
fluorescence (F) and direct silver staining (S)
(images aligned with respect to the geometry of the original gel). The
position of the cysteine replacement in Y8 is indicated. Panel
A, N-terminal cysteine replacements at positions 1 through 14 (lanes 2-16); panel B, C-terminal cysteine
replacements at positions 35 through 48 (lanes 17-27).
Unmodified Y8 is designated YM2 (panel A, lane
1). In each of panels A and B, the
fluorescent image is located directly above the
corresponding silver-stained region of the gel containing Y8.
Panel C, immunoblotting of C-terminal FLAG-tagged Y8
variants either unmodified (FTC2, lane 1) or bearing an
N-terminal substituted cysteine residue (M1C, lane 2) and
detected using anti-FLAG antibodies.
1
through 5 (Fig. 2A, lanes 2-7) and labeled with
FM apparently migrated more slowly in SDS-PAGE than did unmodified Y8
(Fig. 2A, lane 1) or any of the other labeled Y8
cysteine variants. Variants S
1C and M1C co-migrated with an
unidentified, non-fluorescent band of ~8-9 kDa (Fig. 2A,
lanes 2 and 3, lower panel), whereas the labeled Y8 variants P2C through V5C each migrated with an apparent
mobility slightly slower than that of unmodified Y8 (Fig. 2A, lanes 4-7, lower panel). The
apparent alteration to molecular weight was not due to binding of the
FM moiety, as the size shift was observed after silver staining
regardless of the presence or absence of FM (data not shown). For each
of the S
1C and M1C variants, a second broad silver-stained band of
apparent equal molecular weight to unmodified Y8 (see Fig.
2A, lanes 1-3, lower panel) was
identified, suggesting the presence of two distinct species of Y8 in
these samples. Moreover, immunoblotting of the Y8(M1C)-FLAG variant
(bearing a C-terminal fused FLAG epitope) also showed the presence of
two Y8 species (Y8 and Y8*), one having mobility comparable with that
seen for FTC2 mitochondria expressing cysteine-less Y8-FLAG and one of
slower mobility, respectively (Fig. 2C, lanes 1 and 2). To assess the relative accessibilities of cysteines
at the N and C termini of Y8, the intensity of labeling by FM for each
Y8 variant, P2C through T14C and L36C through L48C, was analyzed and
compared with the intensity of the silver-stained protein (Fig.
3). The data for Y8 variants S
1C and
M1C were not analyzed because of the apparent presence of two distinct
proteolytically processed variants.
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Fig. 3.
Relative accessibility of residues at the N
and C termini of Y8. The intensity of FM fluorescence
("fluorescent intensity") for each reactive cysteine replacement at
either the N terminus (panel A) or C terminus (panel
B) of Y8 was quantified and compared with the intensity of silver
staining ("protein intensity") for each sample. Fluorescent
intensity was divided by protein intensity to relate the amount of
fluorescent signal from FM to the amount of protein present, and the
values for each individual residue averaged from at least three
independent experiments to generate the term "accessibility." The
position of the cysteine substitution in Y8 is indicated. Unmodified
Y8, corresponding to background levels of fluorescence, is designated
YM2.
1 through 9 displayed strong reactivity with
FM compared with cysteines at positions 10 through 14 (Fig. 3A; see also Fig. 2A, lanes 1-16). As
stated above, neither extended incubation with FM nor higher FM
concentrations increased the reactivity of residues beyond position 10. Moreover, conditions involving a shorter period of incubation (1 h)
with FM, during which the Y8 variant P2C was maximally labeled,
resulted in decreased fluorescent intensity for labeled cysteines
beyond position 10 and a complete absence of labeling for Y8 variants
L13C and T14C (data not shown). These results demonstrate that
cysteines at positions
1 through 9 are in a highly exposed location,
extending into the intermembrane space of the mitochondrion. Residues
beyond position 10 are located closer to the lipid bilayer, where the environment becomes less favorable for reaction between FM and the
introduced thiol moiety. Therefore, residues at positions 13 and 14 are
very close to the lipid boundary but when substituted by cysteine are
still reactive with FM, thus demonstrating their location on the outer
face of the inner mitochondrial membrane.
as a marker for F1-sector proteins and monoclonal antibodies against the FLAG epitope to demonstrate the presence of Y8 in the membrane preparations. After treating isolated mitochondrial membranes with guanidine HCl the amount
of detectable subunit
was reduced by 45% compared with untreated
mitochondria (Fig. 4, lanes 1 and 2). Degradation of subunit
was evident as
cross-reactive protein bands of higher apparent mobility in each
sample. Y8 remained associated with membranes after treatment (Fig. 4,
lane 4) and its detection was enhanced using anti-FLAG
antibodies compared with untreated mitochondria (Fig. 4, lane
3). Thus, guanidine HCl treatment of the mitochondrial membrane
was able to partially remove at least the
subunit of the
F1 sector of mtATPase while not causing depletion of
membrane-embedded Y8.
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Fig. 4.
Partial removal of the F1 sector
of mtATPase by treatment with guanidine HCl. Mitochondria isolated
from strain FTC2 expressing C-terminal FLAG-tagged Y8 were incubated
with guanidine HCl (G-HCl). The proteins were separated by
SDS-PAGE (15% acrylamide) and transferred to PVDF membranes. Membranes
were probed with antibodies against the mtATPase subunit (lanes 1 and 2) as a marker for the presence of
the F1 sector, or antibodies against the FLAG epitope
(lanes 3 and 4) to demonstrate the presence of Y8
in the membrane. The positions of size standards are indicated at the
left of the figure.
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Fig. 5.
Reactivity of selected introduced cysteine
residues in the C terminus of Y8 after removal of the F1
sector by guanidine HCl treatment. Mitochondrial membranes either
treated with guanidine HCl (Gu HCl) (+) or not ( ) were
incubated with FM. Proteolipids were extracted and separated by Tricine
SDS-PAGE (18% polyacrylamide) and analyzed by fluorescence
(F) and direct silver staining (S) (images aligned with
respect to the geometry of the original gel). The position of the
cysteine replacement in Y8 is indicated. Sources of mitochondria were:
YM2, expressing unmodified Y8 (lanes 1 and
2); Y8(I35C) (lanes 3 and 4); Y8(L36C)
(lanes 5 and 6); Y8(V40C) (lanes 7 and
8); Y8(S46C) (lanes 9 and 10).
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Fig. 6.
Cross-linking of the N and C termini of Y8
using APDP or APA-Br. Mitochondria isolated from cells expressing
NHAY8 cysteine variants were incubated with either APDP or APA-Br, the
excess cross-linker removed, and the samples were exposed to UV
irradiation. Mitochondrial membranes were then dissolved, proteins were
separated by SDS-PAGE (15% acrylamide) and transferred to PVDF
membranes. Cross-linked products (bands I thorough
VIII) were detected using anti-HA antibodies. The presence
(+) or absence ( ) of either APDP or APA-Br is indicated. Sources of
mitochondria were as follows. Panel A, NHAY8(L4C)
(lanes 1-2 and 9-10), NHAY8(F7C) (lanes
3-4 and 11-12), and NHAY8(M10C) (lanes
5-6 and 13-14). Panel B, NHAY8(F44C)
(lanes 1-2 and 7-8) and NHAY8(L48C)
(lanes 3-4 and 9-10). Cysteine-less Y8
(panel A, lanes 7-8 and 15-16, or
panel B, lanes 5-6 and 11-12) is
designated WT. The positions of size markers are indicated
at the left of the figure.
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Fig. 7.
Identification of cross-linked products
involving Y8. Mitochondrial proteins isolated from cells
expressing NHAY8 cysteine variants were separated by SDS-PAGE (15%
acrylamide) and transferred to PVDF membranes. Cross-linked products
detected previously (Fig. 6, A and B) were probed
using antibodies against mtATPase subunits f (panel A), b
(panel B), d (panel C), or 6 (panel
D). The presence (+) or absence ( ) of cross-linker, source of
mitochondria, and position of size markers are indicated.
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Fig. 8.
Disulfide bridge formation between NHAY8
cysteine variants (F7C) and (M10C) with the endogenous Cys-23 of
subunit 6. Mitochondria isolated from strains expressing NHAY8
variants (F7C or (M10C) were incubated with CuCl2.
Mitochondrial membranes were dissolved, proteins were separated by
SDS-PAGE (15% polyacrylamide) and transferred to PVDF membranes.
Cross-linked products were detected as previously described using
antibodies against the HA epitope (panel A), or mtATPase
subunit 6 (panel B). The presence (+) or absence ( ) of
cross-linker, source of mitochondria, and position of size markers are
indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Schematic diagram suggesting the arrangement
of some subunits in the stator stalk of yeast mtATPase. A putative
arrangement of subunits in the stator stalk of mtATPase is shown based
on data presented here and elsewhere. Subunits b, f, 6, and 8 are
shown; the N and C termini are indicated except for subunit d whose
topology is not known. Only two of at least five transmembrane stems of
subunit 6 are shown. Cross-links are shown as dotted black
lines (demonstrated in this study) or open arrows (from
other work).
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. R. A. Stuart (Marquette University, Milwaukee) for providing antibodies directed against subunits e, g, i/j, and k, and Dr. J. Velours (Institut de Biochimie et Genetique Cellulaires du CNRS, Université Victor Segalen, Bordeaux) for providing antibodies against subunits f, h, and 6.
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FOOTNOTES |
---|
* This work was supported by the Australian Research Council.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.
Present address: Lady Davis Institute for Medical Research, Jewish
General Hospital, Montreal QC H3T 1E2, Canada.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia. Tel.: 61-3-9905-3782; Fax: 61-3-9905-3726; E-mail: rod.devenish@med.monash.edu.au.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M300967200
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ABBREVIATIONS |
---|
The abbreviations used are: mtATPase, mitochondrial F1F0-ATP synthase; APA-Br, p-azidophenacyl bromide; APDP, N-(4-p-azidosalicylamidobutyl)-3'-(pyridyldithio)-propionamide; CHD, central hydrophobic domain; FM, fluorescein 5-maleimide; Y8, yeast mtATPase subunit 8; HA, hemagglutinin; PVDF, polyvinylidene difluoride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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