From the Department of Molecular Pathology,
University of Dundee, Ninewells Medical School, Dundee DD1 9SY, United
Kingdom,
Dunn Human Nutrition Unit, Wellcome Trust, Cambridge
CB2 2XY, United Kingdom, and the ** Division of Biology, California
Institute of Technology, Pasadena,
Los Angeles, California 91125 .
Received for publication, September 5, 2000, and in revised form, October 26, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutations in human mitochondrial DNA are a well
recognized cause of disease. A mutation at nucleotide position 8993 of
human mitochondrial DNA, located within the gene for ATP synthase
subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this
mutation in control nuclear backgrounds, two different cell lines were
transformed with mitochondria carrying NARP mutant mitochondrial DNA.
Transformant cell lines had decreased ATP synthesis capacity, and many
also had abnormally high levels of two ATP synthase sub-complexes, one
of which was F1-ATPase. A combination of metabolic
labeling and immunoblotting experiments indicated that assembly of ATP
synthase was slowed and that the assembled holoenzyme was unstable in
cells carrying NARP mutant mitochondrial DNA compared with control
cells. These findings indicate that altered assembly and stability of
ATP synthase are underlying molecular defects associated with the NARP
mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is
also compromised.
ATP synthase (or complex V) is the enzyme of aerobic ATP
production. It is located in the inner mitochondrial membrane of eukaryotic cells together with four respiratory chain enzymes that
generate the proton motive force, which in turn drives ATP synthesis.
ATP synthase comprises a rotary catalytic portion, F1-ATPase, whose structure has been solved (1), a
transmembrane portion F0, and two stalks that link
F1 and F0. Two of the subunits of the
F0 portion of ATP synthase, subunits 6 and 8 (or subunit a
and A6L), are encoded in mitochondrial DNA in all animal cells. Specific inhibition of mitochondrial translation (including subunits 6 and 8) by drug treatment leads to accumulation of two ATP synthase assembly intermediates and a concomitant decrease in holoenzyme in
human cultured cells (2).
One of the earliest disease-associated point mutations of
mtDNA1 to be described was
localized to ATP synthase subunit 6 gene, hereafter called A6 (3). The
mutation, a thymine to guanine transversion at nucleotide position 8993 of human mtDNA, hereafter termed T8993G, predicts substitution
of a highly conserved leucine by arginine at amino acid position 156. The mutation was found in a family presenting with neurogenic muscle
weakness, ataxia, and retinitis pigmentosa, a syndrome termed NARP.
There was good correlation between mutant load and disease severity
(3). This was further documented when it was shown that very high
levels of T8993G mutant mtDNA were associated with a severe
neurodegenerative disease of childhood (maternally inherited Leigh
syndrome, or MILS) (4). The T8993G mtDNA mutation is found in ~15%
of patients with a mitochondrial disorder whose disease has been
clearly linked to a point mutation in
mtDNA.2 Considered
collectively, mitochondrial disorders are among the commonest
neurological diseases; therefore the mutation is of considerable
clinical importance.
In the current structural model of mitochondrial ATP synthase, the
enzyme represents a rotary motor (1). A6 forms part of one of the
stators, and subunit c forms the rotor (5). The T8993G mtDNA mutation
predicts an arginine for leucine substitution in the fourth helix of
A6, a region that is believed to interact with subunit c. A second
point mutation at nucleotide position 8993 changes leucine to proline
and is associated with a similar phenotype in patients (6). Thus, it is
likely that any amino acid substitution in this region that induces a
conformational change will perturb holoenzyme activity, assembly, or stability.
The T8993G mtDNA mutation did not appear to alter ATP hydrolysis
activity (4) but did decrease ATP synthesis in digitonin-permeabilized cells (7). Subsequently, Wallace and co-workers (8) use 143B
osteosarcoma cells that lack mtDNA ( The standard cell culture medium in this study was Dulbecco's
modified Eagle's medium (DMEM) containing 4.5 g/liter glucose, 110 mg/liter pyruvate, with 10% fetal bovine serum. Tissue culture reagents were purchased from Life Technologies, Inc. The osteosarcoma 143B TK Enucleation of cells was achieved by inverting 35-mm tissue culture
plates, 70-90% confluent, in 95% DMEM, 5% fetal bovine serum with
10 µg/ml cytochalasin B (Calbiochem) and centrifuging at 7,000 × g for 20 min. The resultant cytoplast lawn was incubated for 3 h at 37 °C with ~8 × 105
Blue native electrophoresis (BN-PAGE) and second dimension SDS-PAGE
were performed using the method of Schagger and Von Jagow (13) and
Schagger et al. (14). Mitochondrial samples were prepared by incubating 5 × 106 cells in 200 µl of
phosphate-buffered saline with 2 mg/ml digitonin for 10 min on ice. The
solution was centrifuged at 12,000 × g for 4 min at
4 °C, and the resultant crude mitochondrial pellet was washed once
with phosphate-buffered saline, re-centrifuged, and stored at
Pulse-chase experiments were performed as described previously. (16).
Exponentially growing cells in DMEM without methionine (ICN) were
incubated with [35S]methionine at a final concentration
of 20 µCi/ml. After chase times of 0, 1, 3, 6, and 18, h cells were
harvested and used to prepare crude mitochondrial fractions (17) for
two-dimensional BN-PAGE. The gels were fixed, treated with
AmplifyTM (Amersham Pharmacia Biotech) according to the
protocol of the manufacturer, dried, and exposed to x-ray film for
1-24 h at The rate of ATP synthesis in cybrids was determined using the method of
(18). Briefly, cells were harvested and resuspended at 1 × 106 cells/ml in incubation buffer including 20 µg/ml
digitonin. Permeabilized cells were incubated with succinate (5 mM) and rotenone (4 µg/ml) for 15 min at 37 °C.
Reactions were stopped by the addition of perchloric acid. After
incubation on ice for 2 min, samples were centrifuged at 13,000 × g for 2 min, and the supernatants were neutralized with 2 M KOH, 0.6 M MOPS. The samples were
re-centrifuged, and 1-5-µl aliquots of the final supernatant
combined with 50 µl of ATP monitoring reagent (Bio-Orbit, Turku,
Finland). The amount of light detected in a luminometer was converted
to moles of ATP with reference to ATP standards after deducting the
signal obtained from the corresponding Growth rates were assessed either by direct counting of trypsinized
cells on a Neubauer counting chamber or using a tetrazolium salt
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) that
acts as a vital dye. (20). Cells were grown in DMEM with 4.5 g/liter
glucose or DMEM with 0.9 g/liter galactose substituted for glucose.
Intact cell oxygen consumption rates were determined in a Clark-type
oxygen electrode from 500-1000 µl of 5 × 106
cells/ml in RPMI 1640 medium without glucose (Life Technologies). Oxygen consumption rates were expressed as fmol of
O2/min/cell. The addition of carbonyl cyanide
p-chlorophenylhydrazone or carbonyl cyanide
p-trifluoromethoxyphenylhydrazone during an experiment led
to an increase in the rate of oxygen consumption in all cells tested
(data not shown), indicating that the assay was measuring coupled
respiration. DNA extraction, amplification, electrophoresis, blotting,
and hybridization were as described (39). For quantification of
the level of mutant mtDNA, DNA was extracted from ~5 × 106 cells and digested with AvaI, and the
restriction fragments were separated on 1% agarose gels. After
Southern blotting, filters were probed with total purified human mtDNA.
Mitochondrial translation products were labeled specifically by
incubating 106 cells with 250 µCi/ml
[35S]methionine (PerkinElmer Life Sciences) for
30-60 min in the presence of 10 µg/ml emetine, as described
previously (21).
Mitochondria carrying NARP mutant mtDNA were transferred from
human fetal fibroblasts to lung carcinoma or osteosarcoma cells that
lacked endogenous mtDNA by cell-cytoplast fusion. The donor mitochondria from fetal fibroblasts contained exclusively mutant (T8993G) mtDNA, and sequencing of the genes encoding ATP synthase subunits 8 and 6 revealed no other novel mutations (data not shown). Mitochondrial transformant cells (cybrids) were selected by their ability to grow in the absence of uridine, in contrast to
Immunoblotting of BN-PAGE and two-dimensional BN-PAGE/SDS-PAGE (Figs.
1 and 2, respectively) with
F1-ATPase antibody revealed an abnormal amount of
sub-complexes of mitochondrial ATP synthase in some lung carcinoma NARP
cybrids and all osteosarcoma NARP cybrids examined. These sub-complexes
are F1-ATPase and a sub-complex, denoted V*, which contains
F1-ATPase and an unknown number of copies of subunit c (2,
9). Sub-complex V* was present in
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
o cells) as
recipients of mitochondria carrying T8993G mtDNA. They found that
mitochondria with T8993G mtDNA, isolated from this control nuclear
background, had reduced state III respiration, indicative of decreased
ATP synthase activity. In another study, muscle mitochondria harboring
T8993G mtDNA were shown to contain sub-complexes of ATP synthase,
raising the possibility that the underlying defect in this disease was
holoenzyme assembly or stability (9). Here we demonstrate for
the first time that mutant subunit A6 of ATP synthase is linked to
impaired assembly of complex V in human cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cells and cybrids were supplemented with 100 µg/ml bromodeoxyuridine. The
o cells derived from the
osteosarcoma 143B cell line (143B.206) and the lung carcinoma cell line
(A549.B2) were in addition supplemented with 50 µg/ml uridine. The
absence of mtDNA from both these cell lines has been shown previously
by Southern blotting and polymerase chain reaction (10, 11).
o cells. The addition of 50% w/v polyethylene glycol
1500, 45% DMEM, 5% Me2SO induced cell-cytoplast fusion.
After 1 min, the cells were washed twice in 90% DMEM, 10%
Me2SO and three times in DMEM alone and incubated overnight
in 90% DMEM, 10% fetal bovine serum without uridine. Putative
transformant cells were re-plated on 90-mm dishes in 90% DMEM, 10%
fetal bovine serum without uridine. Individual colonies were picked
~14 days later using glass rings. Cytoplast-
o
cell fusion was performed between NARP fetal fibroblasts after enucleation and osteosarcoma or lung carcinoma
o cells.
Transformant osteosarcoma cybrids carrying mtDNA molecules derived from
NARP fetal fibroblasts were designated 206.8993. Equivalent lung
carcinoma cybrids were denoted B2.8993. In addition, cybrids carrying
mtDNA from a control subject were generated by the same protocol and
designated 206.con (osteosarcoma cybrids) or B2.con (lung carcinoma cybrids).
70 °C. Immediately before electrophoresis, the mitochondrial
pellet was resuspended in 100 µl of 1.5 M 6-aminohexanoic acid, 50 mM Bis-Tris, pH 7.0, with 20 µl of 10%
n-dodecyl maltoside and incubated on ice for 15 min. After
centrifugation at 12,000 × g for 20 min at 4 °C,
the supernatant was mixed with 10 µl of 5% Serva Blue G in 1 M 6-aminohexanoic acid, and equal amounts of protein, as
determined by the Bradford method (15), were added to each lane of a
5-13% gradient gel.
70 °C. Alternatively, in some instances labeled protein
was transferred to Hybond-C membrane and exposed to x-ray film, as for
the dried gels, after which the membrane was blocked and immunoblotted
with antibody to subunits of F1-ATPase. A Molecular
Dynamics Personal Densitometer SI was used to quantify the relative
amounts of each complex.
o cells
(osteosarcoma or lung carcinoma). Thus, the values obtained reflect ATP
synthesis activity that was specific to oxidative phosphorylation (OP).
Histochemical staining of ATP hydrolysis activity in blue native
polyacrylamide gels was performed according to Zerbetto et
al. (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
o cells, which are auxotrophic for uridine (10).
Screening of transformant cell lines for the presence of mutant mtDNA
revealed that all cybrids examined contained exclusively T8993G mutant mtDNA (data not shown).
o cells (Fig. 1,
lane 8, and Fig. 2, lane
4) and must therefore lack subunits A6 and A8, which are encoded
in mtDNA. V* is also known to lack subunit b of
F0F1-ATPase (9). Free F1-ATPase has
been described previously in
o cells (23) and is known
to accumulate together with V* in cells where mitochondrial translation
has been inhibited (2). Interestingly, a sub-complex of ATP synthase
has been crystallized recently that comprises F1-ATPase and
a ring of 10 copies of subunit c (22); however, it is not known if this
ATP synthase derivative and V* are equivalent. All the NARP cybrid cell
lines contained at least some fully assembled complex V (Figs. 1 and
2). As the cybrids were homoplasmic for the T8993G NARP mutation, the
complex V holoenzyme in these cells must contain mutant subunit 6.
View larger version (47K):
[in a new window]
Fig. 1.
Mitochondrial membrane fractions prepared
from NARP cybrids contained increased levels of ATP synthase
sub-complexes. Proteins from crude mitochondrial preparations were
separated by BN-PAGE on a 5-12% polyacrylamide gradient gel. Protein
was transferred to solid support and immunoblotted with human
F1-ATPase antibodies. V, ATP synthase
holoenzyme, F1, F1-ATPase; V*, a
sub-complex of complex V that includes F1-ATPase and
subunit c. Lane 1, A549 lung carcinoma cells; lanes
2 and 3, control lung carcinoma cybrids; lane
4, lung carcinoma cybrid clone B2.8993 B; lanes 5-7,
sub-clones of cybrid B2.8993B; lane 8, lung carcinoma
(A549.B2) o cells. Control cybrids were generated by
fusing cytoplasts derived from fibroblasts of a normal control subject
with lung carcinoma
o cells. B2.8993 denotes the
transformant cybrid lines produced by fusing lung carcinoma
o cells (A549.B2) with cytoplasts derived from NARP
fetal fibroblasts.
View larger version (114K):
[in a new window]
Fig. 2.
ATP synthase appeared structurally normal in
some lung carcinoma NARP cybrids, whereas complex V sub-complexes were
always present in osteosarcoma NARP cybrids. Each panel
is a mitochondrial membrane sample separated in the first dimension by
BN-PAGE (left to right) and subsequently in a second dimension 12%
denaturing SDS-PAG (top to bottom). Protein was transferred to solid
support and immunoblotted with the same F1-ATPase
antibodies used in Fig. 1. Panel 1, 143B osteosarcoma
mitochondrial; panels 2 and 3, typical
osteosarcoma NARP cybrids (two of five screened) showing increased
amounts of F1-ATPase and V*. Panel 4,
mitochondria derived from 206 o osteosarcoma cells.
Panels 5-8, four lung carcinoma NARP cybrids, two of which
appeared structurally normal (panels 7 and 8),
whereas two displayed abnormally high levels of F1-ATPase
and V* (panels 5 and 6).
Re-cloning a NARP lung carcinoma cybrid gave rise to sub-clones with an identical pattern of sub-complexes to the parental cell line (Fig. 1), suggesting that the population of cells was homogenous. That is, the result argues against the idea that some cells contained high levels of sub-complexes, whereas others contained exclusively holoenzyme.
Sub-complexes of complex V were present at very low levels or
undetectable in three of five NARP lung carcinoma cybrids (clones A, D,
and E), two of which are shown in Fig. 2. Where no sub-complexes of
complex V were detected, the total amount of complex V holoenzyme was
similar in NARP cybrids and control cells, suggesting that there was no
significant alteration in the amount of complex V in NARP cybrids. The
interclonal variability, in the amount of complex V sub-complexes,
among the B2.8993 cybrids may reflect differences in the nuclear gene
composition or activity (involving, e.g. assembly factors,
chaperones, or nuclear-encoded subunits) among lung carcinoma
0 cells. Such nuclear heterogeneity has been observed
previously for osteosarcoma
0 cells (24).
There was some experiment-to-experiment variation in the amount of
sub-complexes for a given clone. The extent of the variation is shown
in Fig. 3 for osteosarcoma clone 206.8993 A. Note that the relative amounts of all three complexes, complex V
holoenzyme, V*, and F1-ATPase, varied not merely the
ratio of holoenzyme to sub-complexes. The most common result is shown
in Fig. 3, panel 1, where the proportions of holoenzyme, V*,
and free F1-ATPase were 75, 12, and 13%, respectively. In
the most extreme case, free F1-ATPase accounted for
approximately half the total H+-ATPase (47%), whereas ATP
synthase holoenzyme represented only 37% of the total (Fig. 3,
panel 3). No such variation was observed in control cells,
where holoenzyme always accounted for at least 95% of
H+-ATPase. These results indicate either that the phenotype
fluctuates over time in NARP cybrids or, more likely, that mutant
containing complex V is less stable than wild-type ATP synthase. All
the NARP cybrid cell lines remained homoplasmic mutant throughout the
course of the study. Both the interclonal variability among the lung
carcinoma NARP cybrids and the sensitivity to the conditions of
isolation and sample preparation of the holoenzyme from the same NARP
cybrid point to the critical role of leucine 156 in the assembly and
stability of the ATP synthase complex.
|
As a further test of the possible effects of the T8993G mutation,
[35S]methionine pulse-chase experiments were performed
followed by two-dimensional BN-PAGE of labeled proteins. ATP synthase,
F1-ATPase, and V* were distinguishable (Fig.
4), and their relative representation could be deduced by comparing the labeling of and
subunits of
F1-ATPase, since these subunits are constituents of all
three complexes. First, lung carcinoma NARP cybrids A and D were
analyzed, as these did not give appreciable amounts of steady-state
sub-complexes. After a 1-h chase, labeled sub-complexes were detected
in lung carcinoma NARP cybrid cells, whereas these were largely absent from control cells. In particular, V* was detectable in lung carcinoma NARP cybrids, whereas labeled V* was not seen in control cells (Fig.
4A, panels 1-3). V* was not detectable by
immunoblotting in any of the mitochondrial preparations from these lung
carcinoma NARP cybrids or controls (Fig. 4B), a finding that
established the labeled V* and F1-ATPase as assembly,
rather than breakdown, intermediates. We conclude that the transition
from V* sub-complex to ATP synthase holoenzyme is impeded in cells
carrying mutant A6. In cells that had been incubated for 3-18 h after
removal of [35S]methionine, almost all the labeled
and
subunits were incorporated into fully assembled complex V in
both controls and lung carcinoma NARP cybrids (Fig. 4A,
panels 4-6, and data not shown).
|
A similar assessment of osteosarcoma NARP cybrids also revealed
differences between immunoblotting and metabolic labeling analyses.
After chases of up to 3 h, the ratio of sub-complexes to
holoenzyme was higher for newly synthesized
[35S]methionine-labeled complexes (66:34) than for the
steady-state level (30:70) determined by blotting with
F1-ATPase antibody (Fig. 5,
A and B). Thus, although all the newly
synthesized (radiolabeled) and
subunits had been incorporated
into ATP synthase holoenzyme in control cells after 3 h, one-third
remained as sub-complexes in osteosarcoma cybrids carrying NARP mutant
mtDNA. After an 18-h chase, the ratio was similar by both methods in
osteosarcoma NARP cybrid and the control cells (Fig. 5, A
and B). These findings indicate that the assembly defect was
common to both nuclear backgrounds, yet was more marked in the
osteosarcoma than the lung carcinoma background given that labeled
sub-complexes, which could not be ascribed to disassembly, were
detected after a 3-h chase only in the former cell type.
|
Measurement of ATP synthesis in digitonin-permeabilized cells indicated
a decrease of approximately one-third in lung carcinoma NARP cybrids
compared with the parental control cell line (Fig. 6). As stated above, some of the cybrids
analyzed (B2.8993A and -D) contained few if any sub-complexes on
BN-PAGE analysis, like the control cells. Therefore, the ATP synthesis
capacity of holoenzyme containing mutant A6 must itself be impaired.
The ATP synthesis capacity of osteosarcoma NARP cybrids was
approximately half that of control cells (Fig. 6), suggesting that the
mutation may be more deleterious in the osteosarcoma nuclear background
than that of A549 lung carcinoma cells.
|
The F1-ATPase inhibitory protein (IF1) is
believed to regulate ATP synthase (25, 26); therefore, we tested
whether there was a discernible difference between the amounts of
IF1 in control, NARP, or o cells. No
significant difference was observed (Fig.
7). IF1 was found to be
associated with sub-complexes of ATP synthase as well as with the
holoenzyme (Fig. 7). Nevertheless F1-ATPase consistently displayed higher in-gel ATP hydrolysis activity than holoenzyme (Fig.
8), suggesting that subunits of the
F0 portion of ATP synthase restrict ATP hydrolysis.
|
|
Although assays of holoenzyme integrity and the permeabilized cell
assay clearly indicated defects of complex V, the T8993G mtDNA mutation
had no appreciable phenotypic effect upon intact lung carcinoma or
osteosarcoma cybrids. Specifically, there was no significant decrease
in oxygen consumption of coupled intact cells carrying T8993G mtDNA
compared with controls (Fig. 9). Nor was
there any significant increase in lactate-to-pyruvate ratio in spent
medium (data not shown). The growth rate of NARP and control cybrids
over periods of 7-9 days were indistinguishable, even in medium where
galactose was substituted for glucose (data not shown). In contrast,
o cells and cells with high levels of
"A3243G" mutant mtDNA died in galactose
medium.3 There was no
measurable effect on mitochondrial translation in osteosarcoma cell
cybrids with or without T8993G mtDNA (data not shown). In other
experiments, incubation with reagents that induce oxidative stress
(hydrogen peroxide and menadione) failed to differentiate NARP mutant
and control cybrids. Finally, the cellular ATP:ADP ratio decreased
dramatically in
o lung carcinoma cells incubated for 30 min in the absence of glucose, whereas NARP cybrids maintained a ratio
similar to control cybrids for at least 4 h (data not shown).
Thus, the decrease in ATP synthesis capacity (Fig. 6) and increase in
complex V sub-complexes (Fig. 2) in disrupted NARP cells are either of
no consequence to growth, even under regimes that favor expression of a
functional OP system, or else these in vitro observed
abnormalities are compensated in intact cells.
|
The absence of a marked OP phenotype in intact NARP cybrids was
mirrored in cybrids containing partially duplicated mtDNA (27). In
contrast, earlier studies of A8344G (21) and A3243G mutant mtDNA
(28-30) and partial mtDNA deletions (31) demonstrate clear deleterious
effects upon OP and mitochondrial translation of intact cybrid cells.
Therefore, putative pathological mtDNA mutations cannot be excluded as
a cause of disease based on absence of an OP phenotype in cultured cells.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human cells lacking mtDNA have been used widely to study the
effects of putative pathological mtDNA mutations (e.g. Refs. 8, 21, 24, and 27-32). The genetic outcome of fusing cytoplast and
o cells is to transfer mtDNA to a new nuclear
background. Thus, mtDNA can be isolated from its host nuclear DNA, and
any mitochondrial dysfunction in the transformant cells can be ascribed
to mtDNA, where appropriate controls are in place. In this report,
mtDNA carrying a presumed pathological mutation in subunit 6 of ATP synthase was transferred to two control nuclear backgrounds to assess
its effects on mitochondrial structure and function. In both nuclear
backgrounds tested, T8993G mutant mtDNA was associated with decreased
complex V assembly and decreased ATP synthesis capacity. Because the
effects were observed with NARP mutant mtDNA in two nuclear
backgrounds, they can with confidence be attributed to the mutation.
Despite these abnormalities there was no marked phenotype in intact
cells with mutant ATP synthase. These findings are consistent with what
is known of the T8993G mtDNA mutation and its associated diseases. The
mutation resides in a gene encoding an essential subunit of ATP
synthase and could therefore be expected to affect the intrinsic
activity or amount of holoenzyme. Nevertheless, the effects of the
mutation must necessarily be subtle, first, because substantial
impairment of ATP synthase would be incompatible with life and, second,
because the NARP mutation is highly tissue-specific in its effects. For
instance, muscle pathology is absent in NARP, whereas it is present in
association with a number of other pathological mtDNA mutations.
Early studies assumed that the NARP/MILS (maternally inherited Leigh syndrome) T8993G mutation decreased ATP synthesis capacity directly by impairing proton flux through the F0 portion of the enzyme, i.e. caused a decrease in intrinsic enzyme activity. The finding that the mutation was associated with increased levels of sub-complexes of complex V suggested an alternative explanation, namely that perturbed assembly or stability could lead to a decrease in the total amount of holoenzyme (9). The results reported here indicate that sub-complexes of complex V are often present in isolated mitochondria carrying the mutant form of A6 associated with NARP, yet irrespective of this, ATP synthesis capacity was decreased. Thus, it can be concluded that the function of ATP synthase holoenzyme containing mutant A6 is impaired.
The metabolic labeling experiments indicated that NARP-containing
cybrids were impaired in complex V assembly (Figs. 4 and 5).
Nevertheless, it is unlikely that all the antibody-detected sub-complexes (Figs. 1-3) represent assembly intermediates; some almost certainly arose via disassembly, given the considerable variation in the ratio of sub-complexes to holoenzyme that was observed
(Fig. 3). Therefore, we posit that ATP synthase-containing mutant A6 is
not only assembled less efficiently than wild type but also that it is
less stable. Instability of mutant-containing holoenzyme might explain
much of the reported variation (30-95%) in the degree of impairment
of ATP synthesis capacity (Refs. 8 and 33 and this report) and some of
the phenotypic difference between intact and disrupted cells. Finally,
formation of sub-complexes from disrupted holoenzyme is a reasonable
explanation for the observation that there was little complex V
holoenzyme in muscle mitochondrial preparations of NARP patients that
displayed no muscle pathology (9), i.e. it was likely the
result of the disassembly. In summary, to reconcile the apparently
disparate findings in studies of the T8993G NARP mutation, we propose
that complex V-containing mutant A6 has 70% normal ATP synthesis
capacity in intact cells, yet the enzyme is assembled less efficiently and is less stable than that of wild-type cells. Ultimately, it will be
necessary to develop a sensitive assay of ATP synthase for intact cells
to determine the true extent of enzyme dysfunction in NARP.
Recently it was reported that IF1 was not associated with
F1-ATPase of a fibroblast 0 cell line (34),
a result that is apparently at odds with the finding reported here. The
discrepancy could reflect differences between
0 cell
lines, although a more plausible explanation is that the osmotic shock
procedure used by Garcia et al. (34) had a different effect
on the mitochondria of
+ and
0 cells.
Indeed, the apparent absence of IF1 may simply reflect the
low yield of F1-ATPase obtained by this procedure, as the
and
subunits of F1-ATPase were in low abundance in
the
0 cell H+-ATPase preparation of Garcia
et al. (34).
The combined ATP synthase abnormalities associated with NARP cybrids
might become critical, for example, in particular neuronal cell types
or genetic backgrounds, if the cellular environment accentuated the
assembly or stability defects or increased ATP synthase turnover. In
this context, it is noteworthy that there are differences in brain and
muscle in the expression of isoforms of subunit c with which A6
interacts (35). Furthermore, IF1 is expressed at relatively
high levels in developing rat brain compared with muscle (36). This
observation suggests that ATP hydrolysis needs to be strictly
controlled in developing brain and thereby offers an explanation of how
increased levels of sub-complexes of complex V resulting from the
presence of the T8993G mtDNA mutation might cause tissue-specific
metabolic failure.
![]() |
ACKNOWLEDGEMENTS |
---|
I. Holt and L. Nijtmans acknowledge debt to the late Dr. Coby Van den Bogert and the late Professor Anita Harding. We thank Professor H. T. Jacobs for comments on the manuscript and Dr. John Walker who kindly provided IF1 antibody.
![]() |
FOOTNOTES |
---|
* This work was supported by the European Union in the form of a Training and Mobility of Researchers fellowship (to L. N.), the Scottish Hospital Endowments Research Trust, and the Medical 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.
§ Current address: Section for Molecular Biology, Dept. of Molecular Biology, University of Amsterdam, Kruislan 318, 1098 SM Amsterdam, the Netherlands.
¶ Contributed equally to this work.
A Royal Society University Research Fellow (1992-1999). This
study was initiated when I. Holt was a Lucille Markey Visiting Fellow
in the laboratory of G. Attardi. To whom correspondence should be
addressed: Dunn Human Nutrition Unit, Wellcome Trust-MRC Bldg., Hills
Rd. Cambridge, CB2 2XY, UK. Tel.: 44 12 23 25 28 40; Fax: 44 12 23 25 28 45; E-mail: ih@mrc-dunn.cam.ac.uk.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M008114200
2 M. Zeviani, personal communication.
3 N. Hance and I. J. Holt, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: mtDNA, mitochondrial DNA; NARP, neurological muscle weakness, ataxia, and retinitis pigmentosa; BN-PAGE, blue native-polyacrylamide gel electrophoresis; Bis- Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; DMEM, Dulbecco's modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid; OP, oxidative phosphorylation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628[CrossRef][Medline] [Order article via Infotrieve] |
2. | Nijtmans, L. G., Klement, P., Houstek, J.,., and van den Bogert, C. (1995) Biochim. Biophys. Acta 1272, 190-198[Medline] [Order article via Infotrieve] |
3. | Holt, I. J., Harding, A. E., Petty, R. K., and Morgan-Hughes, J. A. (1990) Am. J. Hum. Genet. 46, 428-433[Medline] [Order article via Infotrieve] |
4. | Tatuch, Y., Christodoulou, J., Feigenbaum, A., Clarke, J. T., Wherret, J., Smith, C., Rudd, N., Petrova-Benedict, R., and Robinson, B. H. (1992) Am. J. Hum. Genet. 50, 852-858[Medline] [Order article via Infotrieve] |
5. | Elston, T., Wang, H., and Oster, G. (1998) Nature 391, 510-513[CrossRef][Medline] [Order article via Infotrieve] |
6. | de Vries, D. D., van Engelen, B. G., Gabreels, F. J., Ruitenbeek, W., and van Oost, B. A. (1993) Ann. Neurol. 34, 410-412[Medline] [Order article via Infotrieve] |
7. | Tatuch, Y., and Robinson, B. H. (1993) Biochem. Biophys. Res. Commun. 192, 124-128[CrossRef][Medline] [Order article via Infotrieve] |
8. | Trounce, I., Neill, S., and Wallace, D. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8334-8338[Abstract] |
9. | Houstek, J., Klement, P., Hermanska, J., Houstkova, H., Hansikova, H., Van den Bogert, C., and Zeman, J. (1995) Biochim. Biophys. Acta 1271, 349-357[Medline] [Order article via Infotrieve] |
10. | King, M. P., and Attardi, G. (1989) Science 246, 500-503[Medline] [Order article via Infotrieve] |
11. | Bodnar, A. G., Cooper, J. M., Holt, I. J., Leonard, J. V., and Schapira, A. H. (1993) Am. J. Hum. Genet. 53, 663-669[Medline] [Order article via Infotrieve] |
12. | Klement, P., Nijtmans, L. G., Van den Bogert, C., and Houstek, J. (1995) Anal. Biochem. 231, 218-224[CrossRef][Medline] [Order article via Infotrieve] |
13. | Schagger, H., and von Jagow, G. (1991) Anal. Biochem. 199, 223-231[Medline] [Order article via Infotrieve] |
14. | Schagger, H., Cramer, W. A., and von Jagow, G. (1994) Anal. Biochem. 217, 220-230[CrossRef][Medline] [Order article via Infotrieve] |
15. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
16. | Nijtmans, L. G., Barth, P. G., Lincke, C. R., Van Galen, M. J., Zwart, R., Klement, P., Bolhuis, P. A., Ruitenbeek, W., Wanders, R. J., and Van den Bogert, C. (1995) Biochim. Biophys. Acta 1270, 193-201[Medline] [Order article via Infotrieve] |
17. | Nijtmans, L. G., Taanman, J. W., Muijsers, A. O., Speijer, D., and Van den Bogert, C. (1998) Eur. J. Biochem. 254, 389-394[Abstract] |
18. | Wanders, R. J., Ruiter, J. P., and Wijburg, F. A. (1993) Biochim. Biophys. Acta 1181, 219-222[Medline] [Order article via Infotrieve] |
19. | Zerbetto, E., Vergani, L., and Dabbeni-Sala, F. (1997) Electrophoresis 18, 2059-2064[Medline] [Order article via Infotrieve] |
20. | Mosmann, T. (1983) J. Immunol. Methods 65, 55-63[CrossRef][Medline] [Order article via Infotrieve] |
21. | Chomyn, A., Meola, G., Bresolin, N., Lai, S. T., Scarlato, G., and Attardi, G. (1991) Mol. Cell. Biol. 11, 2236-2244[Medline] [Order article via Infotrieve] |
22. |
Stock, D.,
Leslie, A. G.,
and Walker, J. E.
(1999)
Science
286,
1700-1705 |
23. |
Buchet, K.,
and Godinot, C.
(1998)
J. Biol. Chem.
273,
22983-22989 |
24. | Chomyn, A., Lai, S. T., Shakeley, R., Bresolin, N., Scarlato, G., and Attardi, G. (1994) Am. J. Hum. Genet. 54, 966-974[Medline] [Order article via Infotrieve] |
25. | Klein, G., Satre, M., Dianoux, A. C., and Vignais, P. V. (1980) Biochemistry 19, 2919-2925[Medline] [Order article via Infotrieve] |
26. | Penin, F., Di Pietro, A., Godinot, C., and Gautheron, D. C. (1988) Biochemistry 27, 8969-8974[Medline] [Order article via Infotrieve] |
27. |
Holt, I. J.,
Dunbar, D. R.,
and Jacobs, H. T.
(1997)
Hum. Mol. Genet.
6,
1251-1260 |
28. | King, M. P., Koga, Y., Davidson, M., and Schon, E. A. (1992) Mol. Cell. Biol. 12, 480-490[Abstract] |
29. | Chomyn, A., Martinuzzi, A., Yoneda, M., Daga, A., Hurko, O., Johns, D., Lai, S. T., Nonaka, I., Angelini, C., and Attardi, G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4221-4225[Abstract] |
30. |
Dunbar, D. R.,
Moonie, P. A.,
Zeviani, M.,
and Holt, I. J.
(1996)
Hum. Mol. Genet.
5,
123-129 |
31. | Hayashi, J., Ohta, S., Kikuchi, A., Takemitsu, M., Goto, Y., and Nonaka, I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10614-10618[Abstract] |
32. | Dunbar, D. R., Moonie, P. A., Jacobs, H. T., and Holt, I. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6562-6566[Abstract] |
33. |
Baracca, A.,
Barogi, S.,
Carelli, V.,
Lenaz, G.,
and Solaini, G.
(2000)
J. Biol. Chem.
275,
4177-4182 |
34. |
Garcia, J. J.,
Ogilvie, I.,
Robinson, B. H.,
and Capaldi, R. A.
(2000)
J. Biol. Chem.
275,
11075-11081 |
35. | Gay, N. J., and Walker, J. E. (1985) EMBO J. 4, 3519-3524[Abstract] |
36. |
Sangawa, H.,
Himeda, T.,
Shibata, H.,
and Higuti, T.
(1997)
J. Biol. Chem.
272,
6034-6037 |
37. |
Cabezon, E.,
Butler, P. J.,
Runswick, M. J.,
and Walker, J. E.
(2000)
J. Biol. Chem.
275,
25460-25464 |
38. | Lopez-Mediavilla, C., Vigny, H., and Godinot, C. (1993) Eur. J. Biochem. 215, 487-496[Abstract] |
39. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1990) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |