From the Department of Pediatrics, Nijmegen Center
for Mitochondrial Disorders, University Hospital Nijmegen St. Radboud,
6500 HB Nijmegen, the Netherlands and § Institute of
Molecular Biology and
Institute of Neuroscience, University of
Oregon, Eugene, Oregon 97403
Received for publication, October 31, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Complex I defects are one of the most
frequent causes of mitochondrial respiratory chain disorders.
Therefore, it is important to find new approaches for detecting and
characterizing Complex I deficiencies. In this paper, we introduce a
new set of monoclonal antibodies that react with 39-, 30-, 20-, 18-, 15-, and 8-kDa subunits of Complex I. These antibodies are shown to aid
in diagnosis of Complex I deficiencies and add understanding to the
genotype-phenotype relationships of different mutations. A total of 11 different patients were examined. Four patients had undefined Complex I defects, whereas the other patients had defects in NDUFV1, NDUFS2 (two
patients), NDUFS4 (two patients), NDUFS7, and NDUFS8. We show here that
Western blotting with these antibodies, particularly when used in
conjunction with sucrose gradient studies and enzymatic activity
measurements, helps distinguish catalytic versus assembly defects and further distinguishes between mutations in different subunits. Furthermore, different mutations in the same gene are shown
to give very similar subunit profiles, and we show that one of the
patients is a good candidate for having a defect in a Complex I
assembly factor.
Disorders of mitochondrial energy metabolism occur in humans with
a frequency of ~1 in 10,000 live births (1). Most are caused by the
dysfunction of one or more of the enzyme complexes of oxidative
phosphorylation (OXPHOS).1
Isolated enzymatic deficiency of the first OXPHOS complex,
NADH:ubiquinone oxidoreductase (EC 1.6.99.3) or Complex I, is one of
the most frequent causes of mitochondrial respiratory chain disorders
(2). Complex I is the first multiprotein complex of the OXPHOS system (3) and participates in the formation of a proton gradient across the
inner mitochondrial membrane coupled to transfer of electrons from NADH
to ubiquinone. This proton gradient provides part of the proton-motive
force used for ATP production. Complex I is the largest of the
respiratory chain complexes, made up of seven different subunits
encoded on mitochondrial DNA (mtDNA; ND1-6 and ND4L) and 35 or more
different subunits encoded by nuclear genes (4, 5). Together, these
subunits form a complex with an estimated molecular mass of 900,000 daltons (3). Mutations in both the mitochondrial and nuclear encoded
genes are known to cause Complex I deficiencies (6). However, in
addition to the structural genes, there may be additional genes
encoding proteins required for the assembly of a functional Complex I. So-called "assembly factors" involved in assembly of Complex IV and
the ATP synthase have already been reported (7-9). Mutations in
SURF1, an assembly factor required for full assembly of Complex
IV, has been shown to cause cytochrome c oxidase deficiency
in many of the reported cases of Leigh's disease (7, 10, 11).
The genes for all of the components of Complex I have now been
identified (12), opening up the possibility of genetic approaches for
diagnosis. However, such an analysis would not identify Complex I
deficiencies caused by mutations in assembly factors until these factors are identified and even then would not yield sufficient information to understand the genotype-phenotype relationships of the
various mutations that can occur. Therefore, in addition to genetic
analysis, it is important to have protein-based approaches to detecting
and characterizing complex I deficiencies. Here, we introduce a
monoclonal antibody set that will be useful in this regard. We show
that Western blotting with these antibodies, particularly when used in
conjunction with sucrose gradient studies and enzymatic activity
measurements, distinguishes catalytic versus assembly
defects. In the latter class, patient samples can be classified by
their assembly profiles, which appear to be representative of which
subunit is mutated.
Purification of Bovine Heart Complex I--
Biochemically
purified bovine heart Complex I as well as the flavoprotein,
iron-sulfur protein, and hydrophobic protein subfractions of Complex I
isolated as described previously (13-15) were kindly supplied by Dr.
Youssef Hatefi (The Scripps Institute, La Jolla, CA). Immunopurified
bovine heart Complex I was generated by solubilizing bovine heart
mitochondria in 1% N-dodecyl- Cell Lines--
MRC5 fibroblasts were obtained from the American
Type Culture Collection, and MRC5-Rho0 fibroblasts were derived from
the MRC5 fibroblasts by culturing the cells in permissive medium
supplemented with 50 ng/ml ethidium bromide as described previously
(17).
Patient fibroblasts were obtained from skin biopsies of young children
in whom an isolated Complex I deficiency has been confirmed in muscle
tissue as well as in cultured fibroblasts, using the slightly modified
method of Fischer et al. (18). The phenotypes and genotypes
of the patients included in this study have been extensively described
by Loeffen et al. (2). Control fibroblasts were obtained
from postcircumcision tissue from a child in the same age range in whom
biochemical enzyme analyses revealed normal results.
Genetic Characterization of Patient Cell Lines--
All patients
included in this study were screened for the presence of DNA
alterations in each of the known nuclear-encoded "structural" genes
of Complex I as described previously (19, 20). Mutations were found in
7 of the 11 patients as listed in Table I. mtDNA was also screened for
deletions and point mutations that have previously been shown to cause
Complex I deficiency. These are T14484C, G14459A (mutations in the
mitochondrial complex I subunit ND6), G11778A (mutation in the
mitochondrial complex I subunit ND4), T4160C and G3460A (mutations in
the mitochondrial complex I subunit ND1), T8993G/C (mutation in the
mitochondrial ATPase subunit 6) and T8356C, A8344G, A4317G, T3271C, and
A3243G (mutations in the mitochondrial tRNA genes). Large mtDNA
rearrangements were ruled out by a previously developed long-template
PCR technique (21). None of the above mtDNA alterations were found.
Monoclonal Antibodies--
The monoclonal antibodies used in
this study were developed at the University of Oregon (Eugene, OR).
They were generated by immunizing mice with purified bovine Complex I
as described previously (22). The newly generated monoclonal antibodies
(mAbs) were sequentially screened for 1) binding to purified bovine
Complex I adsorbed to polystyrene; 2) binding specifically to a single subunit in denaturing Western blots of bovine Complex I; 3) binding to
a single subunit in denaturing Western blots of the flavoprotein, iron-sulfur protein, or hydrophobic protein subfractions of bovine Complex I; 4) binding to a single subunit in denaturing Western blots
of immunopurified bovine Complex I; 5) binding to a single subunit in
denaturing Western blots of human mitochondria; and 6) reactivity and
mitochondrial localization in immunohistochemistry of human
mitochondria. Immunohistochemistry was carried out as described
previously (17). The monoclonal antibody concentrations used were:
anti-Complex I-39 kDa, anti-Complex I-15 kDa, anti-Complex I-8 kDa,
anti-Complex IV Va, and anti-Complex V- Overexpression of Complex I Subunits in Eacherichia Coli--
On
the basis of estimated molecular weights and with which Complex I
subfraction (flavoprotein, iron-sulfur protein, or hydrophobic protein)
each antibody reacted, a list of possible Complex I antigens was
compiled for each antibody. The cDNA of two selected Complex I
subunits (NDUFA9 and NDUFS3) was then amplified by PCR from a human
heart cDNA library (CLONTECH) using the forward
and reverse primers (Life Technologies, Inc.) 5'-TAT ATC ATG AGC CAT
CAT CAT CAT CAT CAC ATG GCG GCT GCC GCA CAA TCC-3' and 5'-CAG CCG GAT CCT CGA GCA TAT GGC TCT AAA TGT TGA CGG TCT TGG CC-3'; and 5'-TAT ATA
CCA TGG GCC ATC ATC ATC ATC ATC ATG AGA GCG CCG GGG CCG ACA CGC-3' and
5'-GCG CGC GCC ATA TGC TAC TTG GCA TCA GGC TTC TTG TCT-3',
respectively. The resultant PCR products were subcloned into the pET15b
vector (Novagen) using BspHI-NdeI and
NcoI-NdeI (New England Biolabs) restriction sites
respectively. BL21-DE3 cells (Novagen) were transformed with the
plasmids, and when the cells reached an absorbance of 0.6, they were
induced for 3 h with 1 mM
isopropyl-1-thio- Fibroblast Culture and Mitochondrial Protein
Isolation--
Human control and patient fibroblasts were grown in
M199 (Life Technologies), 5 mg/liter Tween 20 medium with 10%
fetal calf serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin.
Approximately 30 × 106 cells were harvested at 95%
confluence after mild trypsinization (3-5 min) with 2-3 ml 0.25%
trypsin solution/175-cm2 (5 × 106 cells)
cell culture. Cells were resuspended in 50 ml 10% fetal calf
serum-PBS. Cells were rinsed three times with 1% fetal calf serum-PBS
as well as with PBS and finally frozen at Western Blot Analysis of Mitochondrial
Proteins--
Approximately 5 µg/lane mitochondrial protein,
dissolved in SDS-polyacrylamide gel electrophoresis-Tricine sample
buffer (Bio-Rad) containing 2% Sucrose Gradient Centrifugation--
Mitochondria (1 mg) from
control MRC5 fibroblasts and three patient cell lines (patients 1, 7, and 11) were pelleted (10,000 × g, 10 min, 4 °C)
and resuspended at a protein concentration of 5 mg/ml in 100 mM Tris/HCl, 1 mM EDTA, pH 7.5, 1 µg/ml
pepstatin,1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride, 1% LM. The mitochondria were incubated in this solution for
20 min on ice with stirring before any insoluble membranes were
pelleted again by centrifugation (10,000 × g, 10 min,
4 °C). The supernatant was layered on top of a discontinuous sucrose
gradient composed of 250 µl of 35% sucrose, 500 µl of 30%
sucrose, 750 µl of 27.5% sucrose, 1 ml of 25% sucrose, 1 ml of 20%
sucrose, and 1 ml of 15% sucrose. All sucrose solutions contained 100 mM Tris/HCl, pH 8.0, 0.05% LM, 1 µg/ml pepstatin,1
µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride. The
gradient was then centrifuged overnight at 4 °C (128,000 × g, 16.5 h, SW 50.1). The sucrose gradient was
fractionated from the bottom of the tube into 500-µl fractions, which
were frozen at Antibody Characterization--
The antigen used to generate
monoclonal antibodies was beef heart Complex I purified according to
Hatefi (13). Screening involved an assay for binding to native Complex
I, Western blotting, and/or immunohistochemistry. Biochemically
purified and immunopurified bovine heart Complex I, biochemically
purified iron-sulfur protein, flavoprotein, and hydrophobic protein
subfractions of bovine Complex I and human fibroblast cell lines from
controls and patients were used in the screening. Unequivocal
identification of two of the antibodies was also made by Western
blotting after overexpressing the candidate human subunit antigens in
E. coli. To date, seven monoclonal antibodies meet our
standards for long-term culturing, i.e. they react with
biochemically purified bovine Complex I, immunopurified bovine Complex
I, solubilized human mitochondria, and whole-cell extracts to generate
a single band in Western blot. Where two or more antibodies were
obtained to a particular subunit, that which worked best in
immunohistochemistry or effectively immunoprecipitated Complex I, or
both, was chosen. Table II summarizes the information on the antibodies
used in this study.
Patient Characterization--
Fibroblasts were cultured from 11 patients in whom an isolated enzymatic Complex I deficiency had been
confirmed in muscle tissue as well as cultured fibroblasts. In seven of
the patients, the pathogenic mutation was identified genetically, and
for four patients the genetic defect was not identified. The residual
Complex I activities of the 11 patient fibroblast cell lines ranged
from 35 to 85%. Specifics of each patient are provided in Table
I.
Confirmation of Isolated Complex I Deficiency by Western
Blotting--
Mitochondria were isolated from each of the patient
fibroblast cell lines, a control skin fibroblast cell line, and normal and Rho0 MRC5 fibroblasts (a lung fibroblast cell line). Samples of
each were examined by Western blotting with mixtures of antibodies, including ones specific to the 39-kDa subunit of Complex I, the 70-kDa
subunit of succinate dehydrogenase (Complex II), core II protein of
Complex III, subunit II of cytochrome c oxidase (Complex IV), subunit IV of Complex IV, the Variations in Complex I Assembly Identified by Western
Blotting--
The mitochondrial samples of the 11 patient fibroblasts
were examined for levels of six different subunits of Complex I
(referred to by their apparent molecular weights as listed in Table
II). For the most part, mAbs to the 30-, 20-, 15-, and 8-kDa subunits were used as an antibody mixture along
with porin, and the amounts of the 39- and the 18-kDa subunits were
quantitated relative to porin separately. A representative Western blot
and a bar graph of the levels of the six components of Complex I in the
different samples are shown in Fig. 2. A
significant reduction in the levels of one or more components of the
complex was seen in all but one of the patient samples; that is,
patient 7, with a mutation in NDUFV1. The patterns of subunit loss were
similar in patients 3 and 4, each of which has a different mutation in
NDUFS4. Similarly, the pattern of subunit loss was the same in patients
5 and 6, each with a different mutation in the same subunit, NDUFS2.
Patients 9 and 10, both of which have unidentified mutations, show
remarkable similarity in the pattern of subunit loss. This pattern most
closely resembles that of patients 3 and 4. In Rho0 cells, where there is an absence of the mitochondrially encoded subunits of Complex I, a
different pattern from any of the patient samples is observed. In this
case, the levels of the 20- and 18-kDa subunits are as low or lower
than those of the 15- and 8-kDa subunits. Subunit amounts were lowest
in patient 11, identifying this as a likely candidate for a mutation in
an assembly factor (see below).
The relationship between the loss of each subunit as detected by
Western blot analysis and the residual Complex I activity is shown in
Fig. 3. In each panel, the dashed
line represents what would be expected if there were a perfect
correlation between loss of subunit and loss of activity. As shown in
Fig. 3, the levels of the 39- and 30-kDa subunits most closely track
the loss of activity. However, in most cases the levels of the 20- and 18-kDa subunits are higher than predicted from the activity effects, whereas the levels of the 15- and 8-kDa subunits are lower than the
residual levels of enzymatic activity.
Sucrose Gradient Analysis of Patient Cell Lines--
Mitochondria
from three cells lines, i.e. from patient 7 with a mutation
in NDUFV1, patient 1 with a mutation in NDUFS7, and patient 11, with an
unknown mutation, were each dissolved in 1% LM and subjected to
sucrose gradient centrifugation using a discontinuous gradient. In this
gradient, the five complexes of OXPHOS are separated by size, and each
can be identified by Western blotting of the fractions with monoclonal
antibodies. Densitometric scans of the Western blots can then be made,
and, for convenience, the relative expression levels of each subunit in
the various fractions can be expressed as a percentage of the highest
intensity band in the gradient. Fig. 4
shows the distribution in the gradient of the Va subunit of cytochrome
c oxidase as well as the 39- and 20-kDa subunits of Complex
I for the three patient cell lines and a control of MRC5 fibroblasts.
Complex I, with a molecular weight of close to 900,000, elutes before
the other respiratory chain complexes after gradient separation. The
39- and 20-kDa subunits of patients 1 and 7 elute at a position similar
to that of control Complex I, indicative of complete or near complete assembly. However, in patient 11, the 39- and 20-kDa subunits migrate
in subcomplexes of ~200 and 500 kDa, respectively, and there is also
a free subunit (eluting in fraction 9). Thus, in patient 11, assembly
of Complex I is poor.
Monoclonal antibodies are finding widespread use in detecting and
analyzing mitochondrial disorders (17, 24). In the case of cytochrome
c oxidase, a panel of antibodies has been developed, which
reveals different patterns of assembly of the enzyme complex (25), and
this is providing a good indicator of which gene has been mutated,
including not only structural components but assembly factors as well.
Here, we show the utility of mAbs in the study of Complex I disorders.
Six different mAbs were used to examine 11 different patients in which
OXPHOS enzyme activity measurements had identified an isolated Complex
I deficiency. In seven of these, the mutations had been determined by
extensive gene sequencing. In the other four, a mutation has not yet
been identified. Screening with an antibody mixture containing an mAb
against at least one subunit of each of the five OXPHOS complexes
supports the conclusion from enzymatic data that all are deficient in
Complex I alone. It is important to note that the antibody screen to
localize the defect by the complex or complexes involved is relatively
rapid and easily performed and requires much less sample than enzymatic assays.
The reaction of the six mAbs against Complex I used here was variable,
and several different patterns of steady-state levels of subunits could
be distinguished. For two subunits, NDUFS4 and NDUFS2, two patient cell
lines were available with different mutations in the same gene. In both
cases, the subunit profiles resulting from the two different mutations
were essentially the same.
In general, the subunits behaved as three classes. The levels of the
39- and 30-kDa subunits varied in the same way, as did the 20- and
18-kDa subunits, whereas the 15- and 8-kDa subunits are a third class.
In most patients, the levels of the 20- and 18-kDa subunits were higher
than the levels of functional complex as measured by enzymatic
activity, whereas the levels of the 15- and 8-kDa subunits were lower.
Interestingly, the Rho0 cells seem to behave differently, because the
15- and 8-kDa subunits are present in higher amounts relative to the
20- and 18-kDa subunits.
The implication of the data in Fig. 3 is that the steady-state levels
of fully assembled Complex I depend on expression levels of all of the
subunits being examined here. When any one subunit is mutated, the
levels of assembled Complex I are reduced. The lower levels of the 15- and 8-kDa subunits in relation to activity could indicate more lability
of these subunits after assembly of the complex. One of the patient
cell lines, patient 11, had very low levels of all of the subunits
examined and values significantly lower than expected based on activity
measurements. It could be that enzymatic activity was overestimated or
that the complex is more labile to detergent solubilization with a
resulting proteolysis of polypeptides because of the mutation. On the
basis of the antibody data, patient 11 is the most likely to involve a
mutation in an assembly factor for Complex I. The levels of subunits
are low, and these subunits are not in a fully assembled complex based on the sucrose gradient data. The comparison of subunit profiles as
shown here allows patients to be sorted as in genetic complementation studies, so that with wider screening of patients, a group of possible
Complex I assembly factor mutants can be collected for chromosomal
analysis and gene identification, as was done for the SURF1 mutations
of cytochrome c oxidase (8).
Besides a role in diagnosis, the antibody studies here add to the
understanding of genotype-phenotype relationships of mutations already
specified genetically. Patient 7 was shown to carry a mutation, T423M,
in NDUFV1, the flavin-containing subunit of Complex I. The subunit
profile and, more definitively, the sucrose gradient experiments show
that the ~25% loss of activity is due to altered catalytic function
and not a failure to assemble the complex. This is different from other
patients studied here, such as patients 5, 6, and 8, in whom the levels
of all of the subunits probed were significantly decreased, suggesting
a more profound defect.
In summary, we provide evidence of the utility of monoclonal antibody
analysis in the characterization of Complex I deficiencies. It appears
that different assembly profiles occur when different subunits are
mutated. Antibodies to additional subunits of Complex I will be
required to extend the work reported here, and this project is ongoing.
Complex I patients have been reported from many centers studying
mitochondrial disorders. A more comprehensive analysis of the range of
assembly patterns and correlation with site of the mutation will
require collaboration and the sharing of the antibodies and cell lines,
which should be possible.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-maltoside
(LM; Calbiochem), centrifuging twice (10,000 × g, 12 min) to remove insoluble material, passing the supernatant over an
immunoaffinity column generated as described previously (16) using the
15-kDa Complex I antibody created in this laboratory, washing with
phosphate-buffered saline (PBS) containing 0.05% LM, and eluting with
100 mM glycine, pH 2.5.
at 2.0 µg/ml, anti-Complex II-70 kDa at 0.15 µg/ml (17), anti-Complex III-core Complex at 0.3 µg/ml, anti-Complex II at 3.0 µg/ml, anti-Complex IV
at 0.5 µg/ml (23), and anti-Complex I-20 kDa and anti-Complex I-25
kDa as twice-diluted hybridoma cell culture supernatants. The
commercially obtained anti-porin antibody (Calbiochem) was diluted
1:120,000.
-D-galactopyranoside. Cells before and
after induction were analyzed by Western blot. Antigens with which
antibodies reacted after but not before induction are listed in Table
II.
80 °C. For mitochondrial
pellets, cells were solubilized in 5 ml homogenization buffer (1 mM EDTA, 0.25 M sucrose, 10 mM
Tris, pH 7.4) containing protease inhibitors (0.5 µg/ml leupeptin,
0.5 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl
fluoride). Cells were repeatedly (three times) homogenized with a
motorized pestle (15-20 strokes), and the postnuclear supernatants
were pooled after centrifugation (10 min, 1500 × g).
Mitochondrial pellets were obtained by centrifugation of the collected
postnuclear supernatants (15 min, 10,000 × g). The
mitochondrial pellets were washed twice (15 min, 10,000 × g) with 2 ml washing buffer (1 mM EDTA, 0.25 M sucrose, 10 mM Tris/HCl, pH 7.5) including
protease inhibitors (0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). Finally, pellets were
saved in 200 µl protease inhibitors/washing buffer and stored frozen
at
80 °C. Protein amounts were estimated by A
280 determination.
-mercaptoethanol (30 min, 37 °C),
were separated on 10-20% gradient polyacrylamide gels in a
Mini-Protein II Apparatus (Bio-Rad). After electrophoresis (100-V
stacking gel and 150-V running gel), proteins were transferred
electrophoretically (2 h, 0.10 A) to 0.45-µm polyvinylidine
difluoride membranes in transfer buffer (10% methanol in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid, pH 11) on
ice. The polyvinylidine difluoride membranes were blocked overnight
with 5% nonfat dried milk powder in Dulbecco's phosphate-buffered
saline (CMF-PBS). Afterward the blots were treated with primary
antibodies diluted in 5% milk CMF-PBS for 2 h. After rinsing the
blot three times with CMF-PBS and 0.05% Tween 20, the blots were
incubated for 2 h with horseradish peroxidase-conjugated goat
anti-mouse IgG + M (heavy and light chain) at 0.2 µg/ml
(Jackson ImmunoResearch) in CMF-PBS. Specific detection of the
secondary antibody was obtained with the chemiluminescent Western
blotting detection reagent ECL Plus (Amersham Pharmacia Biotech) after rinsing the blots with CMF-PBS three times. Fluorescence was quantified using a Storm 860 chemifluorescence imager and the accompanying Molecular Dynamics Imagequant software. Ratios of individual proteins were calculated in relation to the porin signal. The ratios of the
control fibroblast cell line were set to 100%, and the ratios of other
cell lines were reported in comparison with this. The obtained results
represent average values of two to five independent experiments for
each subunit.
80 °C. For Western blotting, 20 µl of each
fraction was loaded per lane.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Genetic and biochemical characteristics of the investigated fibroblast
cell lines
subunit of
F1F0 (Complex V), and porin (as a control for
equal loading of lanes). Fig. 1
summarizes the data with a bar graph in which the levels of each
complex are quantitated by determining the amount of the component
subunit in each patient cell line in relation to that found in control
skin fibroblasts. From the bar graph, it can be seen that the levels of
the 39-kDa subunit of Complex I, but not that of any of the other
OXPHOS subunit probed, are diminished in most of the patient cell
lines. This is different from the MRC5-Rho0 mitochondria, in which
loss of mtDNA leads to an absence of subunit II of Complex IV and
lower levels of the core II protein of Complex III.
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Fig. 1.
Isolated Complex I deficiency in patient cell
lines as revealed by Western blots. Western blot signals were
quantitated, and the levels of the Complex I 39-kDa subunit
(CI-39), Complex II 70-kDa subunit (CII-70),
Complex III core 2 subunit (CIII-Core 2), Complex IV subunit
IV (CIV-IV), and Complex V subunit (CV-alpha)
in normal and Rho0 MRC5 fibroblasts and the 11 patient cell lines in
relation to the control skin fibroblasts (C) were plotted.
All subunits were set to 100% for the control fibroblasts. All lanes
were standardized using porin as the control for equal loading.
Monoclonal antibodies used in this study
View larger version (89K):
[in a new window]
Fig. 2.
Variations in Complex I assembly identified
by Western blot. A, representative Western blot showing
the levels of each indicated polypeptide in normal and Rho0 MRC5
fibroblasts, control skin fibroblasts, and the 11 patient cell lines.
B, Western blot signals were quantitated, and the levels of
the indicated Complex I subunits in normal and Rho0 MRC5 fibroblasts
and the 11 patient cell lines in relation to the control skin
fibroblasts were plotted. All subunits were set to 100% for the
control fibroblasts. All lanes were standardized using porin as the
control for equal loading.
View larger version (30K):
[in a new window]
Fig. 3.
Relationship between loss of Complex I
subunits and residual Complex I activity. For each patient and the
control fibroblasts, the level of each of the indicated subunits as
detected by Western blot was plotted in relation to the residual
Complex I activity. In each panel, the dashed line
represents what would be expected if there were a perfect correlation
between loss of subunit and loss of activity.
View larger version (25K):
[in a new window]
Fig. 4.
Western blot analysis of three different
patient cell lines and a control MRC5 cell line after sucrose gradient
centrifugation. Shown are gradients of patient 1 ( ), patient 7 (
), patient 11 (×), and control MRC5 fibroblasts (
). In each
case the darkest intensity band for each antibody and each sample is
set to 100%. Shown are the distributions of the Va subunit of Complex
IV (A), the 39-kDa subunit of Complex I (B), and
a 20-kDa subunit of Complex I (C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We greatly appreciate the technicians of the Nijmegen Center for Mitochondrial Disorders biochemistry group for measurements of the OXPHOS activities in skeletal muscle and cultured skin fibroblasts. We also thank Frans Trijbels and Rob Sengers for continuous support and are grateful to all colleagues who provided the patient cell lines.
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FOOTNOTES |
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* This work was supported in part by Prinses Beatrix Fonds Grant 97-0111 (to J. A. S. and L. P. v. d. H.), National Institutes of Health Grant HL24526 (to R. A. C.), and the Fonds Beoefening Wetenschap foundation of the Department of Pediatrics of the University Medical Center Nijmegen. R. H. T. and B. J. H. contributed equally to this work.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.
¶ Supported by National Institutes of Health Training Grant GM07759.
** To whom correspondence should be addressed: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403. Tel.: 541-346-5881; Fax: 541-346-5891; E-mail: rcapaldi@oregon.uoregon.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M009903200
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ABBREVIATIONS |
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The abbreviations used are:
OXPHOS, oxidative
phosphorylation;
mtDNA, mitochondrial DNA;
LM, N-dodecyl--D-maltoside;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody;
CMF-PBS, Dulbecco's phosphate-buffered saline.
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