From the Institut für Physiologische Chemie der
Universität München, 80336 München, Germany and the
§ Zentrum der Biologischen Chemie,
Universitätsklinikum Frankfurt, 60590 Frankfurt, Germany
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ABSTRACT |
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The subunit composition of the mitochondrial ATP
synthase from Saccharomyces cerevisiae was analyzed using
blue native gel electrophoresis and high resolution SDS-polyacrylamide
gel electrophoresis. We report here the identification of a novel
subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open
reading frame of 127 base pairs (ATP18), which encodes for
Su j, was identified on chromosome XIII. Su j does not display sequence
similarity to ATP synthase subunits from other organisms. Data base
searches, however, identified a potential homolog from
Schizosaccharomyces pombe with 51% identity to Su j of
S. cerevisiae. Su j, a small protein of 59 amino acid
residues, has the characteristics of an integral inner membrane protein
with a single transmembrane segment. Deletion of the ATP18
gene encoding Su j led to a strain ( Yeast mitochondrial ATP synthase (1) is similar to the
corresponding bovine enzyme (2, 3) regarding its polypeptide composition, but there are also differences. All components of the
bovine catalytic sector of the ATP synthase (F1),
i.e. subunits The experiments described in this paper focus on the reassessment of
the polypeptide composition of the yeast ATP synthase using a different
isolation technique, namely blue native electrophoresis (BN-PAGE).2 BN-PAGE is a
microscale technique for the separation of the multiprotein complexes
of oxidative phosphorylation directly from isolated mitochondrial
membranes (21). Combined with SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) in a second dimension, an overview on the protein subunits
of all oxidative phosphorylation complexes is obtained in a
two-dimensional gel (22). Using this two-dimensional electrophoretic
technique, we observed in the ATP synthase the presence of a previously
undetected protein in the 6-7-kDa range. N-terminal protein sequencing
revealed it to be a novel subunit of the ATP synthase, subunit j (Su
j). Su j is encoded by a gene termed here ATP18 and has no
apparent bovine counterpart.
Materials--
Aminocaproic acid (6-aminohexanoic acid) and
imidazole were from Fluka, Tricine and Serva Blue G (Coomassie Blue
G-250) were from Serva, and phenylmethylsulfonyl fluoride was from
Sigma. Hydroxyapatite was prepared as described recently (23).
Yeast Strains and Growth Conditions--
For construction
of the
The resulting yeast strain Isolation of Mitochondrial Membranes--
About 5 g (wet
weight) of sedimented cells, 5 ml of glass beads (0.25-0.5 mm), and 5 ml of sucrose buffer were vortexed for 10 min in a 50-ml tube. After
dilution with sucrose buffer, the sedimented glass beads were removed,
and the supernatant was centrifuged for 20 min at 1,250 × g. Mitochondrial membranes were then collected by
centrifugation for 30 min at 18,000 × g, taken up with
sucrose buffer at a protein concentration of 10-30 mg/ml, and stored
at
For the analysis of the submitochondrial localization of Su j, intact
mitochondria were isolated according to previously published methods
(26).
Electrophoretic Techniques--
BN-PAGE was performed as
described previously (22) with the following modifications.
Mitochondrial membranes (400 µg of protein) were sedimented by
centrifugation for 10 min at 100,000 × g. The pellet
was suspended with 40 µl of 50 mM NaCl, 2 mM
6-aminohexanoic acid, 1 mM EDTA, 50 mM
imidazole HCl, pH 7.0, and 1.0 µl of 0.5 M
phenylmethylsulfonyl fluoride in Me2SO was added. Membrane
protein complexes were solubilized by the addition of Triton X-100 (9.6 µl from a 10% (w/v) stock solution, 2.4 g of Triton X-100/g of protein). After centrifugation for 20 min at 100,000 × g, the supernatant was supplemented with 5 µl of a
Coomassie Blue G-250 dye suspension (5% Serva Blue G (w/v) in 750 mM 6-aminohexanoic acid) and immediately applied to a
1.6-mm acrylamide gradient gel for analytical BN-PAGE (1-cm gel well,
linear 4-13% acrylamide gradient gel overlaid with a 4% sample gel).
For SDS electrophoresis, the Tricine-SDS-PAGE (27) or the Laemmli
system (28) was used. Two-dimensional electrophoresis (BN-PAGE/Tricine-SDS-PAGE), staining, and densitometric quantification were performed as described previously (29, 30).
Isolation of ATP Synthase, Separation of Subunits, and N-terminal
Sequencing--
All steps were performed at 4 °C, and the pH values
of all buffers were adjusted to 4 °C unless otherwise indicated.
Mitochondrial membranes from the W303-1A strain (50 mg of protein) were
washed with a 4-fold volume of buffer 1 (50 mM NaCl, 2 mM 6-aminohexanoic acid, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM
imidazole HCl, pH 7.0) and collected by centrifugation for 60 min at
100,000 × g. The pellet was homogenized in 2.35 ml of
buffer 1, and 0.6 ml of Triton X-100 (20% w/v) was added (2.4 g of
Triton X-100/g of protein). After centrifugation for 60 min at
100,000 × g, the supernatant (2.6 ml) was adjusted to
150 mM Na+ phosphate and loaded onto a 3-ml
hydroxyapatite column equilibrated with buffer 2 (0.05% Triton X-100,
2 mM 6-aminohexanoic acid, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 150 mM
Na+ phosphate, pH 7.7). Hydroxyapatite-bound ATP synthase
was washed at room temperature with 1 column volume of buffer 3 (0.1%
Triton X-100, 333 µM egg yolk phospholipid, 200 mM Na+ phosphate, pH 7.3) and eluted with
buffer 4 (0.1% Triton X-100, 333 µM egg yolk
phospholipid, 300 mM Na+ phosphate, pH 7.3).
One ml of the fraction with highest ATP hydrolysis activity (about 30%
of the total yield, 0.5 mg of total protein) was supplemented with 200 mM 6-aminohexanoic acid and loaded onto a 3-mm-thick
preparative gel for BN-PAGE. The major blue band comprising ATP
synthase, which was visible during BN-PAGE, was excised and cut into 4 pieces. A stack of these 4 pieces was processed by Tricine-SDS-PAGE in
a second dimension and electroblotted onto Immobilon P membranes (30).
The transferred proteins were sequenced directly using a 473A protein
sequencer (Applied Biosystems) or after incubation in a 1:1 (v/v)
mixture of trifluoroacetic acid and methanol (24 h at 37 °C)
for deformylation (31).
Catalytic Activities--
Oligomycin-sensitive ATP hydrolysis
was measured at 25 °C using an assay coupled to the oxidation of
NADH. Shortly before the test, 0.25 mM NADH, 1 mM phosphoenolpyruvate, 2.5 units/ml lactate dehydrogenase,
and 2 units/ml pyruvate kinase were added to the test buffer (250 mM sucrose, 50 mM KCl, 5 mM
MgCl2, 2 mM NaCN, 20 mM Tris/HCl,
pH 7.5). The reaction was started with protein without detergent and
stopped by the addition of 25 µg of oligomycin from a 5 mg/ml
stock solution in Me2SO.
Antibody Production--
Antisera against the C-terminal region
of Su j were raised in rabbits against a chemically synthesized peptide
CRFAKGGKFVEVD that had been coupled to activated ovalbumin (Pierce).
Miscellaneous--
Hypotonic swelling and carbonate extraction
of mitochondria were performed as described previously (32, 33).
Protein determination was performed according to Bradford (34) and a
Lowry protocol in the presence of SDS (35).
Isolation of ATP Synthase and Analysis Protein Subunit
Composition--
The ATP synthase binds stronger to hydroxyapatite
than most other mitochondrial proteins of yeast. Therefore
hydroxyapatite chromatography is an efficient technique for its
isolation. After this first purification step, some of the known ATP
synthase subunits can already be recognized in SDS-PAGE (Fig.
1A, lanes 3-5).
The same fractions of the hydroxyapatite column were also applied to a
gel for BN-PAGE. The S. cerevisiae ATP synthase (Fig.
1B, lanes 3-5) is slightly smaller than the
bovine ATP synthase that was loaded in parallel. BN-PAGE was then
repeated on the preparative scale using the hydroxyapatite fraction
with the highest amount of ATP synthase complex (Fig. 1B,
lane 4). The band of ATP synthase was excised, and the
subunit composition of this complex was analyzed further by SDS-PAGE.
N-terminal protein sequencing of the resolved proteins (Fig.
1C) showed the presence of known subunits of the ATPase and
one additional protein that was termed subunit j, Su j. To exclude the
possibility that this protein may represent a contamination of the ATP
synthase, a two-dimensional resolution of the sample from Fig. 1,
lane 4, was performed. The two-dimensional gel clearly shows
that Su j fits the pattern of established subunits of the complex (Fig.
1D). Because no streaking of this protein was observed, we
conclude that Su j is a true constituent of the ATP synthase
complex.
Subunit j could not be removed from the ATP synthase isolated by
hydroxyapatite chromatography by adding 7 g of Triton X-100/g of
protein and application to BN-PAGE. After the addition of Triton X-100
and 2 M urea and application to BN-PAGE, most of the ATP synthase was dissociated into the individual subunits. The residual fraction of holo-ATP synthase still contained Su j (data not shown).
Polypeptide Composition of ATP Synthase--
Direct Edman
degradation of the proteins transferred to Immobilon P or after
deformylation (Su 8 and Su 9) confirmed the presence of known subunits
of ATP synthase. The mature subunits Su j Is an Integral Mitochondrial Inner Membrane
Protein--
Using the obtained N-terminal sequence of Su j, a search
for an open reading frame corresponding to a 6.5-kDa protein in the yeast genome data base was performed. A 177-base pair sequence on
chromosome XIII was identified with the potential to encode for a
protein of 59 amino acid residues (Fig.
2A). This gene encoding for Su
j was termed ATP18. We identified a potential homolog in Schizosaccharomyces pombe, a hypothetical protein of 6.8 kDa. This potential Su j homolog in S. pombe was 51%
identical to the Su j of S. cerevisiae. The hydropathy plots
for both proteins were very similar and suggested them to be membrane
proteins with a single transmembrane domain (Fig. 2B).
A peptide corresponding to the C-terminal region of the protein was
used to raise antibodies against Su j. Su j was localized to
mitochondria by immunostaining. It was inaccessible to added protease
in intact mitochondria (Fig. 2C). Disruption of the outer membrane by hypotonic swelling rendered Su j sensitive to the added
protease. In addition, Su j was resistant to alkaline extraction and
therefore most likely is an integral membrane protein (Fig. 2C).
In summary, Su j is a protein anchored to the inner membrane by a
single transmembrane segment at its N terminus and has an Nin-Cout orientation.
Deletion of the ATP18 Gene Leads to Spontaneously Arising
Rho Su j Is Required for the Stable Expression of Other Subunits of the
F0 Sector--
The nature of the association of Su j with
the inner membrane and its predicted orientation in the membrane would
suggest Su j to be a subunit of the F0 sector of the
F1F0-ATP synthase. We therefore asked whether
the expression of Su j was required for the stable expression of other
F0 sector subunits. Mitochondria from the The subunit composition of the yeast mitochondrial
F1F0-ATP synthase was analyzed using the
combined techniques of BN-PAGE and high resolution Tricine-SDS-PAGE. We
present evidence here for the existence of a novel ATP synthase
subunit, Su j. A homologue in the purified bovine ATP synthase complex
has so far not been reported. The presence of an open reading frame in
S. pombe with 51% amino acid sequence identity to the
S. cerevisiae Su j suggests that Su j represents a general
component of eukaryotic ATP synthases.
The novel Su j protein appears to represent a bona fide subunit of the
ATP synthase. Su j purified with the ATP synthase after BN-PAGE. As
this technique resolves proteins by their native molecular mass (22),
Su j is unlikely to be a contaminant of the ATP synthase; this would
require it to have the same native size. No other polypeptides, which
could not be assigned to the ATP synthase complex according to the
amino acid sequence, were present in the purified ATP synthase fractions. Su j was observed to be tightly bound to the ATP synthase. Treatment of the isolated complex under conditions that led to its
almost complete dissociation resulted in a residual fraction of
holo-ATP synthase, which still contained Su j. We therefore suggest
that Su j is required for the structural integrity of the ATP synthase
complex. Consistent with this view, Su j appears to be an essential
subunit of the yeast ATP synthase complex. Deletion of the gene
encoding Su j (ATP18) gave rise to a respiratory-deficient phenotype and loss of measurable oligomycin-sensitive ATP synthase activity.
The tight binding of subunit j to the isolated yeast ATP synthase
raises the question as to why subunit j was not previously identified
in other ATP synthase preparations. The previous use of SDS gels
probably did not yield sufficient resolution of the smaller subunits of
the complex. As shown here, the use of high resolution Tricine-SDS-PAGE
has optimized the separation of the yeast ATP synthase subunits in the
molecular mass range of Su j. Furthermore, although we used mild
solubilization with Triton X-100 and BN-PAGE as a one-step procedure,
subunits g and e (Tim11) or a potential homologue of bovine subunit F6
were not found in association with the ATP synthase complex. Notably,
recent variation of the conditions for protein solubilization and
BN-PAGE led to the isolation of an ATP synthase with three more bound
proteins, including subunits e (Tim11)
and subunit g. The analysis of the role of these proteins for the
structure and function of ATP synthase will be discussed separately
(37).
In conclusion, we demonstrate here that Su j is an integral inner
membrane protein, spanning the membrane once in an
Nin-Cout orientation. The membrane association
of Su j is compatible with it being a subunit of the F0
sector of the ATP synthase. We are currently investigating the
association of Su j with other known F0 sector subunits.
su j) completely
deficient in oligomycin-sensitive ATPase activity and unable to grow on
nonfermentable carbon sources. The presence of Su j is required for the
stable expression of subunits 6 and f of the F0 membrane
sector. In the absence of Su j, spontaneously arising rho
cells were observed that lacked also ubiquinol-cytochrome c
reductase and cytochrome c oxidase activities. We conclude
that Su j is a novel and essential subunit of yeast ATP synthase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
,
,
,
,
, and the inhibitor
protein (IF1), have homologous counterparts in
Saccharomyces cerevisiae (4-9). The structural similarity
extends to subunits of the membrane sector (F0) and second
stalk, i.e. subunit a (Su a or Su 6) (10), Su b (11), Su c
(proteolipid or Su 9) (12), which is mitochondrially encoded in yeast
but nuclearly encoded in mammals, Su d (13), oligomycin sensitivity
conferring protein (14), Su 8 (15), and Su f (16). In addition, a gene
encoding a putative homolog of subunit g of the bovine ATP synthase has
been identified in the yeast genome (Su
g).1 The corresponding
protein, however, has not been observed in the isolated ATP synthase
yet. A yeast homolog of bovine subunit e has also been reported
recently (17). Subunit e, also known as Tim11, was originally reported
as being a component of the mitochondrial inner membrane import
machinery (18); however, it has subsequently been shown to be a
membrane-bound subunit of the ATP synthase complex (17). Although in
mitochondria it is associated with the ATP synthase complex and can be
co-immunoprecipitated with subunits of the F1 sector (17),
subunit e, like subunit g, has not been identified yet in the purified
ATP synthase enzyme. A yeast homologue to bovine subunit F6 (19) so far
has not been reported. Conversely, a novel subunit, subunit h, has been
found recently in yeast ATP synthase (20), which appears not to be related to any of the known subunits of bovine or other ATP synthases.
EXPERIMENTAL PROCEDURES
su j::HIS3 yeast strain, introduction of
the HIS3 gene resulting in a partial deletion and disruption of the ATP18/Su j gene was performed as follows.
The HIS3 gene was amplified from the plasmid pFA6a-HIS3MX6
(24) using the following primers: S1,
5'-GTTTAACATACGACGACAGATTAATTGATTGGATTGTACTGCCATGCGTACGCTGCAGGTCGAC-3' (corresponding to nucleotides
43 to +3 of the
ATP18/Su j gene locus and 18 nucleotides of the
multiple cloning site of pFA6a-HIS3MX6 from the 5' flanking region of
the HIS3 gene); and S2,
5'-TGGATCATTGATAAATTCCTTCGTGTTAGAAGAAAGGTCAGCAGCATCGATGAATTCGAGCTCG-3' (corresponding to nucleotides +90 to +132 of the
ATP18/Su j gene and 19 nucleotides of the
multiple cloning site of pFA6a-HIS3MX6 from the 3' flanking region of
the HIS3 gene). The resulting polymerase chain reaction
product was transformed into the haploid yeast strain W303-1A using the
lithium acetate method (25), and HIS3 positive clones were
selected. Correct integration of the HIS3 marker into the
ATP18/Su j locus was confirmed by polymerase
chain reaction using oligonucleotides that primed upstream and
downstream of the disrupted ATP18 gene.
su j, the corresponding
wild-type W303-1A, and
su f were grown in YPGal medium
supplemented with 0.5% lactate at 30 °C (26). Cells were harvested
by centrifugation at 1,800 × g, washed three times
with sucrose buffer (250 mM sucrose, 5 mM
6-aminohexanoic acid, 10 mM Tris/HCl, pH 7.0), and used
directly for preparation of mitochondrial membranes.
80 °C.
RESULTS
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Fig. 1.
Isolation and polypeptide composition of ATP
synthase from S. cerevisiae. A, enrichment of ATP
synthase by hydroxyapatite chromatography is shown. Lane 1, flow-through; lane 2, wash with buffer 3; lanes
3-5, fractions eluted with buffer 4. Equal volumes of the
fractions (10 µl each) were analyzed by Tricine-SDS-PAGE using a 10%
acrylamide gel. B, resolution of the same fractions as in
A by BN-PAGE. The oxidative phosphorylation complexes from
solubilized bovine heart mitochondria (BHM) were used for
molecular mass calibration in a linear 4-13% acrylamide gradient gel.
C, polypeptide composition of ATP synthase purified by
preparative BN-PAGE of fraction 4 and electroelution of the ATP
synthase band. The polypeptides were resolved by Tricine-SDS-PAGE (14%
acrylamide, 6 M urea gel) (see "Experimental
Procedures"). Proteins were stained with Coomassie Blue G-250.
D, resolution of fraction 4 by BN-PAGE (B)
followed by Tricine-SDS-PAGE in the second dimension. Silver stain, gel
type as in C. OSCP, oligomycin sensitivity
conferring protein; INH1, inhibitor protein.
and
were found to be 4 and
14 amino acids, respectively, shorter than described in protein data
bases (Table I). The presence of subunit 6 was confirmed by Western blotting and subsequent immunodecoration using a specific antiserum. The novel Su j was directly accessible to
Edman degradation, and a sequence of 13 N-terminal amino acids could be
obtained (Table I).
Proteins identified in isolated ATP synthase of S. cerevisiae
). Small letters
in the sequences indicate amino acids that were not identified. The
masses of the mature proteins do not include N-terminal modifications.
AA, number of amino acids.
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Fig. 2.
Sequence and submitochondrial localization of
Su j from S. cerevisiae and comparison to a S. pombe
homolog. A, alignment of the sequences of Su j
from S. cerevisiae (S.c.) and potential homolog
from S. pombe (ORF S.p.) (O13931, EMBL).
Identical (connecting line) and similar residues
(colons) between sequences (36) are indicated. B,
hydropathy profiles of Su j from S. cerevisiae and
homologous open reading frame (ORF) from S. pombe. C, submitochondrial localization of Su j is
shown. Left panel, mitochondria and mitoplasts were
generated by hypotonic swelling and incubated for 30 min on ice in the
presence or absence of proteinase K (PK) (200 µg/ml).
Right panel, mitochondria were subjected to alkaline
extraction (0.1 M Na2CO3, pH 11.5)
for 30 min on ice. The sample was divided; one-half was directly
trichloroacetic acid-precipitated (T, total), and the other
was separated by centrifugation (60 min at 226,000 × g) into pellet (P) and supernatant (S)
fractions, prior to trichloroacetic acid precipitation. All samples
were analyzed by SDS-PAGE and Western blot analysis. Specific antisera
were used against cytochrome c peroxidase (CCPO)
and cytochrome b2
(Cytb2), both soluble proteins of the
intermembrane space; Mge1p, a matrix localized soluble
protein; AAC, ADP/ATP carrier protein, an integral inner
membrane protein; and Su j.
Cells--
To test whether the presence of Su j is
essential for the activity of the F1F0-ATP
synthase, the gene encoding Su j was deleted. The resulting yeast
strain
su j was respiratory incompetent, as it could no
longer grow on the nonfermentable carbon source glycerol, in contrast
to its isogenic wild-type strain (Fig.
3). Enzymatic measurement of the
F1F0-ATP synthase activity confirmed the loss
of oligomycin-sensitive ATPase activity in isolated
su j
in contrast to the wild-type control (results not shown); therefore Su
j seemed to be an essential subunit of the yeast
F1F0-ATP synthase. However, comparison of the
mitochondrial proteins of the
su j and
su f
strains by BN-PAGE (Fig. 4) and
two-dimensional resolution (not shown) revealed that not only the ATP
synthase but also cytochrome oxidase (complex IV) and
ubiquinol-cytochrome c reductase (complex III) were below the limit of detection (<10% as compared
with wild-type W303-1A). The reduced levels of complex III and IV can be explained by the fact that a spontaneous transition of the
su j cells to the rho
state was
observed.3 Interestingly a
similar formation of rho
cells was observed in the
su f strain, as reported by Spannagel et al.
(16).
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Fig. 3.
Su j is essential for growth on
nonfermentable carbon sources. Yeast strains su j
and corresponding isogenic wild-type W303-1A grown on YPD
(glucose-containing) medium were resuspended in sterile water at
a concentration of 10 A578/ml. A dilution series
was generated by serially diluting this suspension 10-fold each time. 2 µl of each of the resulting dilutions were spotted onto a YPG
(glycerol-containing) plate (spots 1-5) and were incubated
at 30 °C for 2 days.
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Fig. 4.
BN-PAGE of Triton X-100-solubilized
mitochondria from S. cerevisiae wild-type strain (W303-1A)
and the deletion strains su j and
su f.
ATP synthase (complex V) and cytochrome oxidase (complex
IV) are missing in both deletion strains. An additional loss
of ubiquinol-cytochrome c reductase (complex
III), which gives rise to a broad band not detectable in
BN-PAGE, was revealed after resolution by SDS-PAGE in a second
dimension (not shown). BHM, bovine heart mitochondria.
su
j strain were analyzed by Western blotting for the presence of
various subunits of the ATP synthase (Fig.
5). In the absence of Su j, subunits 6 and f of the F0 sector were not detectable. The
-subunit
of the F1 sector was reduced in the
su j
strain. Levels of other mitochondrial marker proteins, such as
cytochrome c peroxidase and Mge1p, were not altered in the absence of Su j.
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Fig. 5.
The presence of Su j is essential for the
stable expression of F0 sector subunits. Mitochondria
(50 µg of protein) isolated from the su j strain and
corresponding isogenic wild-type strain W303-1A were subjected to
SDS-PAGE and analyzed by Western blotting for the presence of marker
proteins, as indicated. CCPO, cytochrome c
peroxidase; Mge1p, a matrix-localized soluble protein.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Jean Velours
(Université Bordeaux, France) for the generous gift of the
antisera against ATPase subunits f and 6 and the su f
yeast strain. We thank Sandra Weinzierl and Monika Krampert for
excellent technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 184 (Teilprojekt B2) (to R. A. S.) and Sonderforschungsbereich 472 (Teilprojekt P11) (to H. S.).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.
The protein sequence data have been submitted to the SWISS-PROT data base with accession number P81450 (ATPase j chain).
¶ To whom correspondence should be addressed. Tel.: 49-89-5996-295; Fax: 49-89-5996-270; E-mail: stuart{at}bio.med.uni-muenchen.de.
1 EMBL accession number Z71255, SWISS-PROT accession number Q12233.
3 I. Arnold, K. Pfeiffer, W. Neupert, R. A. Stuart, and H. Schägger, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: BN, blue native; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine..
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