From the Instituto de Biologia Molecular e Celular,
Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto,
Portugal, the § Department of Plant Physiology, Lund
University, Box 117, S-22100 Lund, Sweden, the ¶ Institut
für Physiologische Chemie der Universität München,
Goethestrasse 33, D-80336 München, Germany, the
Department of Biology, University of New Mexico, Albuquerque,
New Mexico 87131, and the ** Instituto de Ciências
Biomédicas de Abel Salazar, Universidade do Porto, 4099-003 Porto, Portugal
Received for publication, September 7, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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We have inactivated the nuclear gene coding for a
putative NAD(P)H dehydrogenase from the inner membrane of
Neurospora crassa mitochondria by repeat-induced point
mutations. The respiratory rates of mitochondria from the resulting
mutant (nde-1) were measured, using NADH or NADPH as
substrates under different assay conditions. The results showed that
the mutant lacks an external calcium-dependent NADPH
dehydrogenase. The observation of NADH and NADPH oxidation by intact
mitochondria from the nde-1 mutant suggests the existence of a second external NAD(P)H dehydrogenase. The topology of the NDE1
protein was further studied by protease accessibility, in vitro import experiments, and in silico analysis of
the amino acid sequence. Taken together, it appears that most of the
NDE1 protein extends into the intermembrane space in a tightly folded conformation and that it remains anchored to the inner mitochondrial membrane by an N-terminal transmembrane domain.
In nonphotosynthetic eukaryotes, the mitochondrion is the cellular
organelle responsible for producing most of the energy required for
cellular metabolism. The process of oxidative phosphorylation takes
place in the inner mitochondrial membrane, whereby the electrons produced by the oxidation of substrates like NAD(P)H are transported through the electron transport chain to oxygen, coupled to the generation of a transmembrane proton gradient that eventually leads to
ATP synthesis (1). In contrast to mammals, the electron transport
chains of plants and fungi possess several nonproton-pumping NAD(P)H
dehydrogenases for transferring electrons to ubiquinone (2). In the
case of potato tubers, four rotenone-insensitive NAD(P)H dehydrogenases
have been identified in the inner mitochondrial membrane, two with the
catalytic site facing the matrix (3, 4) and two facing the
intermembrane space (5). In mitochondria from Saccharomyces
cerevisiae, where the proton-pumping complex I is not present, the
oxidation of NADH and NADPH is performed exclusively by three
nonproton-pumping enzymes, one facing the matrix and two facing the
intermembrane space (6-8). In addition, the genome analysis of
Synechocystis revealed three open reading frames that
may code for such type II NAD(P)H dehydrogenases (9). On the other
hand, only one external type II NADH dehydrogenase was reported for the
fungus Yarrowia lipolytica (10). Although NAD(P)H
dehydrogenases have been studied for a long time, our understanding of
protein function at the molecular level is still very incomplete. The
cloning of genes encoding several of these rotenone-insensitive NAD(P)H
dehydrogenases from mitochondria of different organisms (7, 10-13)
provides important tools for further research in this field. These
enzymes might constitute a wasteful system acting to prevent the
overreduction of the electron transport components and the production
of reactive oxygen species, but their exact roles remain unclear.
Both proton-pumping and nonproton-pumping NAD(P)H dehydrogenases have
been described in Neurospora crassa mitochondria. In 1970, the presence of at least two different rotenone-insensitive NADH
dehydrogenases in the inner membrane was described (14), one on the
inner side catalyzing the oxidation of matrix NADH and a second on the
outer side accessible to cytosolic NADH and NADPH. It was later
reported that the external NAD(P)H activity is partly
calcium-dependent (15). The internal rotenone-insensitive NADH dehydrogenase is not linked to the formation of ATP and is very
active in the early exponential phase of growth (16). Here we identify
NDE1, previously called p64 (11), as an external calcium-dependent NADPH dehydrogenase and provide evidence
for the presence of at least a second dehydrogenase; both enzymes oxidize reduced pyridine nucleotides from the cytosol of N. crassa cells.
Preparation of Mitochondria--
General manipulation of
N. crassa, including crosses, was performed by standard
procedures (17, 18). The wild-type strain 74-OR23-1A and the
nde-1 mutant were grown in Vogel's minimal medium. The
techniques for the preparation of N. crassa mitochondria and
IO-SMP1 either for oxygen
electrode measurements (4) or for digitonin and import experiments
(19), as well as the isolation of mitochondria from S. cerevisiae wild-type strain D-273-108 (20), have been described.
In Vitro Import into Mitochondria--
The cDNA encoding the
open reading frame of NDE1 was amplified by polymerase chain
reaction using two specific primers and cloned in pCR II-TOPO vector
(Invitrogen). Precursor polypeptides were synthesized in
vitro with a coupled transcription/translation system (Promega) in
the presence of [35S]methionine (21) and imported into
N. crassa or S. cerevisiae mitochondria. Import
reactions were performed in a medium containing 0.5 M
sorbitol, 80 mM KCl, 50 mM HEPES, pH 7.2, 3%
(w/v) bovine serum albumin, 10 mM
Mg(CH3COO)2, 2 mM potassium
phosphate, 2.5 mM EDTA, 1 mM MnCl2,
2 mM NADH, 2 mM ATP, 1% (v/v) ethanol, 2 mM creatine phosphate, 0.1 mg/ml creatine kinase, 1% (v/v)
of rabbit reticulocytes with the relevant radiolabeled precursors, and
0.2 mg/ml mitochondria for 20 min at 25 °C. To abolish the membrane
potential, 0.1 mM valinomycin was added, and creatine phosphate and creatine kinase were omitted. After import, mitochondria were incubated with 30 µg/ml trypsin on ice for 15 min, and the protease activity was stopped by the addition of 800 µg/ml soybean trypsin inhibitor (22). Swelling of yeast mitochondria was achieved in
media without sorbitol.
Disruption of nde-1--
The cDNA encoding NDE1 (11) was
excised from pBluescript by digestion at flanking PvuII
restriction sites, inserted in the EcoRV restriction site of
the pCSN44 vector, and transformed into N. crassa
spheroplasts (18). Individual transformants were selected on hygromycin
B (Sigma) plates (200 µg/ml) and purified by several asexual
transfers in Vogel's minimal medium plus 150 µg/ml hygromycin B. A
single copy transformant was identified by Southern blot analysis of
digested genomic DNA (23, 24) and crossed with the wild-type strain
74-OR23-1A to obtain mutants by repeat-induced point mutations (25,
26). Detection of nde-1 mutants among the progeny of the
cross was carried out by the analysis of Western blots (27) of
mitochondrial proteins with an antiserum against NDE1 (11).
Northern Blot Analysis--
To analyze gene expression, N. crassa mRNA was isolated from conidial (germinating asexual
spores), mycelial (branching hyphae), or perithecial (fruiting body or
sexual) tissues (28). Northern blots (29) were hybridized with random
primer-labeled restriction fragments from four N. crassa
cDNA clones obtained from three cDNA libraries (30). The probes
used were NM1C2, encoding NDE1, and SC3A5, SM1F6, and SP6B10, which
encode the 12.3-, 17.8-, and 21-kDa subunits, respectively, of the
mitochondrial complex I.
Oxygen Consumption--
Respiration was measured
polarographically at 25 °C with a Hansatech oxygen electrode in a
total volume of 1 ml. Assays with mitochondria and IO-SMP contained
0.3-0.5 mg of protein, 0.3 M sucrose, 10 mM
potassium phosphate, pH 7.2, 5 mM MgCl2, 1 mM EGTA, 10 mM KCl, 4 µM carbonyl
cyanide m-chlorophenylhydrazone, and 0.02% (w/v) bovine
serum albumin. For the pH experiments, the reaction medium contained 20 mM MES, 20 mM Tris, 20 mM MOPS, 0.3 M sucrose, 0.1 mM CaCl2, 4 µM carbonyl cyanide m-chlorophenylhydrazone, and 0.02% (w/v) bovine serum albumin adjusted to pH 4.7-9.2 with KOH.
Calcium depletion was achieved with 1 mM EGTA. The assays were initiated by the addition of either 1 mM NADH or 1 mM NADPH. Rotenone and antimycin A were added to final
concentrations of 20 µM and 0.2 µg/ml, respectively.
Integrity of mitochondria and sidedness of IO-SMP were assessed by the
activities of cytochrome-c oxidase (EC 1.9.3.1) and malate
dehydrogenase (EC 1.1.1.37) in the absence and presence of Triton X-100
(31).
Miscellaneous--
Standard procedures were used for cloning,
agarose gel electrophoresis and Southern blotting (32, 33),
polyacrylamide gel electrophoresis (34), protein determination (35),
and the development of rabbit antisera (36). Digitonin solubilization followed by proteinase K treatment (37) and
Na2CO3 extraction (24) of mitochondrial
proteins have been described.
Inactivation of nde-1--
To investigate the specific role of
NDE1, we disrupted the corresponding gene by the generation of
repeat-induced point mutations, an unusual phenomenon that causes
methylation and GC to AT transition mutations of repeated sequences in
Neurospora (38). Briefly, a strain carrying two copies of
the nde-1 gene was generated and crossed with a wild-type
strain to achieve the inactivation of both copies of nde-1
in the duplication strain. Mitochondrial proteins from individual
ascospore progeny were analyzed by Western blotting with antibodies
against NDE1 (11), and a mutant strain lacking the polypeptide was
identified (Fig. 1, compare lanes 1 and 2). Because we initially thought that NDE1 was
acting on the inner side of the inner mitochondrial membrane, oxidizing matrix NADH as an alternative to complex I, we crossed the
nde-1 mutant with several mutants in subunits of complex I. Surprisingly, we obtained double mutants in crosses between
nde-1 and the complex I mutants nuo51 (39),
nuo24 (40), nuo21.3a (26), nuo21.3c (41), nuo20.8 (42), and nuo12.3 (43),
respectively, which display various phenotypes in terms of complex I
assembly and function. Fig. 1, lane 3 depicts the analysis
of the double mutant nde-1/nuo51, which expresses
a nonfunctional complex I. We would expect these double mutants to be
nonviable due to severe deficiency in the oxidation of matrix NADH,
because only complex I and one alternative dehydrogenase were described
in Neurospora (14). Thus, these results represented the
first indication that NDE1 was using cytosolic NAD(P)H as a
substrate.
Thus, despite the fact that complex I is required for sexual
development (44), neither complex I nor NDE1 is essential for Neurospora vegetative growth under standard conditions. We
analyzed the expression of the corresponding genes throughout the life cycle of the fungus and performed a Northern blot analysis of mRNA
from conidia (germinating asexual spores), mycelia (branching hyphae),
or perithecia (fruiting bodies) using cDNAs encoding NDE1 and
complex I subunits as probes. The results demonstrated that all
transcripts, and probably the encoded proteins, are expressed constitutively throughout the Neurospora life cycle (data
not shown). This finding is corroborated by the identification of the
various cDNA clones in three different cDNA libraries,
representing both vegetative and sexual stages of development
(30).2
Characterization of NDE1 Activity--
To characterize the
activity of NDE1, mitochondria and IO-SMP were prepared from wild type
and the nde-1 mutant and tested for different activities
under various conditions. The oxidation rates of NADH and NADPH were
followed polarographically with an oxygen electrode. The
rotenone-insensitive oxidation rates for both substrates at pH 7.2 were
similar in IO-SMP from both strains. In addition, when intact
mitochondria were assayed with NADH at pH 7.2 no significant difference
was found between the two strains. However, NDE1-defective mitochondria
showed very reduced oxidation activity relative to the wild-type
organelles when NADPH was used as substrate. All activities were fully
inhibited by antimycin A (data not shown).
Therefore, we carried out a detailed characterization of exogenous
NAD(P)H oxidation by mitochondria from both strains by following the
oxidation rates of NADH and NADPH in the pH range from 4.7 to 9.2, in
the presence or absence of calcium (Fig.
2). The data were examined by the
statistical analysis of variance-covariance (ANCOVA), using as
covariate the value of pH (45). There was no significant difference in
the pattern of NADH oxidation between mitochondria from the two strains
throughout the pH range, neither in the presence (p = 0.29) nor in the absence of calcium (p = 0.089). NADH
oxidation by both strains was unaffected by calcium at acidic pH,
whereas decreased activity in the absence of calcium was observed under
alkaline conditions (Fig. 2, a and b). The differences were not statistically significant either in wild type
(p = 0.62) or in nde-1 (p = 0.89) in the pH range from 4.7 to 7.4 but were significant both in wild
type (p < 0.001) and nde-1
(p < 0.01) when the pH range from 7.7 to 9.2 was
considered. In contrast, there was a clear difference in NADPH
oxidation. In the presence of calcium, wild-type mitochondria oxidized
NADPH from pH 4.7 to 8.3, whereas mitochondria from the
nde-1 mutant had no activity above pH 7.2. Under calcium
depletion conditions, a drastic reduction in NADPH oxidation rate was
observed in wild-type mitochondria. At pH 8, this activity was totally
dependent on calcium (Fig. 2c). The statistical analysis
revealed that calcium had a highly significant effect on wild-type
activity throughout the pH range (p < 0.001). In
contrast, the oxidation of NADPH by mitochondria from the
nde-1 mutant was not significantly affected by calcium (Fig.
2d), as confirmed by the statistical analysis (p = 0.63). These results clearly indicate that NDE1 is
the external calcium-dependent NADPH dehydrogenase.
Topology of NDE1--
Several approaches were employed to obtain a
more detailed characterization of the topology of NDE1. Fig.
3 displays the accessibility of the
protein to proteinase K upon the fractionation of mitochondria with
increasing concentrations of digitonin. The opening of mitochondrial membranes was monitored by the use of antisera against the
mitochondrial processing peptidase (46), the ADP/ATP carrier (47),
cytochrome c heme-lyase (48), and TOM20 (49) as markers for
the matrix, inner membrane, intermembrane space, and outer membrane,
respectively. TOM20, with domains facing the cytosol, was readily
digested by proteinase K. Total solubilization of the outer
mitochondrial membrane was achieved at a digitonin concentration of
0.2% as attested by the disappearance of cytochrome c
heme-lyase. The inner mitochondrial membrane was solubilized at the
highest digitonin concentration (2%). The behavior of NDE1 parallels
that of cytochrome c heme-lyase; when the outer
membrane was opened at 0.2% digitonin concentration, its exposure to
proteinase K resulted in two resistant fragments. The larger fragment
of 57 kDa remained membrane-bound as shown in Fig. 3, right
panel. In this experiment, a mitochondrial sample incubated with
0.3% digitonin and proteinase K was incubated with
Na2CO3 and resolved into pellet and supernatant
to discriminate between intrinsic and extrinsic membrane proteins. The
mitochondrial processing peptidase and the ADP/ATP carrier were used as
controls.
Preliminary experiments of in vitro import of NDE1 into
mitochondria were performed. In parallel experiments, the precursor of
subunit F1 We have further characterized an NADPH dehydrogenase of the inner
mitochondrial membrane, which, like the proton-pumping complex I, is
constitutively expressed throughout the Neurospora life cycle. The isolation of double mutants lacking these enzymes clearly indicated that the fungus has additional alternative ways of oxidizing NAD(P)H. The combination of data from several strategies indicated that
the NDE1 polypeptide is an intrinsic protein of the inner mitochondrial
membrane with its catalytic site facing the intermembrane space. In
N. crassa, the protein is probably anchored to the membrane by an N-terminal domain (amino acid residues 73-94), the segment displaying the highest probability of being transmembrane, based on
computer prediction analysis. A second predicted transmembrane domain
(residues 173-196) is unlikely, because it would place the two
dinucleotide binding sites (presumably for FAD and NAD(P)H) on opposite
sides of the membrane. It is not clear whether a third predicted
transmembrane domain at the C-terminal end is present (residues
620-639).
Inactivation of NDE1 was a crucial step in elucidating its function.
Comparison of rotenone-insensitive NADH and NADPH oxidation by IO-SMP
from the nde-1 mutant and the wild-type strain revealed a
similar behavior, and NADH oxidation by intact mitochondria from the
two strains was identical. In contrast, NADPH oxidation at
physiological pH was lacking in intact mitochondria from the nde-1 mutant. Thus, we conclude that NDE1 is the external
NADPH dehydrogenase of N. crassa mitochondria. The protein
contains two GXGXXG motifs within The NDE1 protein is homologous to the type II NAD(P)H dehydrogenases of
S. cerevisiae (6-8), but, in contrast to them, it contains
an additional domain predicted to bind calcium (11). This E-F hand
motif is also present in the external NDB protein from
Solanum tuberosum mitochondria (13) and in a putative
NADH:ubiquinone reductase from Arabidopsis thaliana
(AC006234.3). We found that calcium had a similar effect on NADH
oxidation by mitochondria from the nde-1 mutant and wild
type, whereas there were significant differences regarding its effect
on NADPH oxidation in the two strains. Wild-type mitochondria displayed
strong calcium sensitivity, and the presence of the cation was
absolutely required for activity at physiological pH. Because oxidation
of NADPH by nde-1 mitochondria was not affected by calcium,
we demonstrated experimentally that the NADPH oxidation activity of the
NDE1 protein is calcium-dependent. Previous work has
documented a calcium stimulation of NAD(P)H oxidase activities in
Neurospora (15) and plant (53) mitochondria, although the
mechanism by which calcium stimulates activity is not yet clear. It was
suggested that this divalent cation has an important role in avoiding
electrostatic repulsion between the phosphate groups of NADPH and the
catalytic site of the enzyme localized in a net negatively charged
environment like the outer surface of the inner mitochondrial membrane
(54). It is also likely that calcium binding induces conformational
changes that increase the enzyme-substrate affinity.
Following external NADH and NADPH oxidation from acidic to alkaline
conditions (pH 4.7-9.2), two distinct activities were identified in
N. crassa mitochondria. One corresponds to NDE1, which
oxidizes NADPH and is calcium-dependent. The other, which remains active in mutant nde-1 mitochondria, can be
attributed to an NDE2 enzyme that oxidizes NADH throughout the pH range
and is also able to oxidize NADPH at acidic pH. The inability of the putative NDE2 to oxidize NADPH at pH higher than 7.2 may explain the
need for a separate enzyme, NDE1, to regenerate the cytosolic NAD(P)+ pool required in several biosynthetic pathways.
N. crassa also contains at least one internal
rotenone-insensitive NADH dehydrogenase (14). The presence of these
nonproton-pumping alternative NAD(P)H dehydrogenases varies between
different organisms. They might be involved in situations of NAD(P)H
stress, but their specific role is unclear. In addition to metabolic
functions (7, 8, 10), a regulatory role in response to the redox state
of the plastoquinone pool has been suggested in cyanobacteria (9).
This is the first time that the gene for a mitochondrial NADPH
dehydrogenase has been identified and that evidence for at least two
external NAD(P)H dehydrogenases in N. crassa mitochondria has been obtained. The cloning and disruption of the additional rotenone-insensitive NAD(P)H dehydrogenases will help to clarify their
function(s) in mitochondrial and cellular metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of nde-1
mutants. Total mitochondrial proteins (100 µg) from wild
type (lane 1), the nde-1 mutant (lane
2), and the double mutant nde-1/nuo51
(lane 3) were analyzed by Western blotting with an antiserum
against the NDE1 protein and a mixture of antisera against the 51-, 30.4-, 20.8-, and 12.3-kDa subunits of complex I.
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Fig. 2.
Exogenous NAD(P)H oxidation by wild type and
nde-1 mitochondria. Latencies of wild type
(a and c) and nde-1 mitochondria
(b and d) were 86 and 89% for
cytochrome-c oxidase (outer membrane integrity) and 92 and
93% for malate dehydrogenase (inner membrane integrity), respectively.
Open circles, NADH; closed circles, NADH + EGTA;
open squares, NADPH; closed squares, NADPH + EGTA. The results are an average of data collected from at least three
independent preparations of mitochondria.
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Fig. 3.
Visualization of proteins after digitonin
solubilization of mitochondria. In the left panel, 100 µg of mitochondria were solubilized with different digitonin
(DIG) concentrations, treated with proteinase K (except the
first lane), and analyzed by Western blotting with antisera
against NDE1, the mitochondrial processing peptidase (MPP),
cytochrome c heme-lyase (CCHL), the ADP/ATP
carrier (AAC), and the outer membrane protein TOM20. In the
right panel, mitochondria solubilized with 0.3% digitonin
and treated with proteinase K were incubated with
Na2CO3 and separated by centrifugation into
pellet (P) and supernatant (S) before the Western
blotting analysis.
of ATPase (50) was used as control. The precursor of NDE1
was synthesized in vitro and imported into
Neurospora mitochondria with and without a membrane
potential. Import and processing to the mature form was only achieved
in the former case (data not shown), a typical result for an inner
membrane protein (51). Because it is not possible to swell
Neurospora mitochondria, the NDE1 precursor was also
successfully imported into and processed in yeast mitochondria, as
deduced by the resistance of the mature form of the protein to added
trypsin, again only in the presence of a membrane potential (Fig.
4). In addition, and in contrast to
F1
, mature NDE1 was made accessible to the protease in the
mitoplasts generated by the swelling of mitochondria, in agreement with
the results obtained in the digitonin experiments.
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Fig. 4.
Import of Neurospora
precursor polypeptides by yeast mitochondria. NDE1 and F1
precursors were imported into yeast mitochondria in the presence of a
membrane potential, generated by succinate or NADH, respectively. The
precursor of NDE1 was also imported without a membrane potential in the
presence of valinomycin (Val). After import, mitochondria
were incubated with trypsin (TRP) in swelling
(SW) and nonswelling conditions. The samples were
electrophoresed, blotted onto a membrane, and exposed to x-ray film.
p, precursor; m, mature.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sheet-
helix-
sheet structures for binding NAD(P)H and FAD (11). We are
tempted to speculate that the first segment of NDE1 binds NADPH,
because the third Gly in NADPH-binding proteins is generally
replaced by Ser, Ala, or Pro, and a conserved negative charge at the
end of the second
sheet is missing (52) and might be substituted by
an Asn, avoiding the unfavorable interaction between the negatively
charged residue and the negatively charged 2'-phosphate of NADPH (53).
These features are present in the first, and absent from the second, dinucleotide-binding motif of NDE1 (11).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Corália Vicente for the statistical analysis and to Drs. Walter Neupert, Rosemary Anne Stuart, and Michael Brunner for helpful discussions and support.
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FOOTNOTES |
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* This research was supported by Fundação para a Ciência e a Tecnologia from Portugal through research grants (to A. V.) and a fellowship (to A. M. P. M.), by grants from the Swedish Natural Science Research Council (to I. M. M.), and by National Science Foundation Grant MCB-9874488 (to M. A. N.).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.
To whom correspondence should be addressed. Tel.:
351-226074900; Fax: 351-226099157; E-mail: asvideir@icbas.up.pt.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M008199200
2 M. A. Nelson, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: IO-SMP, inside-out submitochondrial particles; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.
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