The C-terminally Truncated NuoL Subunit (ND5 Homologue) of the Na+-dependent Complex I from Escherichia coli Transports Na+*
Julia Steuber
From the
Mikrobiologisches Institut der Eidgenössischen Technischen
Hochschule, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zürich,
Switzerland
Received for publication, February 18, 2003
, and in revised form, May 5, 2003.
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ABSTRACT
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The NADH:quinone oxidoreductase (complex I) from Escherichia coli
acts as a primary Na+ pump. Expression of a C-terminally truncated
version of the hydrophobic NuoL subunit (ND5 homologue) from E. coli
complex I resulted in Na+-dependent growth inhibition of the E.
coli host cells. Membrane vesicles containing the truncated NuoL subunit
(NuoLN) exhibited 24-fold higher Na+ uptake
activity than control vesicles without NuoLN. Respiratory proton
transport into inverted vesicles containing NuoLN decreased upon
addition of Na+, but was not affected by K+, indicating
a Na+-dependent increase of proton permeability of membranes in the
presence of NuoLN. The His-tagged NuoLN protein was
solubilized, enriched by affinity chromatography, and reconstituted into
proteoliposomes. Reconstituted His6-NuoLN facilitated
the uptake of Na+ into the proteoliposomes along a concentration
gradient. This Na+ uptake was prevented by EIPA
(5-(N-ethyl-N-isopropyl)-amiloride), which acts as inhibitor
against Na+/H+ antiporters.
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INTRODUCTION
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Mitochondrial complex I (NADH:quinone oxidoreductase) is the largest
multiprotein complex of the oxidative phosphorylation (OXPHOS) system.
Diminished complex I activity is associated with Parkinson's disease
(1) and aging
(2) and represents the most
frequently encountered inherited defect of the OXPHOS system
(3). Despite the considerable
knowledge on primary sequences, cofactors and assembly
(48),
the mechanism of redox-driven proton transport by complex I and the subunit(s)
that guide the proton through its membranous part are unknown. A promising
approach is to study bacterial counterparts of complex I that are smaller but
possess all subunits required for redox-driven H+ (or
Na+) transport (9).
In particular, a Na+-translocating complex I found in
enterobacteria like Escherichia coli
(10) or Klebsiella
pneumoniae (11,
12) is a useful model to trace
the pathway of the coupling cation, as exemplified by the
Na+-translocating F1F0 ATP synthase
(13).
The L-shaped complex I is composed of a peripheral arm extending into the
bacterial cytoplasm (or the mitochondrial matrix) and a hydrophobic arm that
is embedded in the membrane
(14). In addition to the
L-shaped conformation, a "horse-shoe-conformation" was observed
for E. coli complex I, with two arms arranged side by side
(15). Upon purification, the
peripheral and the membrane arm tend to dissociate
(1618).
Sequence comparisons indicate that the membrane-embedded part of complex I
consists of seven conserved hydrophobic subunits with homologues found on
bacterial or mitochondrial genomes and additional nuclear-encoded subunits in
the case of eukaryotic complex I. One or several of these seven conserved
subunits (NuoA, H, J, K, L, M, N, in the Na+-translocating complex
I from E. coli, or the homologues ND3, 1, 6, 4L, 5, 4, 2 in the
H+-translocating mitochondrial complex I) participate in
Na+ (or H+) transport through complex I. So far, the
cation-translocating subunit(s) of complex I have not been identified. A
likely candidate is the NuoL subunit (ND5 according to bovine nomenclature),
which is related to subunit A of bacterial multicomponent Na+ (or
K+):H+ antiporters
(1922).
Here evidence is presented that the C-terminally truncated NuoL subunit of
E. coli complex I transports Na+.
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EXPERIMENTAL PROCEDURES
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Strains and MediaE. coli K-12 strain DH5
(MBI
Fermentas) was used as host for clonings. Expression of the truncated
nuoL gene was performed using E. coli DH5
, E.
coli strain CP875 (23),
or E. coli strain EP432
(24). E. coli EP432
is resistant to kanamycin and lacks the Na+/H+
antiporters NhaA and NhaB. These antiporters are not related to subunits of
the multicomponent antiporters belonging to the CPA3 family
(22). Cells were grown
aerobically in Luria-Bertani medium without added NaCl in the presence of 10
mM glucose and 50 mM potassium phosphate (pH 7.6).
E. coli EP432 was also grown aerobically in mineral medium
(25) at pH 8.0 with glucose or
glucose plus L-arabinose as indicated. If necessary, 100 µg
ml1 ampicillin or 50 µg
ml1 kanamycin was added. Cells were harvested,
washed once in 10 mM Tris-HCl, pH 8.0, 200 mM KCl, 0.5
mM dithiothreitol and suspended in buffer. Inside-out membrane
vesicles were obtained by French press cell rupture
(26). Washed membrane vesicles
used for Na+ transport measurements were prepared in the anaerobic
chamber under an atmosphere of N2/H2 (95/5%)
(11) in 10 mM
Tris-HCl, pH 8.0, 200 mM KCl, 0.5 mM dithiothreitol,
0.25 M sucrose
(24).
Construction of PlasmidsAll plasmids are pISC1 derivatives,
which contain a ColE1 origin of replication and the araC gene
encoding the activator protein, which induces transcription in the presence of
arabinose (27). PISC vectors
confer ampicillin resistance. Plasmid pISC2 contains a unique NdeI
site downstream of the para promoter. Vector pISC3 is a
pISC2 derivative containing an XbaI/NdeI linker derived from
vector pET-14b (Novagen), which replaces the unique NdeI site of
pISC2 and introduces six histidine residues to the N terminus of the target
protein (28). A 1.1-kb DNA
fragment encoding for the truncated NuoL subunit (amino acids 1369;
NuoLN) of complex I from E. coli was obtained by PCR using
Pfu-DNA-polymerase (New England Biolabs). The primers used were
5'-TTCATATGAACATGCTTGCCTTAACC3-'
(forward) and
5'GCGAATTCTTAACGCAGACCGCCCATCTTG-3'
(reverse). Coding sequences of the nuoL gene are underlined.
NdeI and EcoRI sites (bold letters) and a stop codon
(italics) were introduced by PCR. The PCR product was digested with
NdeI and EcoRI and cloned into pISC2, resulting in vector
pECL3. An NdeI/EcoRI fragment containing the truncated
nuoL gene was obtained from pECL3 and cloned into pISC3, resulting in
vector pECL5. Vector pECL5 was subjected to double-strand sequencing
(Microsynth, Balgach, Switzerland). The sequence obtained was identical to the
nuoL sequence (accession number NP-416781) found on the E.
coli genome (29).
Solubilization and ReconstitutionWashed membrane vesicles
(40 mg protein, 1 ml) from E. coli DH5
transformed with pECL5
(NuoLN) or pISC3 (control) grown on Luria Bertani medium in the
presence of 10 mM glucose were mixed with 9 ml buffer (20
mM Tris-HCl, pH. 8.0, 300 mM KCl, 2 mM NaCl,
9% glycerol) containing 0.1 g dodecylmaltoside (Glykon) and stirred on ice (30
min). After ultracentrifugation, the supernatant containing the solubilized
membrane proteins was added to 2.5 ml nickel-nitrilotriacetic acid agarose
resin (Qiagen) equilibrated with buffer. The slurry was gently shaken
overnight at 4 °C. The resin was centrifuged and washed with 50 ml buffer
containing 0.05% dodecylmaltoside. The resin was packed in a column, and bound
NuoLN was eluted with 5 ml buffer containing 0.05% dodecylmaltoside
and 50 mM imidazole. The enriched NuoLN protein (10 mg)
was reconstituted into liposomes by a dilution method established for complex
I from K. pneumoniae
(11) using E. coli
lipids (150 mg, Avanti). The lipids were mixed with NuoLN (4.5 ml
eluate from the affinity column in 0.05% dodecylmaltoside), and the formation
of proteoliposomes was achieved by drop-wise addition of 90 ml buffer (10
mM Tris-HCl, pH 8.0, 300 mM KCl). The proteoliposomes
were collected by ultracentrifugation and resuspended in buffer to a final
concentration of 60120 mg ml1 lipid.
Na+ TransportNa+ transport
was followed under air at room temperature as described previously
(11). Incubation mixtures
contained in 0.31 ml the reconstituted proteoliposomes (4.5 mg lipid) and 20
µM valinomycin (Sigma) in 10 mM Tris-HCl, pH 8.0, 300
mM KCl. If indicated, 0.1 mM
EIPA1
(5-(N-ethyl-N-isopropyl)-amiloride (Sigma)) was added to the
assay. The reaction was started by the addition of 5 mM NaCl (final
concentration). At different times, samples of 70 µl (1.1 mg lipid) were
applied to a 1-ml plastic syringe containing 0.6 ml Dowex 50 (K+).
The proteoliposomes were immediately eluted with 0.8 ml deionized water. The
eluate containing Na+ entrapped in the vesicles was analyzed by
atomic absorption spectroscopy. In controls performed with 5 mM
NaCl in buffer in the absence of vesicles, Na+ was completely
absorbed to the Dowex columns. Blank values obtained with vesicles in buffer
without NaCl added corresponded to the internal Na+ content of the
liposomes (0.52.4 nmol Na+ per sample after passage through
the Dowex column). Na+ transport into native membrane vesicles was
followed in 0.3 ml 10 mM Tris-Mes, pH 8.0, 200 mM KCl,
2.5 mM MgCl2
(24). The incubation mixture
contained 2 mg of protein and 20 µM valinomycin. The internal
Na+ content of the vesicles was 4 nmol Na+
mg1 protein. The reaction was started by adding 5
mM NaCl, and at different times, aliquots of 70 µl were applied
to the Dowex columns as described above.
Other MethodsOxidation of NADH or deaminoNADH (nicotinamide
hypoxanthine dinucleotide) by membrane vesicles with O2 as electron
acceptor was followed at 340 nm (
340 = 6.22
mM1 cm1).
NADH-induced proton uptake into native membrane vesicles was measured by the
quenching of ACMA (9-amino-6-chloro-2-methoxyacridin) in 1.5 ml of 10
mM Tris-HCl, pH 8.0, 200 mM KCl at 25 °C
(30). The residual
Na+ concentration in the buffer was 0.13 mM.
DeaminoNADH, NADH (potassium salt), and ACMA were obtained from Sigma. Protein
was determined by the bicinchoninic acid method using the reagent obtained
from Pierce.
SDS-PAGE was performed with 10% polyacrylamide according to
(31). The polypeptides were
blotted onto a nitrocellulose membrane (Hybond-C Extra, Amersham Biosciences),
and His-tagged proteins were identified by immunostaining using anti-His
(4) antibodies (Qiagen).
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RESULTS
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The C-terminally Truncated NuoL Subunit Increases the
Na+ Permeability of E. coli MembranesComplex I
(NDH I) from E. coli couples the oxidation of NADH with quinone to
the translocation of Na+
(10). An important goal is to
identify the membranous complex I subunit(s) that participate in cation
transport. A prime candidate is the hydrophobic NuoL subunit of complex I from
E. coli (ND5 in Bos taurus, or Nqo12 in Paracoccus
denitrificans) that exhibits striking sequence similarity to
multicomponent Na+/H+ antiporters. A sequence alignment
of the NuoL subunit from E. coli complex I with the MnhA subunit of
the Na+/H+ antiporter from Staphylococcus
aureus reveals 133 conserved amino acids in NuoL encompassing a total of
613 amino acid residues. Fig. 1
shows the putative topology of the NuoL subunit of E. coli complex I
that is based on phylogenetic analyses and experimental data obtained for NuoL
of Rhodobacter capsulatus complex I
(32). The central region of
the NuoL subunit encompassing transmembrane helices III to XIII exhibits the
highest sequence similarity to Na+/H+ antiporters. The
region from amino acid 1 to 369 encompasses 107 residues of the 133 amino
acids that are fully conserved in the complete E. coli NuoL subunit
and in the MnhA antiporter subunit from S. aureus. This portion of
the NuoL subunit contains nine putative transmembrane helices (I-IX) and the
amphipathic helices (X) and (XI) that are not assumed to traverse the membrane
(32). Truncation of NuoL at
R369 (Fig. 1) results in a
shortened NuoL subunit that includes the conserved parts of the cytoplasmic
loop between helices (XI) and XII. The gene fragment encoding for amino acid
residues 1369 was amplified by PCR and cloned into a low-copy
expression vector that is repressed by glucose and derepressed by
L-arabinose, resulting in vector pECL3. The truncated NuoL subunit
(NuoLN) has a calculated molecular mass of 40,054 Da, compared with
66,440 Da of the NuoL subunit. In addition, a vector encoding for
NuoLN encompassing an N-terminal Histag
(His6-NuoLN) was constructed (pECL5). Detection of
His6-NuoLN by immunostaining revealed that the protein
was predominantly found in membranes of E. coli
(Fig. 2). No signal was
detected in cell-free extracts from E. coli in the absence of plasmid
(not shown). Due to its hydrophobic nature, the truncated NuoL subunit showed
an apparent molecular mass of only 33 kDa on SDS-PAGE.

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FIG. 1. Putative topology of the NuoL subunit (ND5 homologue) of complex I from
E. coli and relationship with subunit A of multicomponent
Na+/H+ antiporters. The topology of the E.
coli NuoL subunit is based on data obtained for NuoL from R.
capsulatus complex I
(32). A total of sixteen
hydrophobic helices are predicted for NuoL, and its N and C termini are
located in the periplasm. The beginning of a putative helix is indicated by
its first N-terminal amino acid residue (E. coli numbering).
Shaded helices contain four or more residues that are conserved in
NuoL from E. coli complex I and in the MnhA antiporter subunit from
S. aureus. The amphipathic helices (X) and (XI) probably do not
traverse the membrane (32).
Truncation of NuoL after arginine 369 results in the shortened
NuoLN polypeptide that includes helices I-(XI) and the conserved
parts of the cytoplasmic loop between helices (XI) and XII. Circles
indicate the positions of mutations in helices XII and XV that result in
diminished function of complex I or the related Na+/H+
antiporter (19,
44).
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FIG. 2. Localization of the C-terminally truncated NuoL subunit
(His6-NuoLN) in E. coli membranes.
His6-NuoLN was detected in cell fractions of E.
coli DH5 after separation by SDS-PAGE and immunostaining using
anti-His antibodies. Lane 1, soluble fraction (15 µg); lane
2, membrane fraction (15 µg); lane 3, solubilized membranes
(7 µg); lane 4, eluate from the affinity column (7 µg).
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The effect of plasmid pECL5 encoding His6-NuoLN on
the growth of E. coli DH5
or E. coli EP432 was
investigated. Cells were grown aerobically at 37 °C in phosphate-buffered
Luria Bertani medium (pH 7.6) containing 10 mM glucose and 20, 110,
or 140 mM KCl or NaCl. The optical density (OD) was followed at 600
nm for 24 h, and synthesis of the truncated NuoL subunit was induced by
addition of 8 mM arabinose at A0.5. A general
trend observed with both E. coli strains was a decrease in final
growth yield in pECL5 cells compared with the control cells at increasing salt
concentrations (Table I). Both
KCl and NaCl diminished the growth yield in the presence of
His6-NuoLN, but NaCl showed a more drastic effect than
KCl. In E. coli DH5
, growth in the presence of
His6-NuoLN was impaired at 140 mM NaCl (final
A1.9, compared with A8.8 with the
control vector). This inhibition of growth was not accompanied by an apparent
change in morphology. Growth of E. coli EP432 transformed with pECL5
encoding for His6-NuoLN was already inhibited at 110
mM NaCl (final A1.0, compared with
A2.8 observed with the control vector). The increased
toxicity of Na+ even in the absence of NuoLN observed
with E. coli EP432 compared with E. coli DH5
is due
to the lack of two Na+/H+ antiporters in strain EP432.
In contrast to E. coli DH5
, Na+-induced growth
inhibition of E. coli EP432 in the presence of
His6-NuoLN was accompanied with an elongation and
aggregation of cells. These morphological changes were not observed with
E. coli EP432 growing in 140 mM KCl in the presence of
NuoLN. A similar association of growth inhibition with cell
elongation was observed with E. coli strain DK8 cells overproducing
the truncated, membrane-bound subunit a of the proton-translocating F1F0
ATPase (33).
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TABLE I Expression of the C-terminally truncated NuoL subunit of complex I
decreases the salt tolerance of E. coli
E. coli DH5 or E. coli EP432 cells transformed
with pECL5 encoding for His6-NuoLN or with the control
vector (pISC3) were grown in phosphate-buffered Luria Bertani medium at pH 7.6
in the presence of 10 mM glucose. Growth was monitored as optical density at
600 nm. Two hours after inoculation (optical density at 600 nm, 0.5), 8 mM
arabinose was added. After 24 h, the final optical densities were recorded.
E. coli EP432 cells transformed with pECL5 and grown in the presence
of 110 mM NaCl were elongated and formed aggregates (*).
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It is concluded that the NuoLN protein promotes ion leakage
through the E. coli membranes, hereby dissipating electrochemical
gradients across the cell membrane and diminishing the growth yield. The data
indicate an increased permeability of the membranes for Na+ induced
by the NuoLN protein. This was further investigated by following
the Na+ influx into membrane vesicles from E. coli EP432
transformed with either pECL5 or pISC3. In the presence of NuoLN,
the initial rate of Na+ uptake was 18 nmol
mg1 protein during the first 10 s, compared with
9 nmol Na+ mg1 observed with the
control membrane vesicles (Fig.
3). The addition of ubiquinone-1 (0.49 mM, oxidized
form) to the assay did not stimulate the rate of Na+ transport by
membrane vesicles containing the truncated NuoLN subunit. In six
independent experiments, the uptake of Na+ was 24-fold
higher in membrane vesicles containing NuoLN compared with the
control (50240 nmol Na+ min1
mg1, corrected for initial Na+ uptake
by control vesicles), demonstrating that NuoLN enhances
Na+ transport through native membranes with initial rates that are
comparable with secondary Na+ transporters. For example, the
purified, reconstituted Na+/H+ antiporter NhaA from
E. coli has a specific activity of 600 nmol Na+
min1 mg1
(34).

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FIG. 3. The C-terminally truncated NuoL subunit of complex I increases the
Na+ permeability of membrane vesicles from E. coli.
Membrane vesicles were prepared from the Na+/H+
antiporter-deficient E. coli strain EP432 transformed with plasmid
pECL5 (His6-NuoLN) or pISC3 (control). Cells were grown
on mineral medium in the presence of 45 mM glucose and 5 mM
L-arabinose at pH 8.0. Na+ transport was followed in the
presence of 20 µM valinomycin and 140 mM KCl. The
reaction was started by adding 5 mM NaCl. Open circles,
membrane vesicles containing His6-NuoLN; closed
circles, membrane vesicles prepared from E. coli EP432
transformed with the control vector. The graph shows representative
data from a total of six experiments.
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An important question is whether the presence of the truncated
NuoLN subunit in the cytoplasmic membrane of E. coli
affects the assembly of complex I. The observed Na+ transport might
be catalyzed by misassembled complex I containing the truncated NuoL subunit.
The specific complex I activity of membrane vesicles was estimated from the
oxidation of deaminoNADH with O2 as terminal electron acceptor. In
contrast to NADH, which is oxidized both by complex I and the alternative,
non-electrogenic NADH dehydrogenase (NDH II), deamino-NADH is preferentially
oxidized by complex I and is used to distinguish between complex I and NDH II
in E. coli membranes
(35). The rates of deaminoNADH
oxidation by control membrane vesicles from E. coli EP432 (0.06
µmol min1 mg1)
were comparable with the rates observed with membrane vesicles containing the
truncated NuoL subunit (0.08 µmol min1
mg1). The rates of NADH oxidation by control
membrane vesicles (0.36 µmol min1
mg1) were essentially identical to the rates
observed with membrane vesicles containing NuoLN (0.38 µmol
min1 mg1). The
NuoLN protein did not diminish the activity of complex I in E.
coli membranes, suggesting functional assembly of complex I despite the
presence of its hydrophobic, truncated NuoL subunit.
Na+-dependent Increase of Proton Permeability in
Membranes Containing the C-terminally Truncated NuoL
SubunitRespiratory proton transport activity was studied in
membrane vesicles from E. coli CP875 containing the
His6-NuoLN protein that were energized by the addition
of NADH as outlined above. E. coli contains two respiratory
NADH:quinone oxidoreductases, the Na+-translocating NDH I or
complex I (10), and the
non-electrogenic NDH II. During aerobic growth, NADH is mainly oxidized by the
NDH II (36), and the quinol
formed is oxidized by terminal oxidases under formation of an electrochemical
proton gradient. E. coli membrane vesicles obtained by French press
cell rupture are predominantly oriented inside-out
(26), and the addition of NADH
in the presence of oxygen results in the build-up of a proton motive force
(inside positive). The NADH-driven proton transport into vesicles is followed
by the quenching of ACMA fluorescence. Vesicles containing
His6-NuoLN showed a strong, NADH-induced quench in the
presence of KCl (20 mM), whereas the addition of NaCl (20
mM) prior to NADH diminished the quench signal by 30%
(Fig. 4, upper panel).
In contrast, the quenching of ACMA fluorescence was hardly affected by NaCl in
membrane vesicles prepared from cells transformed with the control vector
(Fig. 4, lower panel).
The slight increase in proton permeability of the control membranes in the
presence of Na+ was due to the activity of endogenous
Na+/H+ antiporters from E. coli
(24). Similar results were
obtained with E. coli DH5
transformed with pECL3 encoding for
the NuoLN protein (not shown). Note that the rates of NADH
oxidation by membrane vesicles with O2 as terminal electron
acceptor were not affected by NuoLN (0.34 µmol
min1 mg1 in vesicles
containing NuoLN, compared with 0.36 µmol
min1 mg1 in control
vesicles). The diminished proton gradient established during NADH oxidation by
vesicles in the presence of NuoLN and Na+ ions provides
a rationale for the Na+-dependent decrease in growth yield of
E. coli cells producing the truncated NuoL subunit.

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FIG. 4. Increased proton permeability of membrane vesicles containing
NuoLN in the presence of Na+. Membrane vesicles were
prepared from E. coli strain CP875 transformed with plasmid pECL5
(His6-NuoLN) or pISC3 (control). Proton transport into
the vesicles (0.25 mg protein) was estimated by NADH-driven quenching of ACMA
fluorescence. After 60 s, H+ transport was initiated by adding 100
µM NADH in the presence of 20 mM KCl (solid
line) or 20 mM NaCl (dotted line). Upper
panel, membrane vesicles containing His6-NuoLN;
lower panel, control vesicles. The residual Na+
concentration in the assay buffer was 0.13 mM.
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Na+ Transport by Reconstituted
NuoLN and Inhibition by EIPAThe
truncated, His-tagged NuoL subunit from E. coli complex I was
solubilized, enriched by affinity chromatography
(Fig. 2), and reconstituted
into proteoliposomes. Addition of proteoliposomes containing
His6-NuoLN to buffer in the presence of 5 mM
NaCl resulted in a transient accumulation of Na+ inside the
liposomes that was
6-fold higher compared with the control liposomes
(Fig. 5). Internal
Na+ concentrations were calculated assuming a liposome volume of
310 µl per mg lipid
(37). At the start of the
reaction, the Na+ concentration in the lumen of the liposomes was
100200 µM, corresponding to an initial chemical
Na+ concentration gradient (
pNa+) of 80100
mV (inside negative). The experiments were performed without respiratory
substrates added, and the build-up of a transmembrane voltage, 
,
was prevented by valinomycin/K+. The uptake of Na+ by reconstituted
His6-NuoLN was driven by the chemical Na+
concentration gradient (
pNa+). After 30 s, the maximum
Na+ concentration in the interior was 620 mM.
This is in the range of the external Na+ concentration in the
buffer and thus in accord with passive downhill transport of Na+
into the proteoliposomes mediated by His6-NuoLN. The
initial uptake during the first 30 s was followed by an efflux of
Na+ to the values observed with the control proteoliposomes
(Fig. 5). Similar results were
obtained with different preparations of NuoLN and different
concentrations of proteoliposomes in the Na+ transport assay (not
shown).
The inhibition of the truncated NuoLN subunit by EIPA was
investigated. EIPA is an amiloride analogue that inhibits eukaryotic
Na+/H+ antiporters but not NhaA, the main
Na+/H+ antiporter in E. coli
(38). Na+ uptake by
His6-NuoLN was drastically diminished in the presence of
100 µM EIPA, whereas the residual uptake of Na+ by
the control proteoliposomes was not affected by EIPA
(Fig. 5). This result suggests
that the truncated NuoL subunit of complex I from E. coli shares
features with Na+/H+ antiporters.
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DISCUSSION
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Complex I is the largest redox-driven H+ pump in the respiratory
chain. It is composed of a peripheral arm harboring one FMN and up to nine
Fe/S clusters (5), and a
membrane arm containing up to three ubiquinones
(11,
39,
40). Although the peripheral,
NADH-oxidizing fragment of complex I has been studied intensively, the
membrane part is a terra incognita that awaits its functional
characterization. The dissection of the membranous fragment of complex I into
functional units will be a prerequisite to identify the subunits and amino
acid residues that contribute to H+ (or Na+) transport.
The Na+-dependent complex I found in E. coli
(10) and the related
enterobacterium K. pneumoniae
(11,
12) serves as a model for the
mitochondrial, H+-pumping enzyme. The high degree of similarity of
the NuoL subunit (ND5 homologue) from complex I with subunit A from
multicomponent cation/H+ antiporters has led to the speculation
that NuoL provides (part of) a proton channel in complex I
(19). Unfortunately, the
observed sequence similarity contributes very little to the understanding of
H+ (or Na+) transport by complex I, because the
multicomponent cation/H+ antiporters are only poorly characterized
with respect to function and catalytic activity.
Evidence for Na+ transport by the C-terminally truncated NuoL
subunit of complex I is presented here. The truncated NuoL subunit comprises
most of the regions conserved between NuoL and the A subunit of the
multicomponent antiporters belonging to the CPA3 family
(22). Because the truncated
NuoL subunit was produced in E. coli in the presence of endogenous
complex I, one cannot completely exclude that the observed Na+
transport was due to other complex I subunits copurified with
His6-NuoLN on the affinity column. On the other hand,
the solubilization of E. coli membranes with dodecylmaltoside under
the applied conditions of pH and salt concentration cleaves and inactivates
complex I (17). It should also
be noted that the specific complex I activity in native membranes was not
affected by the presence of the truncated NuoL subunit. It is therefore
considered very unlikely that the Na+ transport observed with
reconstituted His6-NuoLN is catalyzed by misassembled
complex I. The translocation of Na+ by the C-terminally truncated
NuoL subunit of complex I from E. coli provides strong evidence for
the participation of this subunit in cation transport through the membranous
fragment of complex I.
The Na+-translocating complex I from K. pneumoniae that
exhibits high sequence similarity to E. coli complex I exclusively
pumps Na+ at a ratio of 2 Na+/2 electrons
(11). Na+ transport
by native complex I generates a transmembrane voltage that is not compensated
by the counter-flow of protons
(12). Therefore, one would
expect unidirectional transport of Na+ by NuoLN. On the
other hand, the results presented herein suggest a Na+-dependent
increase of proton permeability of membrane vesicles in the presence of
NuoLN, which can be interpreted in terms of
Na+/H+ antiport. Na+ uptake by reconstituted
His6-NuoLN was followed by the extrusion of
Na+, similar to results obtained with the reconstituted NhaA
antiporter from E. coli
(34). Na+ transport
by His-NuoLN was drastically diminished in the presence of EIPA, an
amiloride derivative that acts as inhibitor against
Na+/H+ antiporters. Note that EIPA also inhibits NADH
oxidation by bovine complex I
(41). It should be considered
that the presence of NuoLN in E. coli host cells could
result in an increase of the intracellular Na+ concentration, which
in turn leads to the up-regulation of endogenous Na+/H+
antiporters (42). As a
consequence, the Na+/H+ antiporter activity in membrane
vesicles or enriched protein fractions could be higher in E. coli
cells producing NuoLN compared with the control cells. Studies
aimed at the determination of Na+ (or H+) transport
stoichiometries using purified, reconstituted NuoLN are currently
underway in this laboratory.
Biochemical (18) and
structural (43) studies with
bovine complex I suggest that the large ND5 (NuoL) subunit might be situated
at the distal end of the membrane-embedded arm of the L-shaped molecule
together with the ND4 (NuoM) subunit. In complex I from E. coli, the
N-terminal part of the NuoL subunit (helices I-XI) may be part of the
Na+-translocating machinery, but electrogenic Na+
transport by the holo-complex probably requires the C-terminal NuoL domain
(Fig. 6). Studies on
site-directed mutants support a functional role of the C-terminal part of NuoL
(helices XII-XVI) (Fig. 1). In
human complex I, a substitution of glycine 465 (Gly-468 in E. coli
complex I) in helix XIV with glutamate represents a primary mutation that
causes Leber's hereditary optic neuropathy
(44). In the related
Na+/H+ antiporter from Bacillus C-125, mutation
of glycine 382 in helix XII to arginine results in an alkali-sensitive
phenotype, indicating diminished activity of the Na+/H+
antiporter that confers alkali resistance in the wild type
(19).

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|
FIG. 6. Proposed coupling of electron and Na+ transport in complex
I. Only five of thirteen Nuo subunits (A-N) of the E. coli
complex I are shown. The bovine nomenclature is indicated in parentheses.
B, NuoB (PSST); F, NuoF (51 kDa); CD, NuoCD (30 kDa and
49 kDa; these subunits are fused in E. coli complex I); H,
NuoH (ND1); LN, helices I-XI of NuoL (ND5);
LC, helices XII-XVI of NuoL (ND5). Electron
transport starts with the oxidation of NADH by FMN located on the NuoF subunit
in the peripheral arm located in the cytoplasm (or in the mitochondrial
matrix). Electrons are transferred via several FeS clusters to the
high-potential [4Fe-4S] cluster N2 that is located on NuoB (PSST)
(5) or on NuoI (TYKY)
(47). The reduced cluster N2
is oxidized by quinone (Q) bound in close vicinity of the B
and CD subunits and the membranous H subunit
(45) under formation of
quinol. This exergonic reaction provides the driving force for the endergonic
transport of Na+ by the N-terminal part of NuoL
(LN) submitted via the C-terminal NuoL domain
(LC) (shaded arrow). Per NADH oxidized,
two electrons are transferred to quinone, and two sodium ions are transported
from the negatively to the positively charged aspect of the membrane
(11).
|
|
A central question is how the endergonic transport of Na+ (or
H+) by complex I is driven by the exergonic oxidation of NADH with quinone
(Fig. 6). Two quinones (Q6 and
Q8) were found in complex I from K. pneumoniae
(11). A quinone-binding site
was identified in the vicinity of the NuoH (ND1), NuoB (PSST), and NuoD (49
kDa) subunits (45) (bovine
nomenclature in brackets). Based on comparisons of primary sequences and known
high-resolution structures of Q-binding proteins, the binding of Q to the NuoL
(ND5) subunit was postulated from a putative Q binding motif located in helix
(XI) and the loop between helices XII and XIII
(Fig. 1)
(46). Recently, Yagi and
co-workers (41) reported on
the labeling of the ND5 subunit (NuoL homologue) of bovine complex I with a
photoaffinity analogue of fenpyroximate, a specific inhibitor of complex I.
Fenpyroximate is assumed to bind at or close to a quinone binding site. The
labeling of complex I by the photoaffinity analogue of fenpyroximate was
diminished by various complex I inhibitors including amiloride derivatives
like EIPA. This result compares favorably with the observed inhibition of
Na+ transport by the truncated NuoL subunit (ND5 homologue) of
E. coli complex I in the presence of EIPA. Passive transport of
Na+ by the truncated NuoL subunit was not stimulated by the
addition of water-soluble ubiquinone-1 (oxidized form). It will now be
important to analyze whether there is an endogenous quinone bound to the NuoL
subunit of E. coli complex I, and whether it participates in
redox-driven Na+ transport.
 |
FOOTNOTES
|
---|
* This work was supported by a Marie Heim-Vögtlin fellowship from the
Swiss National Science Foundation (to J. S.). The costs of publication of this
article were defrayed in part by the payment of page charges. This 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.: 41-1-6323830; Fax:
41-1-6321148; E-mail:
fritz-steuber{at}micro.biol.ethz.ch.
1 The abbreviations used are: EIPA,
5-(N-ethyl-N-isopropyl)-amiloride; ACMA,
9-amino-6-chloro-2-methoxyacridin. 
 |
ACKNOWLEDGMENTS
|
---|
E. coli strains were kindly provided by Etana Padan, Hebrew
University of Jerusalem (E. coli EP432) and Alan Wolfe, Loyola
University Chicago (E. coli CP875). I thank Peter Dimroth, Swiss
Federal Institute of Technology, Zürich, for his support.
 |
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