The Plant Journal (1999) 20(1), 79±87
Homologues of yeast and bacterial rotenone-insensitive
NADH dehydrogenases in higher eukaryotes: two enzymes
are present in potato mitochondria
Allan G. Rasmusson1,*, AÊ. Staffan Svensson1,
Volker Knoop2, Lutz Grohmann2,² and Axel Brennicke2
1
Department of Plant Physiology, Lund University,
Box 117, S-221 00, Lund, Sweden, and
2
UniversitaÈt Ulm, Allgemeine Botanik,
Albert-Einstein-Allee, D-89069 Ulm, Germany
Summary
Two different cDNAs, homologous to genes for
rotenone-insensitive NADH dehydrogenases of bacteria
and yeast, were isolated from potato. The encoded
proteins, called NDA and NDB, have calculated molecular
masses of 55 and 65 kDa, respectively. The N-terminal
parts show similarity to mitochondrial targeting
peptides and the polypeptides are in vitro imported into
potato mitochondria. Import processing to a smaller
polypeptide is seen for the NDA but not the NDB
protein. After import, NDA is intramitochondrially sorted
to the matrix side of the inner membrane, whereas NDB
becomes exposed to the intermembrane space. Imported
proteins are associated to membranes upon digitonin
permeabilization. On expression in Escherichia coli, NDB
is released from the bacterial membrane in the absence
of divalent cations whereas detergents are necessary for
solubilization of NDA. Both deduced amino-acid
sequences contain the dual motifs for nucleotide binding
with the characteristics of the core criteria, similar to the
bacterial homologues. Unique among NADH dehydro-
genases, the NDB amino-acid sequence contains a non-
conserved insert, which is similar to EF-hand motifs for
calcium binding. Phylogenetic analyses group the
rotenone-insensitive NADH dehydrogenases largely by
species, but suggest ancient gene duplications.
Introduction
Two major types of NADH dehydrogenases (NADH :
ubiquinone oxidoreductases, EC 1.6.5.3) are widely dis-
tributed in the living world. The proton-pumping complex I
of mitochondria and bacteria is a huge multi-subunit
Received 30 June 1999; accepted 10 August 1998.
*For correspondence (fax +46 46 2224113;
e-mail allan.rasmusson@fysbot.lu.se).
²
Present address: BioInside GmbH, Warthestr. 21, D-14513 Teltow,
Germany.
ã 1999 Blackwell Science Ltd
protein of 550±900 kDa carrying as prosthetic groups
FMN, FeS centres and ubiquinone (Friedrich et al., 1995).
The second type, the non-proton-pumping NADH dehy-
drogenases, is characterized as a single polypeptide of
generally around 50 kDa, and FAD as sole identi®ed
prosthetic group (Yagi, 1991). Enzymes of this type have
so far only been characterized in unicellular organisms.
The NDH protein of Escherichia coli (Young et al., 1981) is
similar to the NDI1 of Saccharomyces cerevisiae (de Vries
et al., 1992), whereas the NDH of an alkalophile Bacillus sp.
YN-1 is more closely related to sulphur-reducing ¯avoen-
zymes such as thioredoxin reductases (Xu et al., 1991).
The NDI1 of S. cerevisiae is located on the inner surface
of the inner mitochondrial membrane. This yeast lacks
complex I (Friedrich et al., 1995), and disruption of the ndi1
gene abolishes the mitochondrial oxidation of citric acid
cycle substrates, indicating that NDI1 is the only internal
NADH dehydrogenase (Marres et al., 1991). Mitochondria
of S. cerevisiae also oxidize external, cytoplasmic NADH
(de Vries and Marres, 1987). Two ORFs, YDL085w and
YMR145c, were recently shown to encode proteins asso-
ciated with this activity (Luttik et al., 1998; Small and
McAlister-Henn, 1998).
Complex I is present in all higher eukaryotes, but
internal and external mitochondrial NADH dehydrogenase
activities, insensitive to complex I inhibitors such as
rotenone, are found only in plants and fungi, not in
animals (Friedrich et al., 1995; Mùller, 1997; Rasmusson
et al., 1998b; de Vries and Marres, 1987). Plant mitochon-
dria additionally oxidize NADPH via external and internal
rotenone-insensitive enzymes, both biochemically distinct
from the NADH dehydrogenases (Melo et al., 1996; Roberts
et al., 1995). The external enzymes and the internal NADPH
dehydrogenase but not the internal NADH dehydrogenase
activities require micromolar concentrations of calcium
(Mùller, 1997; Rasmusson and Mùller, 1991). In contrast,
neither calcium dependency nor oxidation of NADPH at
appreciable rates has been observed for the rotenone-
insensitive NADH dehydrogenases from bacteria or yeast
(Yagi, 1991). Many attempts to purify and characterize the
rotenone-insensitive NAD(P)H dehydrogenases of plant
mitochondria have been carried out without a consensus
having been reached on the identities of the respective
enzymes (Mùller, 1997; Mùller and Rasmusson, 1998).
In this study we characterized the ®rst two cDNAs from
multicellular eukaryotes coding for enzymes homologous
to the rotenone-insensitive NADH dehydrogenases of E.
79
80 Allan G. Rasmusson et al.

coli and S. cerevisiae. The results suggest that the NDA
and NDB proteins are internal and external rotenone-
insensitive NADH dehydrogenases of plant mitochondria,
respectively.
Results
Clone pBNDa24 has an ATG starting at position 5 of the
cDNA insert, whereas pBNDb36 has no putative translation
start. In order to clone potentially lacking 5¢ parts of the
mRNA sequences and thus determine translational start
positions, rapid ampli®cation of 5¢ cDNA ends (5¢ RACE)
was carried out from poly(A)+ RNA. The sequences of the

Cloning of potato ndi1 homologous genes

An Arabidopsis thaliana-expressed sequence tag
(Newman et al., 1994), with deduced amino-acid sequence
similarity to the NDI1 of S. cerevisiae, was used as probe to
screen a potato (Solanum tuberosum L. cv. DesireÂe) cDNA
library. Two positive clones, pBNDa24 and pBNDb36
(Figure 1), showed distinct sequence similarities to NDI1,
and were assigned as NDA and NDB types, respectively.
Neither of the two clones was polyadenylated, but a Figure 1. Schematic maps of the isolated potato cDNA clones.
The inserts (grey boxes) and parts of the ¯anking vector sequences of
second isolated cDNA of the nda gene had at its 3¢ end a
the cDNA clones pBNDa24 and pBNDb36 are depicted with the locations
9 bp poly(A) segment immediately downstream of the 3¢ of primers for 5¢ RACE (a1±a3, b1±b3). To construct a full-length cDNA
end of the pBNDa24 cDNA, indicating that the mRNA is clone, the PstI/ScaI fragment of pBNDb36 was exchanged by a segment
derived from 5¢ RACE (white box). Orientation of the clones is indicated
polyadenylated in vivo. Both pBNDa24 and pBNDb36
by the position of T3 and T7 promoters of pBIISK. In-frame stop codons
contain motifs in their 3¢ trailer sequences obeying to 5 upstream of the ATG (arrows) as well as the translation stops are
of 6 positions of the AATAAA consensus for polyadenyla- indicated by asterisks. The positions of the motifs for ADP binding are
indicated by black boxes.
tion signals, as often seen in plants (Wu et al., 1995).

Figure 2. Conserved domain structures of bacterial, yeast and plant NADH dehydrogenase homologues.
The deduced amino-acid sequences of potato NDA and NDB (a), as well as homologous sequences recognized in the databases, were aligned. All known
eukaryotic (i.e. yeast) homologues, three representative bacterial known or putative NADH dehydrogenases, and two related bacterial enzymes are included
in alignment (b). In (a), positively charged residues in the N-terminal regions are marked (+). In (b), conserved and similar amino-acid residues are depicted
as black and grey rectangles, respectively, other residues as horizontal lines, and gaps are blank. Numbering in (b) corresponds to the NDA sequence.
Residues conserved among all the putative NADH dehydrogenase sequences in (b) are underlined in (a). The sequences aligned are (accession number): SC-
YDL, Saccharomyces cerevisiae YDL085w (S67621); SC-YMR, S. cerevisiae YMR145c (S50401); SC-NDI, S. cerevisiae NDI1 (S26704); SP-AL0,
Schizosaccharomyces pombe hypothetical `NADH-ubiquinone' (AL021837); SP-Z99, S. pombe hypothetical `ubiquinone reductase' (Z99260); BS-Z93, Bacillus
subtilis `NADH dehydrogenase' (Z93939); BS-AF0, B. subtilis NADH dehydrogenase-like protein (AF015825); EC-NDH, Escherichia coli NADH dehydrogenase
(D90746); AL-DLD, Acholeplasma laidlawii dihydrolipoamide dehydrogenase (D42653); BB-NOX, Borrelia burgdorferi NADH oxidase (AE001172).

ã Blackwell Science Ltd, The Plant Journal, (1999), 20, 79±87


Figure 3. Conserved and novel cofactor-binding motifs identi®ed in the
plant NADH dehydrogenases.
(a) ADP-binding motifs in the NDA and NDB sequences aligned to the
biochemically characterized NADH dehydrogenases of yeast and E. coli.
The consensus ADP-binding motif of Wierenga et al. (1986) is denoted at
the top: O, basic or hydrophilic residue; Z, hydrophobic residue.
Deviations from the consensus are underlined. Acidic residues potentially
de®ning the end of the second part of the motif are highlighted in grey.
(b) The unique insert of the NDB protein sequence is aligned to calcium-
binding proteins. Consistency of the NDB sequence with the EF-hand
motif (Marsden et al., 1990) is denoted by (+) and (±) signs above the
sequence. The central loop for calcium liganding by the EF-hand motif is
denoted with a continuous line. Amino-acid residues predicted to fold
into an alpha-helical con®guration by PredictProtein are underlined with
dashes. The sequences are (accession numbers): TRC, Meleagris
gallopavo troponin C (P10246); CAM, Schizosaccharomyces pombe
calmodulin (P05933); CTR, Chlamydomonas reinhardtii caltractin
(P05434).
longest NDA 5¢ RACE clones (out of 14) determine 33
additional nucleotides in the 5¢ leading region, which
con®rms the ®rst ATG present in the pBNDa24 cDNA as the
translation start. For the pBNDb36 cDNA, 12 clones derived
by 5¢ RACE reveal 81 upstream nucleotides. An ATG codon
and six additional amino-acid residues complete the
reading frame. The 5¢ leader contains two stop codons in
frame with the ATG (Figure 1).
Protein sequence comparison
The completed reading frames encode the polypeptides
NDA and NDB of 55 and 65 kDa, respectively. The
sequence identity of proteins to the yeast NDI1 and to
one another is 30±40%. In protein database searches,
known and putative NADH dehydrogenases from yeasts
and bacteria as well as several other NAD(H)-utilizing
¯avoenzymes are similar to both NDA and NDB. Alignment
of representative sequences (Figure 2b) reveals highest
conservation in two regions containing motifs for binding
ã Blackwell Science Ltd, The Plant Journal, (1999), 20, 79±87
Mitochondrial NADH dehydrogenases in potato 81
of ADP or ADP-derived nucleotides. Apart from these
motifs, relatively few residues are conserved between all
sequences for known and putative NADH dehydrogenases
(Figure 2). Sequence comparison to dihydrolipoamide
dehydrogenase of Acholeplasma and NADH oxidase of
Borrelia reveals similarity along most parts of the proteins.
However, two regions conserved between the NADH
dehydrogenase sequences, a block immediately down-
stream of the ®rst ADP-binding motif, and the C-terminal
part (Figure 2), deviate or are absent in these related
enzymes and may be involved in additional functions, e.g.
quinone binding and membrane association.
Both ADP-binding motifs of the NDA and NDB se-
quences meet 10 of the 11 criteria postulated by
Wierenga et al. (1986). As in the E. coli NDH protein, the
®rst ADP-binding motifs of the potato sequences obey the
core GXGXXG rule, whereas in the yeast NDI1 the third G
is replaced by a similar amino-acid residue (Figure 3a).
With respect to these motifs, the NDH and NDI1 proteins
are representative for the bacterial and yeast homologues,
respectively (not shown).
Unique among its homologues, the potato NDB protein
sequence contains a long insert located between two
highly conserved blocks (Figure 2). This segment is related
to calcium-binding proteins with a-loop-a EF-hand motifs,
as seen in the primary structure and in the predicted
secondary structure (Figure 3b). The ®rst part of the NDB
insert meets the criteria for a single EF-hand motif
(Marsden et al., 1990). The second part of the insert shares
similarity with EF-hands of other enzymes, but diverges
from the consensus in several positions critical for calcium
coordination (Figure 3).
Phylogenetic analysis of the rotenone-insensitive NADH
dehydrogenases sets eukaryotic homologues apart from
bacterial sequences (Figure 4). Sequences are generally
grouped according to species with statistical signi®cance.
However, very long-terminal branches preclude the un-
equivocal assumption of independent gene duplications in
each species. A close association between one
Synechocystis homologue and those of other bacteria
indicates that here gene duplication occurred before
speciation (Figure 4).
Mitochondrial protein import
The potato NDA and NDB protein sequences, like their
yeast homologues, have N-terminal extensions absent
from bacterial homologues (Figure 2). Apart from a bias
towards Lys instead of Arg as positively charged residues,
the potato N-terminal sequences are very similar to
mitochondrial targeting peptides (von Heijne et al., 1989).
To investigate the targeting and sorting of the gene
products, in vitro import experiments were performed.
For synthesis of the complete NDB protein, the reading
82 Allan G. Rasmusson et al.

frame was assembled from pBNDb36 and a 5¢ RACE clone
(Figure 1). In vitro transcription and translation produced a
polypeptide with an apparent molecular mass of 60 kDa
(Figure 5a), similar to the predicted 65 kDa. Upon incuba-
tion with isolated, actively respiring potato mitochondria,
the polypeptide was imported into the organelles, as
indicated by the induced valinomycin-sensitive proteinase
K protection (Figure 5a). However, no change in polypep-
tide size, indicative for protein processing, was seen upon

Figure 4. Phylogenetic grouping of bacterial, fungal and plant protein

sequences.
The sequence of Borrelia was used as outgroup to root the tree. The tree
shown is obtained with the neighbour-joining method of the PHYLIP
program. Identical tree topology for statistically signi®cant branchings is
obtained with a parsimony approach (not shown). Bootstrap values
(> 80%) are given for signi®cant branchings and the
Schizosaccharomyces dichotomy. Values for the parsimony approach are
given in brackets, where divergent. The sequences analysed are as in
Figure 2, and additionally: Calothrix viguieri putative NADH
dehydrogenase (Y09899); Mycobacterium tuberculosis z83, unknown
(z83859); M. tuberculosis z84, unknown (Z84725); Synechocystis l14, ORF
sll1484 (D90916); Synechocystis r17, ORF slr1743 (D90909); Synechocystis
r08, ORF slr0851 (D90909).

Figure 5. Mitochondrial import and localization of imported proteins.

(a) For import of in vitro-synthesized [35S]methionine-labelled poly-
peptides from NDA and NDB clones, translation products were incubated
with puri®ed potato mitochondria (Imp), with and without the ionophore
valinomycin (Val). Unimported polypeptides were then digested by
proteinase K (PrK), as denoted at the top. Molecular mass markers are in
kDa. Below, an enlarged autoradiograph of the proteins in the three
central lanes for NDA shows more clearly the double band after import.
(b) Scanned SDS±PAGE lanes along the axis of migration (left to right) for
imported polypeptides (I) and after subsequent treatment with proteinase
K (IP) or DTSSP, a membrane-impermeable cross-linker (ID). Like the
control for the intermembrane space, mature FeS-subunit of potato bc1
complex (mFeS), the imported NDB protein is removed by the cross-
linking. Imported NDA and the matrix control, Nicotiana b-subunit of ATP
synthase (mF1b), are resistant. Here the closely migrating NDA precursor
(pNDA) and the processed matured NDA (mNDA) are resolved by their
peak positions. Ticks on the x axes correspond to 2 mm, and on the y axes
to 10 counts in arbitrary units.
(c) After import, proteinase K treatment and re-isolation (IP), mitochondria
were permeabilized with digitonin and centrifuged. The supernatants (S)
and pellets (P) were analysed by SDS±PAGE. The imported NDA and NDB
proteins partition mainly with the membranes.

ã Blackwell Science Ltd, The Plant Journal, (1999), 20, 79±87


Figure 6. NDA and NDB show distinct modes of membrane association in
bacterial cells.
(a) E. coli expressing pET32 constructs for NDA (lanes 1±4) and NDB
(lanes 5±8) were sonicated in high-salt medium (containing 0.5 M NaCl)
and centrifuged. Pellets were consecutively extracted with 1% (w/v)
Triton X-100 and 6 M urea in this medium. Lanes 1 and 5 contain total
cellular protein, lanes 2 and 6 the ®rst supernatants of water-soluble
protein, lanes 3 and 7 Triton extracts, and lanes 4 and 8 urea extracts.
(b) NDB fusion proteins were consecutively extracted under different
ionic conditions. Lanes 1±2, soluble protein released by sonication in
2 mM EDTA in low-salt medium and protein further extracted by high-salt
conditions, respectively; lanes 3±5, protein released by sonication in
1 mM CaCl2 in low-salt buffer, and protein consecutively extracted by
1 mM CaCl2 in high-salt medium, and the high-salt medium of 0.5 M NaCl
without further additives, respectively; lanes 6±8, as for 3±5 but CaCl2
replaced by 1 mM MgCl2 and 0.1 mM EGTA.
For both panels, the samples loaded correspond to 200 ml culture volume,
except for halfed amounts in lanes 1 and 5 in (a). Prestained markers
(Biorad) are in kDa.
import (Figure 5a,b). Also, incubation with potato mito-
chondrial membranes under standard reaction conditions
for matrix processing peptidase (Braun et al., 1992; Knorpp
et al., 1994) produced no detectable truncation of the NDB
polypeptide (not shown).
Transcription and translation of NDA protein from
plasmid pBNDa24 produced a polypeptide with an appar-
ent molecular mass of 52 kDa, close to the calculated
55 kDa. The NDA protein is also readily imported into
mitochondria, and acquires a valinomycin-sensitive resis-
tance to proteinase K. After import, two closely migrating
polypeptides, the smaller being protease resistant, are seen
on autoradiograms and as a double peak in phosphorima-
ger scans (Figure 5a,b). This indicates that a short targeting
peptide has been removed upon import of the NDA protein.
ã Blackwell Science Ltd, The Plant Journal, (1999), 20, 79±87
Mitochondrial NADH dehydrogenases in potato 83
Intramitochondrial localization of proteins
The water-soluble cross-linker 3,3¢-dithiobis-sulfosuccini-
midylpropionate (DTSSP) reacts with free amino groups,
especially Lys side chains. Proteins aggregate upon
treatment, so they can no longer be visualized as discrete
bands by SDS±PAGE. The DTSSP is unable to penetrate
the inner mitochondrial membrane and can react only with
proteins exposed to the external medium or the inter-
membrane space (Knudten et al., 1994). To determine
which side of the inner mitochondrial membrane the
NDA and NDB proteins are sorted to, proteins were
imported and the mitochondria were re-isolated and
treated with DTSSP. The imported mature forms of the
FeS protein of potato complex III, exposed to the
intermembrane space, and the F1b-subunit of tobacco
mitochondrial ATP synthase, exposed to the matrix, were
used as controls (Figure 5b). The mature FeS protein band
disappears completely upon DTSSP treatment, whereas
the F1b protein is resistant to DTSSP treatment to
approximately 70%. The partial disappearance of the F1b
protein band is attributed to a limited disruption of
mitochondria upon re-isolation after the import reaction
(Figure 5b). The similar resistances of the imported mature
NDA protein to DTSSP and proteinase K suggest that this
protein, like the F1b subunit, is localized inside the inner
membrane barrier and is not exposed to the intermem-
brane space. On the other hand, the imported NDB protein
is accessible to DTSSP, as the band in SDS±PAGE
disappears under this treatment. This suggests the NDB
protein is exposed to the intermembrane space, similar to
the FeS protein. Cross-linking in the membrane-disrupting
presence of 0.025% Triton X-100 ef®ciently removed all of
the proteins (not shown).
To test for a potential membrane association of the NDA
and NDB proteins, mitochondria were treated with digito-
nin after protein import, proteinase K treatment and re-
isolation. The digitonin treatment solubilized the imported
F1b polypeptide which was thus found in the supernatant
after centrifugation. However, both the imported NDA and
NDB proteins are resistant to extraction by digitonin and
are retained in the membrane pellet (Figure 5c). This
suggests that both proteins are membrane-attached in
plant mitochondria and are not released under these
conditions.
Bacterial expression of proteins
Tagged NDA and NDB hybrid proteins of 73 and 84 kDa,
respectively, were expressed in E. coli cells. Bacteria were
sonicated and membranes were extracted under various
salt and detergent conditions. The proteins were resolved
by SDS±PAGE, blotted, and speci®cally identi®ed by their
N-terminal S-tags (Figure 6). The NDA hybrid was
84 Allan G. Rasmusson et al.
extracted as a single polypeptide by Triton X-100, but not
by 0.5 M NaCl. Smaller forms of the protein present in the
cells were already solubilized by the salt treatment. The
apparent size of the Triton-extracted protein (65 kDa) is
close to the theoretical value, indicating that the intact
protein is released from its membrane association only by
the detergent, whereas the smaller, water-soluble proteins
are proteolytic degradation products. A subsequent urea
extraction released intact as well as partially degraded
NDA fusion proteins (Figure 6a), indicating that intact
protein and in vivo-generated degradation products had
both been sequestered into inclusion bodies. A different
pattern of membrane association was observed for the
NDB hybrid. A polypeptide of apparently 75±80 kDa is
released as a soluble protein either by high monovalent
salt or by EDTA (Figure 6). No further protein is mobilized
by addition of Triton. However, in the presence of 1 mM
CaCl2 or MgCl2, the protein is not solubilized by cell
disruption or extraction at low or high monovalent salt,
respectively (Figure 6b). The subsequent release of NDB
fusion protein by high-salt medium without CaCl2 or MgCl2
indicates binding to the membranes only in the presence
of the divalent cations.
Discussion
The novel potato NADH dehydrogenase homologues are
associated with the inner mitochondrial membrane
We here report cloning of the ®rst homologues to bacterial
rotenone-insensitive NADH dehydrogenases from higher
eukaryotes, the nda and ndb cDNAs from potato. The
presence of N-terminal signal peptides, in vitro mitochon-
drial import of both proteins and, for the NDA protein,
processing, con®rm mitochondrial localization of the gene
products (Figure 5). The absence of processing seen for
NDB indicates that either it is not recognized by the matrix-
processing peptidase, or it does not have access to the
matrix space. Absence of processing has been reported for
several other plant mitochondrial proteins (Whelan and
Glaser, 1997).
Within the organelle, both NADH dehydrogenases are
associated with the inner mitochondrial membrane, as
indicated by resistance to extraction by digitonin (Figure
5c). At low detergent-to-protein ratios, digitonin solubilizes
the outer membrane from plant mitochondria. Increasing
amounts of detergent also permeabilize the inner mem-
brane releasing the matrix content, and subsequently
solubilize the inner membrane (Douce, 1985). In this
investigation, digitonin suf®cient to release imported F1b
polypeptides from the matrix still did not solubilize the
imported NDA, and solubilized NDB protein only to a small
extent, suggesting that they are attached to the inner
membrane under these conditions (Figure 5c). In conjunc-
tion with the selective access of the water-soluble cross-
linker DTSSP to the NDB protein after import (Figure 5b),
these results indicate that the NDA and NDB proteins are
bound to the internal and external surfaces of the inner
mitochondrial membrane, respectively. The inability of
DTSSP to cross-link the NDA protein from the external side
of the inner mitochondrial membrane (Figure 5b) further
suggests that the NDA protein does not span the
membrane, or exposes only a short segment at the
external surface. Both the E. coli NDH and the S. cerevisiae
NDI1 are bound to their respective membranes by an
unknown mechanism. They need detergents for solubiliza-
tion, although the protein sequences contain no potential
membrane-spanning a-helices (Jaworowski et al., 1981;
Kitajima-Ihara and Yagi, 1998; de Vries and Grivell, 1988;
de Vries et al., 1992). The eukaryotic NDI1 homologues,
including the novel potato NDA and NDB, similarly display
no obvious transmembrane segments (not shown) when
analysed by hydropathy plots or the TMAP program
(Persson and Argos, 1994).
The NDA hybrid protein expressed in E. coli needs
detergent for solubilization, as do the E. coli and yeast
homologues, (Figure 6). However, only the intact protein
appears to be membrane bound. Since the N-terminal S-
tag was used for visualization, the water solubility of the
degradation products suggests that the C-terminus is
necessary for membrane binding. Interestingly, the C-
terminal part present in all sequenced NADH dehydro-
genases is missing from the related, but soluble lipoamide
dehydrogenase (Figure 2). This C-terminal domain is,
however, conserved in the plant NDB. The NDB hybrid
protein appears to be more loosely attached than NDA to
the bacterial membrane, by a mechanism involving
divalent cations (Figure 6b). Possibly, the additional
segment with the EF-hand motif imposes a structural
difference to NDB, which is re¯ected in its speci®c
membrane-binding features. The distinct membrane at-
tachments of the NDA and NDB hybrids are consistent with
previous analyses of plant mitochondria. Inner membrane
vesicles maintain rotenone-insensitive NADH dehydrogen-
ase activity bound to their matrix surface (Rasmusson and
Mùller, 1991), but the external NADH dehydrogenase is
released from the inner membrane when mitochondria are
treated by low-osmolarity sucrose solutions (Douce et al.,
1973). The effect of divalent cations was not investigated,
but it has been suggested that Ca2+ ions may activate the
external NADH dehydrogenase by in¯uencing its mem-
brane interaction (Mùller, 1997).
The NDB hybrid protein (Figure 6) appears to be more
easily solubilized than imported NDB (Figure 5c), which
mainly remains associated with the mitochondrial mem-
branes upon permeabilization by digitonin. The reason for
this may reside in differences in disruption, membrane
composition (mitochondrial versus bacterial) or the pre-
ã Blackwell Science Ltd, The Plant Journal, (1999), 20, 79±87

sence of the N-terminal tags in the hybrid protein. On the
other hand, these same tags do not disturb membrane
binding of the homologous NDA. Nevertheless, the NDB
protein clearly displays a mode of membrane association
different from any of the NADH dehydrogenases analysed
so far.
Although many attempts have been made to isolate
rotenone-insensitive NADH dehydrogenases (Mùller and
Rasmusson, 1998; Mùller, 1997), the lack of clear diag-
nostic properties of the puri®ed proteins makes compar-
ison with the potato NDA and NDB dif®cult. Soluble 58 kDa
dehydrogenases oxidizing both NADH and NADPH with
synthetic electron acceptors have been isolated from
maize (Luethy et al., 1995) and red beetroot (Menz and
Day, 1996). The NDB protein, with an apparent molecular
mass of 60 kDa, may theoretically be the potato orthologue
of these enzymes if the latter are released from mem-
branes similar to the NDB hybrid protein. However, as
outlined below, the sequences of the ADP-binding motifs
within the NDB protein suggest that it should only accept
NADH. Positive identi®cation by peptide sequences of the
58 kDa proteins puri®ed from beetroot and maize would
resolve this question.
The potato enzymes contain novel and familiar motifs for
cofactor binding
Both NDA and NDB contain motifs for binding ADP, or
ADP-derived nucleotides, conserved among their homo-
logues, whereas NDB additionally contains a unique insert
carrying an EF-hand motif potentially involved in calcium
binding (Figures 2 and 3). The EF-hand motif is accom-
panied by a second segment bearing similarities in
primary and predicted secondary structure to EF-hand
motifs of other proteins (Figure 3b). The presence of an EF-
hand motif in potato NDB re¯ects the fact that calcium-
dependent NADH : ubiquinone oxidoreductase activity has
only been observed for plant mitochondria (Mùller and
Rasmusson, 1998; Mùller, 1997), and is consistent with the
potential role of the NDB protein as an external NADH
dehydrogenase.
The sequence similarity between the NADH dehydro-
genases and a dihydrolipoamide dehydrogenase (Figure 2)
suggests that the ®rst and the second ADP-binding motif
bind the ADP moiety of FAD and NADH, respectively. Like
the E. coli NDH, the potato NDA and NDB proteins meet
the criteria for binding these molecules (Figure 3a). The
presence of acidic residues conserved in the C-terminal
parts of the NDA and NDB motifs, downstream of the
variable loop region, indicate that the proteins are unable
to bind NADPH (Lesk, 1995; Wierenga et al., 1986). The
results therefore suggest that the potato NDA and NDB
proteins have substrate speci®cities similar to the NDI1 of
yeast and NDH of E. coli, both of which oxidize only NADH.
ã Blackwell Science Ltd, The Plant Journal, (1999), 20, 79±87
Mitochondrial NADH dehydrogenases in potato 85
Evolutionary implications of distinct functions for
separate NADH dehydrogenases
The endosymbiotic progenitor of mitochondria is expected
to have possessed a set of respiratory NADH : ubiquinone
oxidoreductases, including both a proton-pumping com-
plex I and one or several non-proton-pumping rotenone-
insensitive NADH dehydrogenase(s) for oxidation of NADH
from the bacterial cytoplasm/mitochondrial matrix. In
mitochondria of eukaryotes, yeasts and potato, two
rotenone-insensitive NADH dehydrogenase activities are
consistently seen, one external and one internal. This is
apparently the consequence of the two nda and ndb genes
in potato and the similar triplet of homologues in S.
cerevisiae, where the NDI1 protein is located at the inner
surface of the inner mitochondrial membrane, and the two
additional homologues are localized to the external side
(Luttik et al., 1998). The phylogenetic analyses indicate an
ancient common origin for the eukaryotic NADH dehydro-
genases (Figure 4). In the cyanobacterium Synechocystis,
one homologue is more closely associated to the homo-
logues of Mycobacterium than to the other Synechocystis
genes, indicating that gene duplication and possibly
functional specialization of the encoded enzymes occurred
very early, probably even before the evolutionary separa-
tion of the two species (Figure 4). The analyses give no
indication of a similar association between the internal
enzymes, potato NDA and S. cerevisiae NDI1, nor between
the potato NDB and the two additional homologues of S.
cerevisiae (Figure 4). In the light of the wide phylogenetic
separation of potato and yeast, further eukaryotic homo-
logues are needed to elucidate the evolutionary history of
the nda and ndb genes.
Experimental procedures
Isolation and analysis of cDNA clones
Nucleotides 14±337 of the Arabidopsis EST clone 62A4T7,
accession number T41616 (Newman et al., 1994) were ampli®ed
by PCR using gene speci®c 20mer oligonucleotide primers. The
product was randomly labelled and used to screen a potato
(Solanum tuberosum L., cv. Desiree) cDNA library in lZAPII
(Kobmann et al., 1992) according to Grohmann et al. (1992).
Membranes were washed in 0.6 3 SSC, 0.1% SDS at 42°C.
Total RNA was isolated from potato leaves (cv. Desiree)
according to the methodology described by Chomczynski and
Sacchi (1987). Poly(A)+ RNA was then isolated with oligo(dT)-
magnetic beads (Dynal) and used as templates for rapid
ampli®cation of 5¢ ends (5¢-RACE) with a 5¢/3¢ RACE kit
(Boehringer Mannheim). Primers A1 (5¢-CTCCAAGGTTTC-
AACTCCCTCTGTCACTG-3¢) and B1 (5¢-TGATACCAGAACGGCA-
AGATACTGTGCGG-3¢), speci®c for the cDNAs of pBNDA24 and
pBNDb36, respectively, were used as initiators for the reverse
transcription. After poly(dA) tailing of the cDNAs, the nested
primers A2 (5¢-ACAGCAGGTTGAATCCTTCC-3¢) and B2 (5¢-
TCAATCTTCAGACATTCTGC-3¢) (Figure 1) were used as antisense
oligos in respective ®rst PCR ampli®cations. The reactions were
86 Allan G. Rasmusson et al.
run for 30 cycles at 50°C annealing temperature using the oligo
dT-anchor primer as sense oligo. The products were used as
templates for second rounds of ampli®cations using primers A3
(5¢-CATCCTGCCCATCCAGAACC-3¢) and B3 (5¢-CCCATCCTGTT-
CCAAGCACC-3¢) as gene-speci®c antisense oligos (Figure 1).
These ampli®cations were carried out for 30 cycles at 60°C
annealing temperature and the PCR anchor primer as sense
oligonucleotide.
For construction of plasmid pBNDh50, a clone from the 5¢-RACE
was used as template for PCR. Sense primer was B4 (5¢-
AACTGCAGACAGAAATACTGAGAA-3¢) (Figure 1), complemen-
tary to the 5¢ extreme of the mRNA-derived sequence and adapted
for cleavage by PstI. The ampli®cation was run for 25 cycles at
43°C annealing temperature with B3 as antisense primer. The
product was cut with PstI and ScaI and ligated to pBNDb36 in
which the corresponding, truncated 5¢-terminal part of the cDNA
had been removed using the same enzymes (Figure 1).
General molecular cloning techniques were according to
Sambrook et al. (1989). Sequencing was carried out on both
strands with a T7 sequencing kit (Pharmacia) following the
manufacturer's instructions. The cDNA clones were sequenced
by oligonucleotide primer walking. Sequences were analysed
with the Wisconsin GCG program package, version 8.1.
Alignments were obtained with PILEUP (gap weight 5, gap length
weight 0.1). Phylogenetic trees were calculated on the basis of an
alignment which was reduced to 578 amino acids after excluding
the terminal sequence extensions in some species.
Mitochondrial protein import
Mitochondria were isolated from potato tubers (cv. Desiree)
according to Struglics et al. (1993), with the following modi®ca-
tions. The potato homogenate was strained through 30 mm nylon
gauze and centrifuged for 5 min at 3000 g. The supernatant was
decanted and centrifuged for 20 min at 13 000 g and the pellet was
used directly for the puri®cation of mitochondria. [35S]methio-
nine-labelled protein products of cDNA clones were synthesized
with a T3/T7-rabbit reticulocyte lysate in vitro transcription/
translation system (Promega); 5 ml translation product were used
for mitochondrial import, principally according to Rasmusson
et al. (1998a). Import reactions were incubated at 25°C for 30 min.
The reactions were split: 5 mg ml±1 proteinase K was added to one-
half, and both parts were incubated at 25°C for 15 min.
Phenylmethylsulfonyl ¯uoride (PMSF) was added to 1 mM and
mitochondria were re-isolated by centrifugation through 0.5 ml of
25% (w/v) sucrose. For direct analysis, mitochondria were
resuspended in SDS±PAGE loading buffer (Laemmli, 1970).
SDS±PAGE gels were analysed by phosphorimaging.
Localization of imported proteins
For digitonin extraction of proteinase K-treated mitochondria
after import, the re-isolated mitochondria were resuspended in
2.5 mg ml±1 digitonin, 0.35 M mannitol, 20 mM Tris±HCl pH 7.4,
6 mM EDTA, 1 mM PMSF to a protein : detergent ratio of 8 : 1.
After 20 min at 4°C, solubilization was stopped by fourfold
dilution with the above solution without digitonin. Membranes
were separated from extracted proteins by centrifugation at
15 000 g for 15 min.
For cross-linking of imported proteins, mitochondria were re-
isolated immediately after the import reaction and incubated with
3,3¢-dithiobis-sulfosuccinimidylpropionate (DTSSP) according to
Knudten et al. (1994).
Bacterial expression
The pNDa24 and pNDb36 inserts were cloned into pET32
(Novagen) for expression as hybrid proteins containing, in this
order from the N-terminus, a thioredoxin-tag, a His-tag and an S-
tag. Strain BL21(DE3)pLysS of E. coli was used as host. For
induction of protein synthesis, bacteria grown in rich medium up
to OD600 » 0.5 were incubated with 1 mM isopropyl-beta-D-thioga-
lactopyranoside for 3 h at 37°C. Cells were washed in 50 mM Tris±
HCl pH 8.0, and sonicated for 5 3 5 sec in low-salt medium (20 mM
Tris±HCl pH 8.0, 1 mM PMSF) or high-salt medium (as above plus
0.5 M NaCl and 5 mM imidazole), with supplements added as
indicated in the respective ®gure legends. The homogenate was
centrifuged at 70 000 g for 20 min and the supernatant containing
soluble proteins was decanted. For further extractions, pellets
were resuspended in the medium speci®ed in the legends and
were centrifuged for 30 min as above. All separations were carried
out at 0±4°C. After SDS±PAGE and transfer to nitrocellulose
membranes, S-tag fusion proteins were visualized by S-protein±
horseradish peroxidase conjugate (Novagen) and were detected
with enhanced chemiluminescence (Amersham) following the
manufacturer's recommendations.
Acknowledgements
This study was supported by grants from the Swedish Natural
Science Research Council, Crafoordska Stiftelsen and Kungliga
Fysiogra®ska SaÈllskapet i Lund (to A.G.R), the Bundesministerium
fuÈr Bildung, Wissenschaft, Forschung und Technologie, and the
Deutsche Forschungsgemeinschaft, Bonn (to A.B). We are grate-
ful to Dr H.-P. Braun, UniversitaÈt Hannover, Germany, Professor
N.-H. Chua, Rockefeller University, NY, USA, and the Arabidopsis
Biological Resource Center, Ohio State University, USA, for clone
donation; Dr V. Heiser, Max-Planck-Institut fuÈr Molekulare Genet-
ik, Berlin, Germany and Dr C. Knorpp, Lund University, Sweden,
for valuable discussions.
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