Type II NAD(P)H dehydrogenases are targeted to mitochondria
and chloroplasts or peroxisomes in Arabidopsis thaliana
Chris Carriea, Monika W. Murchaa, Kristina Kuehna, Owen Duncana,
Michelle Barthetb, Penelope M. Smithb, Holger Eubela, Etienne Meyera,
David A. Dayb, A. Harvey Millara, James Whelana,*
aARC Centre of Excellence in Plant Energy Biology, MCS Building M316 University of Western Australia,
35 Stirling Highway, Crawley 6009, Western Australia, Australia
bARC Centre of Excellence in Plant Energy Biology, School of Biological Sciences, University of Sydney, NSW, Australia
Received 21 July 2008; revised 30 July 2008; accepted 31 July 2008
Available online 12 August 2008
Edited by Ulf-Ingo Flu ¨gge
ases (ND) in Arabidopsis are targeted to two locations in the
cell; NDC1 was targeted to mitochondria and chloroplasts, while
NDA1, NDA2 and NDB1 were targeted to mitochondria and
peroxisomes. Targeting of NDC1 to chloroplasts as well as mito-
chondria was shown using in vitro and in vivo uptake assays and
dual targeting of NDC1 to plastids relies on regions in the ma-
ture part of the protein. Accumulation of NDA type dehydrogen-
ases to peroxisomes and mitochondria was confirmed using
Western blot analysis on highly purified organelle fractions. Tar-
geting of ND proteins to mitochondria and peroxisomes is
achieved by two separate signals, a C-terminal signal for peroxi-
somes and an N-terminal signal for mitochondria.
? ? 2008 Federation of European Biochemical Societies. Pub-
lished by Elsevier B.V. All rights reserved.
We found that four type II NAD(P)H dehydrogen-
Keywords: Chloroplast; Mitochondria; Peroxisome; Dual
targeting; Green fluorescent protein; Alternative NAD(P)H
A hallmark of eukaryotic cells is the partitioning of various
biochemical pathways out of the cytosolic milieu and into dis-
crete organelles. Although the compartmentalisation of vari-
ous biochemical functions allows specialisation, it requires
that many functions are duplicated and thus many enzymatic
activities take place in more than one organelle. In the majority
of cases these common functions are performed by different
proteins, encoded by distinct genes, that are each targeted to
a single location in the cell . However, in other cases it ap-
pears that the same function in different organelles is carried
out by the same protein that is targeted to two locations, a
process called dual targeting. This was first reported for gluta-
thione reductase from pea, which is targeted to both mitochon-
dria and chloroplasts . To date, studies in several plants
suggest that more than 30 proteins are dual targeted to mito-
chondria and chloroplasts .
The targeting of proteins is routinely assessed by attaching a
reporter, most often green fluorescent protein (GFP), to the
protein being studied and the intra-cellular distribution of fluo-
rescence measured . This approach is convenient and sensi-
tive and has been used widely to define dual targeting to
mitochondria and chloroplasts [5–8]. However, this approach
has some limitations that depend on the nature of the con-
structs. Firstly, for proteins that may be targeted to two loca-
tions using two signals in different parts of the protein
sequence, GFP fusion to one part of the protein can mask
an adjacent signal – resulting in localisation to only one of
its in vivo destinations. Secondly, targeting ability can be af-
fected by the nature of the passenger protein. This occurs even
for proteins targeted to a single location [9,10], but it seems to
be even more pronounced for dual targeted proteins. In two
independent studies examining the role of the mature protein
for dual targeted proteins to mitochondria and chloroplasts,
both concluded that the passenger or mature protein influ-
enced dual targeting ability [11,12].
Type II NAD(P)H dehydrogenases are typically located on
the mitochondrial inner membrane where they can oxidise
NAD(P)H and are insensitive to the complex I inhibitor rote-
none [13–15]. Seven genes encode putative type II NAD(P)H
dehydrogenases in Arabidopsis, three have been defined as
external (NDB 1, 2 and 4) and three defined as internal
NAD(P)H dehydrogenases (NDA 1 and 2 and NDC1)
[13,14]. The remaining gene encoding a putative external
NAD(P)H dehydrogenase, NDB3, could not be cloned by a
number of groups and thus is either a pseudogene or its expres-
sion is very restricted [13,16]. Previous studies using GFP tag-
ging have shown NDA1, NDA2, NDB1, NDB2 and NDC1 to
be targeted to mitochondria , in vitro mitochondrial uptake
assays have shown NDA1, NDA2, NDB1, NDB2, NDB4 and
NDC1 to be imported into mitochondria , and a number of
studies using Western blot analysis of mitochondrial proteins
and/or cellular fraction with antibodies raised against peptides
from potato NDA1 and NDB1 have all concluded a mitochon-
drial localisation for these proteins [17–19]. Additionally over
two decades of biochemical analysis have shown that the
Abbreviations: AOX, alternative oxidase; GFP, green fluorescent
protein; KAT2, 3-ketoacyl-CoA thiolase; ND, type II alternative
NAD(P)H dehydrogenase; RFP, red fluorescent protein; SSU, Rubi-
sco small subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase;
TIM17-2, translocase of the inner mitochondrial membrane
*Corresponding author. Fax: +61 8 93801148.
E-mail address: email@example.com (James Whelan).
0014-5793/$34.00 ? 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 582 (2008) 3073–3079
activities associated with these proteins are located in mito-
chondria [15,20]. Thus it can be concluded that these proteins
are located in mitochondria.
However the set of proteins predicted to be located in per-
oxisomes by the AraPerox database, with medium to high con-
fidence, identifies three of these mitochondrial type II
NAD(P)H dehydrogenases . Thus we re-assessed the tar-
geting ability of all six NAD(P)H dehydrogenases with the
view that they may also be located in other cellular organelles
in addition to mitochondria.
2. Materials and methods
2.1. Sequence analysis and cloning
The full length coding sequences of NDA1, NDA2, NDB1, NDB2,
NDB4 and NDC1 were cloned as both N- and C-terminal GFP fusions
by Gateway cloning under the control of the 35S CaMV promoter.
Additionally the last 10 amino acids of NDA1, NDA2, NDB1 and
NDB2 were cloned to the C-terminus of GFP. The alternative oxidase
(AOX) targeting signal, the full length targeting sequence of small sub-
unit of 1,5 ribulose bisphosphate carboxylase/oxygenase (SSU Rubi-
sco) and the peroxisomal targeting signal SRL of pumpkin malate
synthase, were fused to red fluorescent protein (RFP) and used as
mitochondrial, chloroplast and peroxisomal controls, respectively
The constructs were used to transform Arabidopsis suspension cul-
ture cells, Arabidopsis seedlings (1–2 weeks old) and onion epidermal
cells by biolistic transformation as previously outlined . Fluores-
cence patterns were obtained 24 h after transformation by visualization
under an Olympus BX61 fluorescence microscope, with excitation
wavelengths of 460–480(GFP) and 535–555(RFP). Emissions were col-
lected for GFP between 495 and 540 and RFP between 570 and 625,
and imaged using the CellRimaging software. To ensure no cross over
in detection of signals AOX-RFP and SSU-GFP were co-transformed
to ensure that the filters were detecting the appropriate signal.
2.2. Determination of subcellular targeting ability
N- and C-terminal GFP-tagged proteins were used to transform
Arabidopsis cell suspension culture, 1–2 week old Arabidopsis seed-
lings and onion epidermal cells by biolistic transformation as previ-
ouslyoutlined .For each
transformations were carried out, the test construct with a mitochon-
drial, plastidic and peroxisomal control. In vitro import assays into
isolated Arabidopsis mitochondria and pea chloroplasts were carried
out as previously outlined [23,25].
construct tobe tested three
2.3. Antibody production and Western blotting
Antibodies were raised in rabbit against NDA1, amino acids 57–236
andthe NDB2 specificpeptide
(ETDDVSKNNIELKIE). The specificity of the antibodies was tested
against recombinant proteins synthesised in a wheat germ translation
lysate according to manufacturers instructions (Roche, Sydney), pro-
grammed to synthesise NDA1, NDA2 and NDB2 by making linear
templates by PCR as per manufactures instructions (Roche, Sydney).
Mitochondria and peroxisomes were purified from 7 day old cell sus-
pension culture using free flow electrophoresis as described by Eubel et
al. . Western blot analysis was carried out against 20 lg of mito-
chondrial and peroxisomal proteins separated by SDS–PAGE as
previously outlined .
at amino acids438–452
Arabidopsis NDA1 SRI
Arabidopsis NDA2 SRI
Poplar NDA1 SRI
Grape NDA1 SRI
Potato NDA1 SRI
Rice NDA1 SRI
Grape NDA2 RIG
Rice NDA2 RIG
Physcomitrella NDA1 SRF
Physcomitrella NDA2 SRF
Chlamydomonas NDA1 SRW
Chlamydomonas NDA2 SLF
Yeast NDI KGL
Yeast NDE1 SSI
Yeast NDE2 SSV
Chlamydomonas NDB1 SRV
Physcomitrella NDB1 RVE
Physcomitrella NDB2 SRM
Physcomitrella NDB3 SRM
Arabidopsis NDB1 SRI
Potato NDB1 SRI
Grape NDB1 SRI
Rice NDB1 SRI
Arabidopsis NDB2 SSI
Grape NDB2 SRI
Grape NDB3 SRI
Arabidopsis NDB4 SSI
Rice NDB2 SSL
Rice NDB3 LCS
Fig. 1. ClustalW alignment of type II NAD(P)H dehydrogenases from a variety of plants and yeast. Alignment of the sequences encoding type II
NAD(P)H dehydrogenases from a variety of plants revealed that several contained putative peroxisomal type I targeting signals (PTSI) at the C-
terminal end of the protein. The predicted strength of the PTSI signal was taken from AraPerox . The predicted ability to be targeted to
mitochondria and/or chloroplasts is shown. Arabidopsis thaliana NDC1 At5g08740;NP_568205, NDA1 At1g07180;NP_563783, NDA2
At2g2990;NP_180560, NDB1 At4g28220;NP_567801, NDB2 At4g05020;NP_180560, NDB4 At2g20800;NP_179673, Solanum tuberosum NDA1
CAB52796, NDB1 CAB52797, Populus trichocarpa NDA1 ABK95883, Vitis vinifera NDA1 CAO21440, NDC1 CAO71655, NDA2 CAO67571,
NDB1 CAO41235, NDB2 CAO16606, NDB3 CAO41237, Saccharomyces cerevisiae NDI NP_013586, NDE1 NP_013865, NDE2 NP_010198, Oryza
sativa NDC1 Os06g11140:BAD35311, NDA1 Os01g61410:NP_915326.1, NDA2 Os07g377730:NP911221.1, NDB1 Os06g47000:BAD45556, NDB2
Os05g26660:AAV43826, NDB3 Os08g04630:XP_480031.1, Chlamydomonas reinhardtii NDC1 ABR53723, NDA1 XP_001698901, NDA2
XP_001702271, NDB1 XP_001703643, Physcomitrella patens NDC1 manually annotated from scaffold 101 of Physcomitrella genome ,
NDA1 manually annotated from scaffold 28 of Physcomitrella genome , NDA2 XP_001769969, NDB1 XP_001766162, NDB2 XP_001759207,
NDB3 XP_001764062. Targeting prediction for all proteins are shown in Supplementary Table 1.
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
A ClustalW alignment of all ND sequences available from
various plant species and yeast revealed the amino acid se-
quence SRI at the C-terminal end of Arabidopsis NDA1,
NDA2 and NDB1 (and NDB3), and in a variety of ND pro-
teins from other plants. Other PTS I type targeting signals,
most notably SRM or SSI, were also found in ND sequences
 (Fig. 1). The fact that these amino acids are not present
in all ND sequences suggests that this tripeptide is not required
for function, opening the possibility that it may play a role in
defining subcellular localisation via its peroxisomal targeting
activity . Analysis of NDC1 sequences from Arabidopsis,
rice and Chlamydomonas reinhardtii predicted plastid-targeting
in all three species based on the N-terminal region (Supple-
mentary Table 1), even though these proteins display very
low levels of sequence identity in this region (data not shown).
Examination of the gene constructs used in a previous study
that indicated an exclusive mitochondrial localisation for these
proteins, revealed that only the N-terminal region was used in
the GFP fusions , amino acids 1–55 for NDA1, 1–60 for
NDA2, 1–59 for NDB1 and NDB2 and amino acids 1–83
3.1. NDC1 is targeted to mitochondria and chloroplasts
The full-length cDNA for NDC1 was placed in front of GFP
and its subcellular localisation examined by particle bombard-
ment. As controls, the cells transformed with the NDC1-GFP
construct were co-transformed either with plastid targeted
RFP using the targeting signal of the small subunit of 1,5
ribulose bisphosphate carboxylase/oxygenase (SSU Rubisco-
RFP) or the mitochondrial alternative oxidase targeting signal
(AOX-RFP). Targeting of NDC1-GFP to chloroplasts was
clearly observed in Arabidopsis suspension cells (Fig. 2A),
the pattern was clearly not identical to AOX-RFP but resem-
bled that of SSU-RFP quite closely. This is in contrast to what
has been previously reported where a mitochondrial localisa-
tion was concluded when the first 83 amino acids of NDC1
was used . However we routinely observed a weaker signal,
similar to the pattern obtained with AOX-RFP. Thus we
tested the targeting ability in a variety of tissues, namely Ara-
bidopsis seedlings and onion epidermal cells. Transformation
of these tissues resulted in the detection of two distinct signals,
a plastid signal evidenced by relatively large organelles, 2–
4 lM in diameter and few in number and smaller organelles,
1 or less lM in diameter typical of a mitochondrial pattern.
The mitochondrial targeting ability of NDC1 that we observed
in this study is consistent with previous results using GFP and
in vitro uptake assays [13,14].
To confirm that NDC1 could target to both chloroplasts and
mitochondria, in vitro uptake assays with isolated Arabidopsis
mitochondria and pea chloroplasts were carried out. Upon
incubation with isolated chloroplasts and mitochondria the
NDC1 precursor protein with a mol mass of 70 kDa was im-
ported into a protease resistant location and processed to a
mature size with a mol mass of 60 kDa (Fig. 2B, lanes 1–3).
Both organelles appeared to process the precursor protein to
the same mature protein, to confirm this import reactions into
mitochondria and chloroplasts were loaded into the same lane
to determine any small difference in mobility, none was
Fig. 2. Subcellular targeting of NDC1 using GFP tagging (A) In vitro uptake assays (B and C). A) The full-length cDNA for NDC1 was fused in
frame with GFP and co-transformed into Arabidopsis cells with mitochondrial targeted RFP (top panel) or chloroplast targeted RFP (bottom
panel). (B) In vitro uptake of NDC1 into isolated mitochondria and chloroplasts. Precursor proteins (lane 1) were incubated with isolated
mitochondria (lane 2) and chloroplasts (lane 3) under conditions that support the uptake of proteins into the respective organelles. Uptake was
assessed by insensitivity to added protease. Both organelles processed the precursor to a mature protein with the same mobility (lane 4). For
mitochondria, uptake was sensitive to the addition of valinomycin (lanes 5 and 6). For chloroplasts uptake was inhibited by addition of CuCl2(lanes
7 and 8). (C) The specificity of import of protein into isolated organelles was confirmed using the precursor of 1,5 bisphosphate carboxylase
oxygenase (SSU) that was only imported into chloroplasts and alternative oxidase (AOX) that was only imported into mitochondria.
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
detected (Fig. 2B, lane 4). As the translation of the precursor
alone also produces a protein with a mol mass of 60 kDa,
likely due to translation initiation at an internal methionine,
such as amino acid 47 in NDC1. Translation initiation at
internal methionine residues is frequently observed with in vi-
tro translation lysates . Thus we confirmed that the prote-
ase resistance was due to import into the respective organelle.
Import into mitochondria was inhibited by the addition of
Fig. 3. Subcellular targeting of NDA1, NDA2, NDB1 and NDB2. GFP was fused to the different proteins at the N- or C-terminal and subcellular
targeting assessed by particle bombardment of Arabidopsis suspension cells, 1–2 weeks old Arabidopsis seedlings and onion epidermal cells with
mitochondrial targeted RFP or peroxisomal targeted RFP as controls. (A) Subcelluar targeting pattern obtained with AOX-GFP, KAT2-GFP and
NDB2-GFP. (B) Subcelluar targeting pattern obtained with NDA1, NDA2 and NDB1 fused to GFP. The position of the GFP and the number of
amino acids used is shown for each construct.
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
valinomycin (Fig. 2B, lanes 5 and 6) , and import into chlo-
roplast inhibited by the addition of CuCl2(Fig. 2B, lanes 7 and
8) . The specificity of import into the respective organelles
was confirmed as the small subunit of 1,5 bisphosphate (SSU)
was only imported into chloroplasts and the alternative oxi-
dase precursor only imported into mitochondria (Fig. 2C, left
3.2. NDA1, NDA2 and NDB1 are targeted to mitochondria and
In order to determine the localisation of the other ND pro-
teins, N- and C-terminal GFP fusions were made followed by
particle bombardment. To determine a mitochondrial and per-
oxisomal pattern chimeric constructs with the AOX and KAT2
linked to GFP were used (Fig. 3A, image series 1 and 2). In the
case of NDB2 attaching GFP to the C-terminal resulted in tar-
geting to mitochondria as evidenced by co-localisation with
AOX-RFP (Fig. 3A, images 3a–3c). Attaching the last 10 ami-
no acids of NDB2 to the C-terminal end of GFP resulted in a
cytosolic localisation for GFP, as evidenced by fluorescence
throughout the cell, in all tissues tested (Fig. 3A, image series
4). In contrast when NDA1, NDA2 and NDB1 were tested in a
similar manner both mitochondrial and peroxisomal targeting
ability was detected. C-terminal fusions gave an exclusively
mitochondrial localisation, based on co-localisation with
AOX-RFP (Fig. 3B, images 1a–1c, 3a–3c and 5a–5c). This is
consistent with the mitochondrial targeting ability previously
observed with these proteins . However when the last 10
amino acids of NDA1, NDA2 and NDB1 were placed at the
C-terminal region of GFP peroxisomal targeting was observed
(Fig. 3B, images 2d–2f, 4d–4f and 6d–6f). The peroxisomal tar-
geting ability of these constructs was also detected in Arabid-
opsis seedlings and onion cells (Fig. 3B, images 2g and 2h, 4g
and 4h and 6g and 6h). Thus we concluded that these proteins
were targeted to peroxisomes in addition to mitochondria.
NDB4 targeted GFP to mitochondria as previously reported
(Supplementary Fig. 1) .
To confirm the dual location of NDA1 in mitochondria and
peroxisomes we raised antibodies against NDA1, expected to
be located in both locations from results above, and NDB2,
expected to be located only in mitochondria from results
above. We confirmed that the NDA1 and NDB2 antibodies
did not cross react with the other antigen by over-expression
of the respective proteins in an in vitro translation lysate prob-
ing with Anti 6-His antibodies that detected both proteins,
Anti A1 antibodies that detected only NDA1 and Anti B2 anti-
bodies that detected only B2 (Fig. 4A). As the NDA1 antibody
was raised against a fragment of the NDA1 protein on 180
Fig. 4. Western blot analysis of mitochondrial and peroxisomal fractions probed with various antibodies. (A) Confirmation of NDA1 and NDB2
antibodies. Wheat germ lysate (20 lg) programmed to synthesise each of the ND proteins was separated by SDS–PAGE, blotted to a nitrocellulose
membrane and probed with antibodies raised against NDA1 and NDB2 to confirm that they detected their target antigens. (B) As A except that
translation lysate programmed to synthesis b-glucuronidase (GUS), NDA1 and NDA2. (C) 20 lg of mitochondria or peroxisomes purified by free
flow electrophoresis were separated by SDS–PAGE, blotted to a nitrocellulose membrane and probed with antibodies as indicated. The antibody
used is indicated to the right of the panel and the apparent mol mass of the cross reacting protein indicated in the left in kDa. Note that for NDA1
and NDA2 the precursor size of the protein is detected when probing in vitro synthesised protein whereas the mature size of the protein is detected
when probing organelle fractions.
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
amino acids that displayed 83% sequence identity with the cor-
responding region of NDA2 we tested if the NDA1 antibody
cross reacted with NDA2. No cross reactivity was detected
with full length in vitro synthesised NDA2 (Fig. 4B).
Highly purified mitochondria and peroxisome fractions were
isolated from Arabidopsis cells  and proteins separated by
SDS–PAGE and subjected to Western blotting. In control
experiments, we used antibodies against proven markers of
mitochondria (TIM17-2 (Translocase of the Inner Mitochon-
drial membrane; ), and peroxisomes (KAT2 (3-ketoacyl-
CoA thiolase; [22,30]). These antibodies reacted strongly with
mitochondrial and peroxisomal fractions, respectively, and
much more weakly with the other fraction, indicating a small
degree of cross-contamination between the fractions (Fig.4B).
Densitometric analysis revealed the KAT2 signal in mitochon-
dria was ?5% of that detected in peroxisomes, whilst the
TIM17-2 signal in peroxisomes was ?1–2% of the signal that
could be detected in mitochondria (when the blot was overex-
Probing with antibodies raised against NDA1 resulted in the
detection of a single protein band with an apparent molecular
mass of 55 kDa, in both mitochondrial and peroxisome frac-
tions ( Fig. 4B). The blots indicated that there was more
NDA1 protein in the peroxisomal fraction than in the mito-
chondrial one, confirming that these proteins are found in both
compartments. Probing mitochondrial and peroxisomal frac-
tions with antibodies raised against the NDB2 specific peptide
produced a band only in the mitochondrial fraction (Fig. 4B),
confirming that it can target to mitochondria but not to per-
oxisomes. Importantly, this latter result also shows that the
very small amount of cross-contamination between the two
isolated fractions cannot explain the dual localisation of the
NDA1 signal. Thus the Western blot results confirm the
In this study we have shown that four ND proteins, NDA1,
NDA2, NDB1 and NDC1 are dual targeted. The dual target-
ing ability of ND proteins was overlooked in previous GFP
studies due to a number of technical parameters, namely the
nature of the GFP-protein constructs used in each study. In
the case of NDC1, it appears that the mature protein sequence
is required for its dual localisation by GFP (Fig. 2), as ob-
served for other dual targeted proteins [12,31]. The dual target-
ing of NDA1, NDA2 and NDB1 to mitochondria and
peroxisomes is dictated by two distinct signals. In the case of
the NDAs, the apparent Mr of the mature protein observed
in peroxisomes and mitochondria was identical (Fig. 4C). As
NDA proteins are processed upon import into mitochondria
, this strongly suggests that they are also processed upon
import into peroxisomes. It has been shown previously that
peroxisomes recognise N-terminal PTS2 type targeting signals
that are removed upon import and the processing of the NDA
proteins could be carried out by the same peptidase as both
NDA1 and NDA2 have a cysteine residue at amino acids 35
and 38, respectively, which defines the processing site by this
peptidase . Alternatively, the NDA proteins may be pro-
cessed by pitrilysin-like metallopeptidase present in peroxi-
somes . These enzymes belong to the same family of
proteases as the mitochondrial processing peptidase .
The mitochondrial pattern obtained with GFP with NDA1,
NDA2 and NDB1 differed slightly to that obtained from
AOX-RFP. Close examination of the merged images revealed
that the GFP fluorescence appeared at the periphery of the
mitochondrion, thus the GFP and RFP fluorescence co-local-
ise, but are not identical. A similar pattern of GFP fluores-
cence is routinely obtained when using outer membrane
mitochondrial proteins in humans and Arabidopsis [34,35].
This pattern may be due to the fact that GFP attached to
the C-terminal of an inner membrane protein will not be
?pulled? into mitochondria. The C-terminal of the ND proteins
may be located in the intermembrane space and thus never en-
ter the mitochondrial matrix. Thus the GFP attached to the C-
terminal end of these proteins remains outside the mitochon-
drion. Using only the N-terminal predicted targeting region re-
sults in a typical mitochondrial pattern as previously observed
, as the default targeting information for mitochondria dic-
tated a matrix location . Secondary signals dictate the in-
tra-organelle location and topology of proteins, such as
transmembrane regions and the location of positive residues
relative to transmembrane regions .
The cellular role of various ND proteins now needs to be
re-evaluated in light of their dual localisation. For instance
NDC1 gene expression is enhanced by light treatments 
but the protein is also known to be halved in abundance in
plastoglobules during high light treatment . So what im-
pact does this transcriptional light response have on the mito-
chondrial pool of NDC1 protein? Likewise, Western blot
analysis with potato mitochondria revealed changes in
NDA protein in a diurnal manner , and it is now unclear
how much of this may be attributed to a mitochondrial func-
tion as opposed to a peroxisomal function, or differential
contamination of mitochondria with peroxisomes. Further,
loss of or over-expression of potato NDB1 has been shown
to alter NADPH/NADH ratio in cells , but this may be
related to its activity in peroxisomes rather than mitochon-
Seven genes encode alternative ND proteins in Arabidopsis,
two NDA like proteins, four NDB type proteins and a single
NDC type protein, the latter proposed to be derived from
the cyanobacterial ancestor that gave rise to the plastid endo-
symbiosis . It is tempting to speculate from the prediction
of targeting ability of these proteins from a variety of plants
(Fig. 1, Supplementary Table 1) that genes encoding single
NDA and NDB type proteins underwent duplication followed
by acquisition of additional targeting signals by some proteins.
In the case of NDC it may have acquired dual targeting ability
upon transfer of the gene from the organelle to the nucleus, or
alternatively a location specific signal subsequently acquired
dual targeting ability over time.
Acknowledgements: This work was supported by an Australian
Research Council Grant DP0664692, ARC Australian Postdoctoral
Fellowships to M.W.M. and H.E., and an ARC Australian
Professorial Fellowship to A.H.M.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.febslet.2008.
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079
References Download full-text
 Buchanan, B., Gruissem, W. and Jones, R.L. (2002) Biochemistry
and Molecular Biology of Plants, John Wiley & Sons.
 Creissen, G., Reynolds, H., Xue, Y. and Mullineaux, P. (1995)
Simultaneous targeting of pea glutathione reductase and of a
bacterial fusion protein to chloroplasts and mitochondria in
transgenic tobacco. Plant J. 8, 167–175.
 Millar, A.H., Whelan, J. and Small, I. (2006) Recent surprises in
protein targeting to mitochondria and plastids. Curr. Opin. Plant
Biol. 9, 610–615.
 Koroleva, O.A., Tomlinson, M.L., Leader, D., Shaw, P. and
Doonan, J.H. (2005) High-throughput protein localization in
Arabidopsis using Agrobacterium-mediated transient expression
of GFP-ORF fusions. Plant J. 41, 162–174.
 Bhushan, S., Pavlov, P.F., Rudhe, C. and Glaser, E. (2007) In
vitro and in vivo methods to study protein import into plant
mitochondria. Meth. Mol. Biol. 390, 131–150.
 Duchene, A.M. et al. (2005) Dual targeting is the rule for
organellar aminoacyl-tRNA synthetases in Arabidopsis thaliana.
Proc. Natl. Acad. Sci. USA 102, 16484–16489.
 Mackenzie, S.A. (2005) Plant organellar protein targeting: a
traffic plan still under construction. Trends Cell Biol. 15, 548–554.
 Silva-Filho, M.C. (2003) One ticket for multiple destinations: dual
targeting of proteins to distinct subcellular locations. Curr. Opin.
Plant Biol. 6, 589–595.
 Dabney-Smith, C., van Den Wijngaard, P.W., Treece, Y.,
Vredenberg, W.J. and Bruce, B.D. (1999) The C terminus of a
chloroplast precursor modulates its interaction with the translo-
cation apparatus and PIRAC. J. Biol. Chem. 274, 32351–32359.
 Pfanner, N., Muller, H.K., Harmey, M.A. and Neupert, W.
(1987) Mitochondrial protein import: involvement of the mature
part of a cleavable precursor protein in the binding to receptor
sites. EMBO J. 6, 3449–3454.
 Chew, O., Rudhe, C., Glaser, E. and Whelan, J. (2003) Charac-
terization of the targeting signal of dual-targeted pea glutathione
reductase. Plant Mol. Biol. 53, 341–356.
 von Braun, S.S., Sabetti, A., Hanic-Joyce, P.J., Gu, J., Schleiff, E.
and Joyce, P.B. (2007) Dual targeting of the tRNA nucleotidyl-
transferase in plants: not just the signal. J. Exp. Bot. 58, 4083–
 Elhafez, D., Murcha, M.W., Clifton, R., Soole, K.L., Day, D.A.
and Whelan, J. (2006) Characterization of mitochondrial alter-
native NAD(P)H dehydrogenases in Arabidopsis: intraorganelle
location and expression. Plant Cell Physiol. 47, 43–54.
 Michalecka, A.M., Svensson, A.S., Johansson, F.I., Agius, S.C.,
Johanson, U., Brennicke, A., Binder, S. and Rasmusson, A.G.
(2003) Arabidopsis genes encoding mitochondrial type II
NAD(P)H dehydrogenases have different evolutionary origin
and show distinct responses to light. Plant Physiol. 133, 642–652.
 Rasmusson, A.G., Soole, K.L. and Elthon, T.E. (2004) Alterna-
tive NAD(P)H dehydrogenases of plant mitochondria. Annu.
Rev. Plant Biol. 55, 23–39.
 Escobar, M.A., Franklin, K.A., Svensson, A.S., Salter, M.G.,
Whitelam, G.C. and Rasmusson, A.G. (2004) Light regulation of
the Arabidopsis respiratory chain. Multiple discrete photorecep-
tor responses contribute to induction of type II NAD(P)H
dehydrogenase genes. Plant Physiol. 136, 2710–2721.
 Rasmusson, A.G. and Agius, S.C. (2001) Rotenone-insensitive
NAD(P)H dehydrogenases in plants: immunodetection and dis-
tribution of native proteins in mitochondria. Plant Physiol.
Biochem. 39, 1057–1066.
 Svensson, A.S. and Rasmusson, A.G. (2001) Light-dependent
gene expression for proteins in the respiratory chain of potato
leaves. Plant J. 28, 73–82.
 Geisler, D.A., Johansson, F.I., Svensson, A.S. and Rasmusson,
A.G. (2004) Antimycin A treatment decreases respiratory internal
rotenone-insensitive NADH oxidation capacity in potato leaves.
BMC Plant Biol. 4, 8.
 Geisler, D.A., Broselid, C., Hederstedt, L. and Rasmusson, A.G.
(2007) Ca2+-binding and Ca2+-independent respiratory NADH
and NADPH dehydrogenases of Arabidopsis thaliana. J. Biol.
Chem. 282, 28455–28464.
 Reumann, S., Ma, C., Lemke, S. and Babujee, L. (2004)
AraPerox. A database of putative Arabidopsis proteins from
plant peroxisomes. Plant Physiol. 136, 2587–2608.
 Carrie, C., Murcha, M.W., Millar, A.H., Smith, S.M. and
Whelan, J. (2007) Nine 3-ketoacyl-CoA thiolases (KATs) and
acetoacetyl-CoA thiolases (ACATs) encoded by five genes in
Arabidopsis thaliana are targeted either to peroxisomes or cytosol
but not to mitochondria. Plant Mol. Biol. 63, 97–108.
 Murcha, M.W. et al. (2007) Characterization of the preprotein
and amino acid transporter gene family in Arabidopsis. Plant
Physiol. 143, 199–212.
 Pracharoenwattana, I., Cornah, J.E. and Smith, S.M. (2005)
Arabidopsis peroxisomal citrate synthase is required for fatty acid
respiration and seed germination. Plant Cell 17, 2037–2048.
 Thirkettle-Watts, D., McCabe, T.C., Clifton, R., Moore, C.,
Finnegan, P.M., Day, D.A. and Whelan, J. (2003) Analysis of the
alternative oxidase promoters from soybean. Plant Physiol. 133,
 Eubel, H., Lee, C.P., Kuo, J., Meyer, E.H., Taylor, N.L. and
Millar, A.H. (2007) Free-flow electrophoresis for purification of
plant mitochondria by surface charge. Plant J. 52, 583–594.
 Murcha, M.W., Elhafez, D., Millar, A.H. and Whelan, J. (2005)
The C-terminal region of TIM17 links the outer and inner
mitochondrial membranes in Arabidopsis and is essential for
protein import. J. Biol. Chem. 280, 16476–16483.
 Reumann, S. et al. (2007) Proteome analysis of Arabidopsis leaf
peroxisomes reveals novel targeting peptides, metabolic pathways,
and defense mechanisms. Plant Cell 19, 3170–3193.
 Seedorf, M. and Soll, J. (1995) Copper chloride, an inhibitor of
protein import into chloroplasts. FEBS Lett. 367, 19–22.
 Footitt, S., Cornah, J.E., Pracharoenwattana, I., Bryce, J.H. and
Smith, S.M. (2007) The Arabidopsis 3-ketoacyl-CoA thiolase-2
(kat2-1) mutant exhibits increased flowering but reduced repro-
ductive success. J. Exp. Bot. 58, 2959–2968.
 Chew, O. and Whelan, J. (2003) Dual targeting ability of targeting
signals is dependent on the nature of the mature protein. Funct.
Plant Biol. 30, 805–812.
 Helm, M. et al. (2007) Dual specificities of the glyoxysomal/
peroxisomal processing protease Deg15 in higher plants. Proc.
Natl. Acad. Sci. USA 104, 11501–11506.
 Glaser, E. and Dessi, P. (1999) Integration of the mitochondrial-
processing peptidase into the cytochrome bc1 complex in plants. J.
Bioenerg. Biomembr. 31, 259–274.
 Stojanovski, D., Koutsopoulos, O.S., Okamoto, K. and Ryan,
M.T. (2004) Levels of human Fis1 at the mitochondrial outer
membrane regulate mitochondrial morphology. J. Cell Sci. 117,
 Lister, R., Carrie, C., Duncan, O., Ho, L.H., Howell, K.A.,
Murcha, M.W. and Whelan, J. (2007) Functional definition of
outer membrane proteins involved in preprotein import into
mitochondria. Plant Cell 19, 3739–3759.
 Neupert, W. and Herrmann, J.M. (2007) Translocation of
proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749.
 Ytterberg, A.J., Peltier, J.B. and van Wijk, K.J. (2006) Protein
profiling of plastoglobules in chloroplasts and chromoplasts A
surprising site for differential accumulation of metabolic enzymes.
Plant Physiol. 140, 984–997.
 Liu, Y.J., Norberg, F.E., Szilagyi, A., De Paepe, R., Akerlund,
H.E. and Rasmusson, A.G. (2008) The mitochondrial external
NADPH dehydrogenase modulates the leaf NADPH/NADP+
ratio in transgenic Nicotiana sylvestris. Plant Cell Physiol. 49,
 Rensing, S.A. et al. (2008) The Physcomitrella genome reveals
evolutionary insights into the conquest of land by plants. Science
C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079