MINIREVIEW
Protein transport in organelles: Dual targeting of proteins
to mitochondria and chloroplasts
Chris Carrie, Estelle Giraud and James Whelan
Australian Research Council Centre of Excellence in Plant Energy Biology, M316, University of Western Australia, Crawley, Australia
The traditional dogma of both cell and molecular
biology, one gene fi one protein fi one location, has
well passed its use-by date in postgenomic biology. It
is clear from the sequencing of several genomes that
the complexity of the proteome exceeds that of the
genome in terms of the number of functional units
(i.e. there are more proteins than genes). This protein
complexity is achieved by a number of means, of
which alternative splicing of genes and protein modifi-
cation are the best characterized to date [1–3].
Another mechanism to increase the complexity of
proteomes is the editing of transcripts (both in nuclear
and organelle genomes) [4,5]. Dual targeting of
proteins does not increase the number of proteins in a
cell, but can expand the function(s) of a protein, in
that a protein located in more than one location, will
presumably function with a distinct biochemical
process in each location. Although the number of
dual-targeted proteins is small in terms of the total
organelle proteomes, it is unclear whether this just
represents the tip of the iceberg. Irrespective of the
total number of dual-targeted proteins present in
mitochondria and chloroplasts (note that, for the
purpose of this minireview, dual targeted refers to
proteins targeted to mitochondria and chloroplasts),
the phenomenon of dual targeting raises interesting

proteins may need to be dual targeted and the future challenges that
remain in this area.
Abbreviations
GFP, green fluorescent protein; GR, glutathione reductase; MPP, mitochondrial processing peptidase; NDC1, type II alternative NAD(P)H
dehydrogenase; RPS16, 16 kDa proteins of the small ribosomal subunit of mitochondria or chloroplasts; SPP, stromal processing peptidase;
Toc, translocase at the outer envelope membrane of chloroplasts; Tom, translocase at the outer mitochondrial membrane.
FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS 1187
Dual targeting was first reported for Pisum sativum
(pea) glutathione reductase (GR) in 1995 [6], and, to
date, as many as 47 different proteins have been
reported to be dual-targeted from seven different plant
species (see Table S1). It is notable that there are also
reports of dual-targeted proteins to chloroplasts and
the nucleus [7], to chloroplasts and the peroxisome
[8,9], and, in Chlamydomonas reinhardtii, to chloro-
plasts and the endoplasmic reticulum [10]. However,
by far the greatest number of dual-targeted proteins
known are targeted to chloroplasts and mitochondria.
With the advent of complete genome sequence infor-
mation and the combined information emerging from
organelle proteome studies [11], green fluorescent pro-
tein (GFP) tagging studies [12], and bioinformatic pre-
diction of subcellular localization [13], the number of
dual-targeted proteins has increased in the last 5 years
such that they can be no longer be treated as an exce-
ption compared to location-specific proteins. Dual
targeting can be achieved via two basic mechanisms
[14,15]: alternative transcription initiation or splicing
and ambiguous targeting signals (Fig. 1). Alternative
transcriptional initiation or splicing represents tran-

and one other location have been reported to have a
lower mitochondrial targeting score using mitoprot
compared to exclusive mitochondrial proteins [20].
This is not observed with proteins dual targeted to
mitochondria and plastids, where the mitoprot score
for many is quite high.
Experimental analyses of dual targeting signals have
also failed to define clear facets that define dual target-
ing ability. The best studied dual targeting signal is
from pea GR [21–23]. Deletion and site-directed muta-
genesis studies reveal that although some regions may
be more important for targeting to one organelle, the
dual targeting signal is overlapping. This is consistent
with studies that have used tandem arrangements of
mitochondrial and chloroplastidic targeting signals and
shown that the passenger protein was targeted to the
location defined by the most N-terminal sequence [24].
In the case of GR, it was concluded that positive resi-
dues throughout the signal and hydrophobic residues
at the N-terminus were important for mitochondrial
import, whereas hydrophobic residues alone had the
greatest affect on chloroplast import [21]. The role of
arginine residues playing a more important role for
mitochondrial import was also observed for three
aminoacyl-tRNA synthetases [19].
It has been reported that Arabidopsis thaliana DNA
polymerase c2 is dual targeted via the use of a non-
AUG start codon (a CUG codon) in translation,
resulting in an additional seven amino acids at the
N-terminus of the protein [25]. Thus, translation from

structs may favour targeting to one organelle compared
to another, especially if tested in a single tissue.
In terms of processing, pea GR is the best studied to
date [23]. Based on mobility in gels, it was concluded
that the processing site was the same in both organelles.
It has been demonstrated that purified mitochondrial
processing peptidease (MPP) and stromal processing
peptidase (SPP) are responsible for processing GR [23].
The processing requirements for MPP appear to be
more stringent in that alterations near the processing
site have a greater inhibitory affect of MPP compared to
SPP. In the case of aminoacyl tRNA synthetases, Glu
aminoacyl-tRNA synthetase was processed at the same
site in both mitochondria and chloroplasts but for Met
and Phe aminoacyl-tRNA synthetases they have differ-
ent processing sites in mitochondria and chloroplasts
[19]. However, because the latter study was carried out
with GFP as a passenger protein, and processing was
not assessed by purified peptidases, processing by chlo-
roplasts may be due to a variety of processing activities
that have been detected in chloroplasts, or due to cryptic
processing sites that can be generated when targeting
signals are fused to reporters [28,29].
Dual targeting signals do not exclusively have to
comprise cleavable N-terminal signals. A protein pro-
duced from a gene encoding the small subunit of ribo-
somes in mitochondria and plastids (RPS16) was
found to be dual targeted in Medicago truncatula and
Populus alba without a cleavable N-terminal targeting
signal [30].

plays a major role in determining partitioning between
mitochondria and chloroplasts [34]. Thus, compared to
location-specific proteins, where many studies show that
the targeting signal is sufficient to support import [35],
albeit the mature protein may affect the efficiency, the
effect of the mature protein on targeting appears to be
more pronounced in the case of dual-targeted proteins.
This likely reflects the fact that dual-targeted proteins
have evolved from proteins that were targeted to a
specific location [30]. Thus, the acquisition of the dual
targeting signal would be a constraint compared to tar-
geting of location-specific proteins to avoid loss of tar-
geting to the ‘parental’ organelle (i.e. the ambiguous
dual targeting signal is a compromise and dual targeting
ability is dependent on the passenger protein). Note
that, with dual targeting signals, different passenger
proteins affect dual targeting ability, but do not appear
to block targeting to both organelles simultaneously.
Usually, targeting to one organelle is maintained. This
is evident with GR and NDC1, when the targeting sig-
nal alone is fused to GFP, dual targeting ability is lost,
although targeting to either plastids only (GR) or mito-
chondria only (NDC1) is maintained [31,32].
Organelle receptors
Unfortunately, little is known about the organelle
receptors that recognize dual-targeted proteins. How-
ever, because the dual-targeted proteins identified to
date would be required in various types of plastids, it
suggests that they may employ different receptors com-
pared to the translocase at the outer envelope mem-

compared to mitochondrial precursor proteins. One
protein that was identified to play a role in the import
of GR into mitochondria was metaxin [38]. This mito-
chondrial outer membrane protein was first identified
in mammals, and was subsequently shown to be part
of the sorting and assembly machinery complex in
yeast (SAM), called Sam35 (also called Tom34 or
Tob35) [39]. Metaxin knockouts have severe affects on
the protein import of all proteins tested in Arabidopsis,
presumably acting indirectly because it plays a role in
import and ⁄ or assembly of b-barrel proteins into the
outer mitochondrial membrane [38,39]. However, using
an alternative method to assess a role in import, the
addition of in vitro synthesized metaxin to import reac-
tion mixtures can compete for the import of GR into
mitochondria, and some but not all other mitochon-
drial proteins tested, suggesting that it plays some role
in import of GR on the cytosolic surface of the outer
membrane. Notably, metaxin was also up-regulated in
abundance in the double and triple tom20 knockout
mutants, where import of mitochondrial precursor
proteins was affected but GR was not [38].
Sorting and regulation
There is no direct experimental evidence demonstrating
that dual-targeted proteins are actively sorted or that
sorting between organelles is regulated. However, there
are several observations that suggest sorting is not
simply a passive process. Most dual-targeted proteins
Dual targeting of proteins C. Carrie et al.
1190 FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS

observed in any one cell [43]. Another study reported
that the GFP fluorescence intensity differs between
experiments with dual-targeted proteins [19]. Such
reports are likely to increase in the future.
One interesting opportunity for regulation of parti-
tioning dual-targeted proteins is the possibility that
mRNA for dual-targeted proteins is targeted to the
organelle surface [23]. Regulation of targeting of
mRNA could result in changes in partitioning. As yet,
there is no evidence for mRNA targeting to mitochon-
dria or plastids, even for mRNA encoding location-
specific proteins.
Why dual target proteins?
Mitochondria and chloroplasts share many common
enzymatic steps that are catalysed by location-specific
proteins [44]. Thus, it is unclear why some and, at this
stage, a relatively small number of activities are carried
out by dual-targeted proteins. Furthermore, in many
cases where a dual-targeted protein exists, location-
specific isoforms also exist. Thus, dual targeting does
not appear to be a strategy of limiting gene number in
the nuclear genome. As outlined previously, dual tar-
geting of proteins appears to have arose before the
monocot ⁄ dicot split [30], and is present in several plant
species (see Table S1). The RPS16 protein gives an
interesting insight into the evolutionarily history of
dual targeting. In both Arabidopsis and Oryza sativa
(rice), the chloroplast genome contains a functional
gene encoding this protein; however, the nuclear
located gene that encodes the mitochondrial protein

steps in a biochemical process may be dual targeted
(e.g. the process of organelle gene expression, proteins
involved in DNA replication, transcription and trans-
lation are dual targeted, and for the ascorbate glutathi-
one cycle, several enzymes are dual targeted) [47]. For
both these processes, location-specific isoforms also
exist for many steps.
The reason for dual targeting a protein may com-
prise a means of inter-organelle communication. Send-
ing the same proteins to both organelles at the same
time ensures that they are both at least capable of
carrying out these functions in a co-ordinated manner.
Organelle genome replication and number may
have also have roles beyond their immediate coding
C. Carrie et al. Dual targeting of proteins
FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS 1191
capacity. In human cancers, the depletion of mitochon-
drial DNA is associated with altered methylation pat-
terns in the nucleus, and restoration of mitochondrial
DNA reverses these changes [48]. Given that epigenetic
regulation can have widespread affects beyond specific
organelle functions [49], in plant cells that contain two
organelles with their own genomes, it may be necessary
at times to co-ordinate the replication and ⁄ or expres-
sion of both organelle genomes.
At the level of the individual functions encoded by
dual-targeted proteins, it is likely that the activities
encoded are required in both organelles at the same
time. Thus, dual-targeted glutamine synthetase plays a
role in assimilating ammonia that is produced in the

mammalian and plant Tom20s not only revealed the
molecular details of binding [54,55], but also prompted
the hypothesis of an elegant example of convergent
evolution [54,56] because plant and mammalian
Tom20s are not orthologous. One of the most compel-
ling questions concerning dual targeting is how the
proteins are partitioned between both organelles?
Thus, a better understanding of the role of any cyto-
solic factors involved, and whether they play any regu-
latory role, would explain how each organelle obtains
the appropriate amount of protein. An understanding
of why dual targeting occurs will require the evolution-
arily history of dual targeting to be determined in
more detail in terms of when it arose and whether it is
conserved. A complete understanding of dual targeting
also requires an understanding of why it occurs. This
is probably best achieved by converting dual-targeted
proteins to location-specific isoforms and assessing
organelle function. For dual-targeted proteins that
have location-specific isoforms, it is not clear whether
the dual-targeted isoform has taken on new functions
(neofunctionalization) or whether expression is special-
ized (subfunctionalization). Promoter swapping studies
between dual and location-specific isoforms may also
be informative for assessing the function of dual-
targeted proteins.
Acknowledgement
Work on dual targeting by J.W. is supported by an
Australian Research Council grant DP0664692.
References

9 Reumann S, Babujee L, Ma C, Wienkoop S, Siemsen
T, Antonicelli GE, Rasche N, Lu
¨
der F, Weckwerth W
& Jahn O (2007) Proteome analysis of Arabidopsis leaf
peroxisomes reveals novel targeting peptides, metabolic
pathways, and defense mechanisms. Plant Cell 19,
3170–3193.
10 Levitan A, Trebitsh T, Kiss V, Pereg Y, Dangoor I &
Danon A (2005) Dual targeting of the protein disulfide
isomerase RB60 to the chloroplast and the endoplasmic
reticulum. Proc Natl Acad Sci USA 102, 6225–6230.
11 Heazlewood JL, Verboom RE, Tonti-Filippini J, Small
I & Millar AH (2007) SUBA: the Arabidopsis subcellu-
lar database. Nucleic Acids Res 35, D213–D218.
12 Koroleva OA, Tomlinson ML, Leader D, Shaw P &
Doonan JH (2005) High-throughput protein localization
in Arabidopsis using Agrobacterium-mediated transient
expression of GFP-ORF fusions. Plant J 41, 162–174.
13 Cho SH, Chung YS, Cho SK, Rim YW & Shin JS
(1999) Particle bombardment mediated transformation
and GFP expression in the moss Physcomitrella patens.
Mol Cells 9, 14–19.
14 Karniely S & Pines O (2005) Single translation–dual
destination: mechanisms of dual protein targeting in
eukaryotes. EMBO Rep 6, 420–425.
15 Peeters N & Small I (2001) Dual targeting to mitochon-
dria and chloroplasts. Biochim Biophys Acta 1541, 54–63.
16 Dinkins RD, Majee SM, Nayak NR, Martin D, Xu Q,
Belcastro MP, Houtz RL, Beach CM & Downie AB

the dual targeted precursor protein of glutathione
reductase in mitochondria and chloroplasts. J Mol Biol
343, 639–647.
24 de Castro Silva Filho M, Chaumont F, Leterme S &
Boutry M (1996) Mitochondrial and chloroplast target-
ing sequences in tandem modify protein import specific-
ity in plant organelles. Plant Mol Biol 30, 769–780.
25 Christensen AC, Lyznik A, Mohammed S, Elowsky
CG, Elo A, Yule R & Mackenzie SA (2005) Dual-
domain, dual-targeting organellar protein presequences
in Arabidopsis can use non-AUG start codons. Plant
Cell 17, 2805–2816.
26 Carrie C, Kuhn K, Murch M, Duncan O, Small I,
O’Toole N & Whelan J (2008) Approaches to defining
dual targeted protein in Arabidopsis. Plant J, doi:
10.1111/j.1365-313X.2008.03745.x.
27 Ono Y, Sakai A, Takechi K, Takio S, Takusagawa M
& Takano H (2007) NtPolI-like1 and NtPolI-like2, bac-
terial DNA polymerase I homologs isolated from BY-2
cultured tobacco cells, encode DNA polymerases
engaged in DNA replication in both plastids and mito-
chondria. Plant Cell Physiol 48, 1679–1692.
28 Duby G, Oufattole M & Boutry M (2001) Hydrophobic
residues within the predicted N-terminal amphiphilic
alpha-helix of a plant mitochondrial targeting prese-
quence play a major role in in vivo import. Plant J 27,
539–549.
29 Musgrove BT & Malden NJ (1989) Mediastinitis and
pericarditis caused by dental infection. Br J Oral Max-
illofac Surg 27, 423–428.

37 Jarvis P (2008) Targeting of nucleus-encoded proteins
to chloroplasts in plants. New Phytol 179, 257–285.
38 Lister R, Carrie C, Duncan O, Ho LH, Howell KA,
Murcha MW & Whelan J (2007) Functional definition
of outer membrane proteins involved in preprotein
import into mitochondria. Plant Cell 19, 3739–3759.
39 Neupert W & Herrmann JM (2007) Translocation of
proteins into mitochondria. Annu Rev Biochem 76,
723–749.
40 Martin T, Sharma R, Sippel C, Waegemann K, Soll J
& Vothknecht UC (2006) A protein kinase family in
Arabidopsis phosphorylates chloroplast precursor
proteins. J Biol Chem 281, 40216–40223.
41 Waegemann K & Soll J (1996) Phosphorylation of the
transit sequence of chloroplast precursor proteins.
J Biol Chem 271, 6545–6554.
42 Nakrieko KA, Mould RM & Smith AG (2004) Fidelity
of targeting to chloroplasts is not affected by removal
of the phosphorylation site from the transit peptide.
Eur J Biochem 271, 509–516.
43 Beardslee TA, Roy-Chowdhury S, Jaiswal P, Buhot L,
Lerbs-Mache S, Stern DB & Allison LA (2002) A
nucleus-encoded maize protein with sigma factor activ-
ity accumulates in mitochondria and chloroplasts. Plant
J 31, 199–209.
44 Buchanan B, Gruissem W & Jones RL (2002) Biochem-
istry and Molecular Biology of Plants. American Society
of Plant Physiologists, Rockville, MD.
45 Kabeya Y & Sato N (2005) Unique translation initia-
tion at the second AUG codon determines mitochon-

53 Woodson JD & Chory J (2008) Coordination of gene
expression between organellar and nuclear genomes.
Nat Rev Genet 9, 383–395.
54 Perry AJ, Hulett JM, Likic VA, Lithgow T & Gooley
PR (2006) Convergent evolution of receptors for protein
import into mitochondria. Curr Biol 16, 221–229.
55 Saitoh T, Igura M, Obita T, Ose T, Kojima R, Mae-
naka K, Endo T & Kohda D (2007) Tom20 recognizes
mitochondrial presequences through dynamic equilib-
rium among multiple bound states. EMBO J 26, 4777–
4787.
56 Lister R & Whelan J (2006) Mitochondrial protein
import: convergent solutions for receptor structure.
Curr Biol 16, R197–R199.
Supporting information
The following supplementary material is available:
Fig. S1. Relative transcript abundance of genes encod-
ing proteins dual targeted to mitochondria and plastids
in Arabidopsis.
Table S1. Overview of proteins dual targeted to mito-
chondria and plastids in plants.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
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than missing material) should be directed to the corre-
sponding author for the article.
C. Carrie et al. Dual targeting of proteins
FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS 1195