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 target
Trang 1Protein 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 genefi 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 questions for inter-organelle communication A greater understanding of the process of dual targeting may provide useful insights into the targeting of location-specific proteins to mitochondria or chloroplasts
Keywords
chloroplast; dual targeting; inter-organelle
communication; mature protein;
mitochondria; processing; receptor;
regulation; sorting; targeting signal
Correspondence
J Whelan, Australian Research Council
Centre of Excellence in Plant Energy
Biology, University of Western Australia, 35
Stirling Highway, Crawley 6009, Australia
Fax: +61 8 6488 4401
Tel: +61 8 6488 1749
E-mail: seamus@cyllene.uwa.edu.au
Website: http://www.plantenergy.uwa.
edu.au
(Received 13 August 2008, revised 19
November 2008, accepted 27 November
2008)
doi:10.1111/j.1742-4658.2009.06876.x
As many as fifty proteins have now been experimentally demonstrated to
be targeted to both mitochondria and plastids, a phenomenon referred to
as dual targeting Although the first reported case of dual targeting of a protein was reported in 1995, there is still little understanding of the mech-anism of dual targeting and any similarities or differences with respect to the targeting of location-specific proteins This minireview summarizes dual targeting in terms of signals, passenger proteins, receptors, regulation, why 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.
Trang 2Dual 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 inforinfor-mation 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-scriptional or post-trantran-scriptional events that produce
location-specific targeted proteins [16] This mechanism
of dual targeting will not be discussed further here [17,18] Instead, we provide an overview the signals, proteins, receptors, sorting of dual-targeted proteins and why dual targeting may occur Finally, an outline
of the future challenges in this field is provided, and the insights that may be gained from a greater under-standing of the mechanism of dual targeting for the targeting of location-specific proteins is discussed
Targeting signals and mature proteins Targeting signals
Analysis of dual targeting signals indicates that they are rather similar to plastidic and mitochondrial tar-geting signals, in that they are enriched in positively charged residues, and significantly deficient in acidic residues and glycine [19] However, there are no fea-tures detectable to date that could distinguish them as
a group from location-specific targeted proteins They appear to fall between mitochondrial and chloroplastic targeting signals in terms of arginine and serine con-tent (i.e not as high as in mitochondrial targeting signals) and may be slightly enriched in hydrophobic residues In yeast, proteins targeted to mitochondria
Fig 1 Overview of dual targeting of proteins to mitochondria and chloroplasts in plant cells A gene encoding a dual-targeted protein may: (1) produce two different mRNA molecules via alternative start site for transcription initiation or alternative splicing (blue arrows), where the two mRNA molecules encode location-specific proteins [16], or (2) produce a single mRNA molecule that gives rise to a protein that is dual targeted via an ambiguous targeting signal (black arrows).
Trang 3and 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 targettarget-ing 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 resiresi-dues
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
the standard AUG produces a protein that is targeted
to chloroplasts alone but, by alternative translation site
initiation, the addition of seven amino acids to the
N-terminus results in targeting to mitochondria and
chloroplasts This would represent an elegant
mecha-nism of dual targeting However, experimentally, it is
difficult to demonstrate that alternative translation is
taking place in vivo Analysis of the targeting ability of
DNA polymerase c2 in another study revealed that it
was dual targeted, but from a protein produced from
the standard AUG [26], and thus via an ambiguous
targeting signal rather than alternative translation
initi-ation site Analysis of targeting of DNA polymerase
c2 in different tissues or cell types indicated that the
amount of GFP fluorescence from chloroplasts was
greater than GFP fluorescence from mitochondria
using the AUG start construct [26] However, the
amount of GFP fluorescence from mitochondria using
the CUG start construct was greater in terms of it
being equal in intensity to GFP fluorescence from
plastids Thus, the dual targeting ability of protein
starting at the standard AUG codon may be
over-looked due to differential targeting to both organelles
In tobacco, both DNA polymerases are reported to
be dual targeted from a standard AUG codon [27], even though they also contain the same upstream ‘inframe’ CUG Thus, the addition of the seven amino acids may alter partitioning to allow dual targeting to be observed
As described below in detail, the passenger protein also has large affect on dual targeting, and different con-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]
Mature proteins Analysis of the role of the mature protein in dual target-ing in several studies reveals that it plays a crucial role
in this process, a facet that is often overlooked Pea GR can only support the targeting of GFP to plastids [31], although it can support the import of phosphinothricin acetyl transferase into both mitochondria and chloro-plasts [6] Assessing the targeting ability of three sequences that have dual targeting ability revealed that, with the pea GR targeting signal, a native mitochondrial passenger protein was only targeted to mitochondria and a native chloroplast passenger protein was only tar-geted to chloroplasts [31] By contrast, the dual target-ing signal of Arabidopsis asparaginyl-tRNA synthetase
Trang 4supported targeting to both locations with the same
pas-sengers [31] The properties of targeting of Arabidopsis
histidyl-tRNA synthetase was intermediate between
these two extremes, whereas the mitochondrial
passen-ger was only targeted to mitochondria and the
chloro-plast passenger protein was dual-targeted [31] Further
evidence that the mature protein plays a role in dual
targeting properties is seen with an alternative
NAD(P)H dehydrogenase (NDC1) GFP is only
tar-geted to mitochondria when the targeting signal of 83
amino acids is used [32], but GFP is targeted to both
mitochondria and chloroplasts when the full protein is
fused to GFP [33] It has also been demonstrated that,
for tRNA nucleotidyltransferase, the mature protein
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
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-brane of chloroplasts (Toc)64 and Toc159 system used
by proteins involved in photosynthesis [36] The
Toc159 family in Arabidopsis consists of four proteins:
Toc159, Toc132, Toc120 and Toc 90 [37] It is possible
that one of these members is specialized in the import
of dual-targeted proteins With references to the
vari-ous import pathways that exist for protein import into
mitochondria and plastids, no experimental studies have been carried out to determine the import and⁄ or sorting pathways used by dual-targeted proteins
In the case of mitochondrial receptors for dual-targeted proteins, it has been shown that a double knockout of the translocase at the outer mitochondrial membrane (Tom), tom20, in Arabidopsis, which still contained one functional Tom20 isoform, had higher rates of import for GR, whereas some mitochondrial precursor proteins were decreased [38] In the triple tom20 knockout mutant, which has severely reduced rates of protein import for several mitochondrial pre-cursor proteins, the import of GR was unaffected com-pared to the wild-type [38] This indicates that GR can utilize a different receptor compared to several mito-chondrial proteins imported via the general and carrier import pathways The same study also assessed the role of other outer membrane proteins on GR import Plant mitochondria contain an outer membrane pro-tein of 64 kDa (OM64) that displays more than 70% amino acid sequence identity with the chloroplast outer envelope receptor Toc64 [23] OM64 did not appear to play any specific role in the import of GR 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
Trang 5contain the sequence motif for phosphorylation by a
cytosolic protein kinase that acts as a guidance
com-plex to the Toc comcom-plex of chloroplasts (see Table S1)
Any regulation of the activity of this complex could
change the partitioning of dual-targeted proteins
between mitochondria and chloroplasts [40,41]
Muta-tion of this site in GR [21], and indeed in
chloroplast-specific targeted proteins [42], does not appear to affect
the amount or rate of import However, in vitro import
assays often employ animal-based translation lysates,
or, even in plant-based systems, translation mixtures
are prepared in advance and frozen In such in vitro
import systems with a single purified organelle, this
system of regulation may be by-passed or go
unde-tected Where else does the protein have to go?
Dual targeting is most commonly detected using
GFP tagged proteins Because the emphasis of many
studies has been to determine that the protein under
study is dual targeted, the reported data tend to show
cells with both mitochondria and chloroplasts that are
clearly visible In a more comprehensive study, we
analysed the fluorescence intensity of several
dual-tar-geted proteins and found that it differed considerably
[26] Thus, in Arabidopsis suspension cells, for some
dual-targeted proteins, targeting to chloroplasts was
most dominant, but the same constructs in onion
epi-dermal cells gave approximately equal GFP labelling
of both mitochondria and chloroplasts Furthermore,
one study reported that, for the dual-targeted protein
sigma factor 2B, targeting to one organelle is only
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 has dual targeting ability Thus, acquisition of dual targeting ability may be a pre-requisite or at least facil-itate the loss of organelle genes Therefore, dual target-ing of proteins may have been more widespread, occurring early in the evolutionarily history of plants Characterization of organelle RNA polymerases in Physcomitrella patens concluded that there is no dual-targeted isoform [45], in contrast to previous studies [46] Thus, it is unclear how widespread and conserved dual targeting is in plant evolution
An examination of proteins that are dual targeted reveals that they differ substantially in functional cate-gorization compared to a genome wide classification (Fig 2; see also Table S1) Dual-targeted proteins appear to be particularly enriched in the categories of cell cycle and DNA synthesis, and protein synthesis (Fig 2; see also Table S1) This may simply be due to the fact that a limited number of dual-targeted pro-teins are known because dual-targeted propro-teins also have significantly less proteins of uncharacterized fun-ction compared to the whole genome (Fig 2) Exami-nation of the expression patterns of dual-targeted proteins across several tissue types and developmental stages reveals that they are relatively static, displaying similar levels of expression across green and not green tissues and across developmental stages ranging from embryonic to senescence (see Fig S1) This suggests that they encode basic but essential functions required
in mitochondria and plastids An examination of the list of dual-targeted proteins also reveals that several 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
Trang 6capacity 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
mitochondria during photorespiration, which
com-prises the best known biochemical process that links
mitochondrial and chloroplast function [50] The fact
that GR and the associated enzymes in the ascorbate
glutathione cycle are dual targeted is not surprising given that mitochondria are the site of ascorbate syn-thesis, and that this cycle plays an important role in both organelles in maintaining cellular redox balance [51] Thus, taking the current list of dual-targeted pro-teins as a whole or at an individual level, the activities provide a direct link between organelle functions, which is not achieved by other communication path-ways, such as retrograde regulation [52,53]
Future challenges The understanding of the mechanism of dual targeting
is at a very early stage compared to that of location-specific proteins Defining the plastidic and mitochon-drial receptor(s) for dual-targeted proteins would represent a major landmark, in that a comparison with the binding of location-specific proteins to their
Fig 2 Functional categorization of proteins dual targeted to mitochondria and chloroplasts in Arabidopsis The number of proteins in each classification is expressed as a percentage of the total number of proteins in each set and compared to the functional classification of all pro-teins in Arabidopsis An asterisk (*) indicates a significant difference at a 99% confidence interval compared to the whole genome using a chi-squared test.
Trang 7cognate receptors may reveal what is required for dual
targeting, and also what is required to avoid
mis-sort-ing of proteins between mitochondria and chloroplasts
The structures of binding of targeting peptides to
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
1 Kazan K (2003) Alternative splicing and proteome
diversity in plants: the tip of the iceberg has just
emerged Trends Plant Sci 8, 468–471
2 Siuti N & Kelleher NL (2007) Decoding protein
modifi-cations using top-down mass spectrometry Nat
Meth-ods 4, 817–821
3 Witze ES, Old WM, Resing KA & Ahn NG (2007)
Mapping protein post-translational modifications with
mass spectrometry Nat Methods 4, 798–806
4 Nishikura K (2006) Editor meets silencer: crosstalk
between RNA editing and RNA interference Nat Rev
Mol Cell Biol 7, 919–931
5 Takenaka M, Verbitskiy D, van der Merwe JA,
Zehr-mann A & Brennicke A (2008) The process of RNA
editing in plant mitochondria Mitochondrion 8, 35–46
6 Creissen G, Reynolds H, Xue Y & Mullineaux P (1995) Simultaneous targeting of pea glutathione reductase and
of a bacterial fusion protein to chloroplasts and mito-chondria in transgenic tobacco Plant J 8, 167–175
7 Schwacke R, Fischer K, Ketelsen B, Krupinska K & Krause K (2007) Comparative survey of plastid and mitochondrial targeting properties of transcription fac-tors in Arabidopsis and rice Mol Genet Genomics 277, 631–646
8 Sapir-Mir M, Mett A, Belausov E, Tal-Meshulam S, Frydman A, Gidoni D & Eyal Y (2008) Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to per-oxisomes Plant Physiol 148, 1219–1228
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 (2008) Changing transcriptional initiation sites and alternative 5¢- and 3¢-splice site selection of the first intron deploys Arabidopsis protein isoaspartyl methyl-transferase2 variants to different subcellular compart-ments Plant J 55, 1–13
17 Millar AH, Whelan J & Small I (2006) Recent surprises
in protein targeting to mitochondria and plastids Curr Opin Plant Biol 9, 610–615
18 Silva-Filho MC (2003) One ticket for multiple destina-tions: dual targeting of proteins to distinct subcellular locations Curr Opin Plant Biol 6, 589–595
Trang 819 Pujol C, Marechal-Drouard L & Duchene AM (2007)
How can organellar protein N-terminal sequences be
dual targeting signals? In silico analysis and mutagenesis
approach J Mol Biol 369, 356–367
20 Dinur-Mills M, Tal M & Pines O (2008) Dual targeted
mitochondrial proteins are characterized by lower
MTS parameters and total net charge PLoS ONE 3,
e2161
21 Chew O, Rudhe C, Glaser E & Whelan J (2003)
Char-acterization of the targeting signal of dual-targeted pea
glutathione reductase Plant Mol Biol 53, 341–356
22 Rudhe C, Chew O, Whelan J & Glaser E (2002) A
novel in vitro system for simultaneous import of
pre-cursor proteins into mitochondria and chloroplasts
Plant J 30, 213–220
23 Rudhe C, Clifton R, Chew O, Zemam K, Richter S,
Lamppa G, Whelan J & Glaser E (2004) Processing of
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
30 Ueda M, Nishikawa T, Fujimoto M, Takanashi H,
Arimura SI, Tsutsumi N & Kadowaki KI (2008)
Substitution of the gene for chloroplast RPS16 was
assisted by generation of a dual targeting signal
Mol Biol Evol 25, 1566–1575
31 Chew O & Whelan J (2003) Dual targeting ability of
targeting signals is dependent on the nature of the
mature protein Funct Plant Biol 30, 805–812
32 Michalecka AM, Svensson AS, Johansson FI, Agius
SC, Johanson U, Brennicke A, Binder S & Rasmusson
AG (2003) Arabidopsis genes encoding mitochondrial type II NAD(P)H dehydrogenases have different evolu-tionary origin and show distinct responses to light Plant Physiol 133, 642–652
33 Carrie C, Murcha MW, Kuehn K, Duncan O, Barthet
M, Smith PM, Eubel H, Meyer E, Day DA, Millar AH
et al.(2008) Type II NAD(P)H dehydrogenases are targeted to mitochondria and chloroplasts or peroxisomes in Arabidopsis thaliana FEBS Lett 582, 3073–3079
34 von Braun SS, Sabetti A, Hanic-Joyce PJ, Gu J, Schleiff
E & Joyce PB (2007) Dual targeting of the tRNA nucleotidyltransferase in plants: not just the signal
J Exp Bot 58, 4083–4093
35 Glaser E, Sjoling S, Tanudji M & Whelan J (1998) Mitochondrial protein import in plants Signals, sorting, targeting, processing and regulation Plant Mol Biol 38, 311–338
36 Soll J & Schleiff E (2004) Protein import into chlorop-lasts Nat Rev Mol Cell Biol 5, 198–208
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 Arabidopsisphosphorylates 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-drial localization of the phage-type RNA polymerases
in the moss Physcomitrella patens Plant Physiol 138, 369–382
Trang 946 Richter U, Kiessling J, Hedtke B, Decker E, Reski R,
Borner T & Weihe A (2002) Two RpoT genes of
Physcomitrella patensencode phage-type RNA
polyme-rases with dual targeting to mitochondria and plastids
Gene 290, 95–105
47 Chew O, Whelan J & Millar AH (2003) Molecular
defi-nition of the ascorbate-glutathione cycle in Arabidopsis
mitochondria reveals dual targeting of antioxidant
defenses in plants J Biol Chem 278, 46869–46877
48 Smiraglia DJ, Kulawiec M, Bistulfi GL, Ghoshal S &
Singh KK (2008) A novel role for mitochondria in
regu-lating epigenetic modification in the nucleus Cancer
Biol Ther 7, 1182–1190
49 Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD,
Berry CC, Millar AH & Ecker JR (2008) Highly
inte-grated single-base resolution maps of the epigenome in
Arabidopsis Cell 133, 523–536
50 Linka M & Weber AP (2005) Shuffling ammonia
between mitochondria and plastids during
photorespira-tion Trends Plant Sci 10, 461–465
51 Noctor G, De Paepe R & Foyer CH (2007)
Mitochon-drial redox biology and homeostasis in plants Trends
Plant Sci 12, 125–134
52 Rhoads DM & Subbaiah CC (2007) Mitochondrial
ret-rograde regulation in plants Mitochondrion 7, 177–194
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 materials supplied by the authors Any queries (other than missing material) should be directed to the corre-sponding author for the article