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 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. 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- scriptional or post-transcriptional 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). Dual targeting of proteins C. Carrie et al. 1188 FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 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 C. Carrie et al. Dual targeting of proteins FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS 1189 supported 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 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- 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,inArabidopsis, 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 Dual targeting of proteins C. Carrie et al. 1190 FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS contain the sequence motif for phosphorylation by a cytosolic protein kinase that acts as a guidance com- plex to the Toc complex 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 proteins 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 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 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. Dual targeting of proteins C. Carrie et al. 1192 FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS cognate 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. 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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. C. Carrie et al. Dual targeting of proteins FEBS Journal 276 (2009) 1187–1195 ª 2009 The Authors Journal compilation ª 2009 FEBS 1195 . under- standing of the mechanism of dual targeting for the targeting of location-specific proteins is discussed. Targeting signals and mature proteins Targeting. MINIREVIEW Protein transport in organelles: Dual targeting of proteins to mitochondria and chloroplasts Chris Carrie, Estelle Giraud and James