* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Plant mitochondria contain the protein translocase subunits TatB
Survey
Document related concepts
Hedgehog signaling pathway wikipedia , lookup
Cytokinesis wikipedia , lookup
Protein (nutrient) wikipedia , lookup
Cell membrane wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Protein phosphorylation wikipedia , lookup
Signal transduction wikipedia , lookup
Protein moonlighting wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Endomembrane system wikipedia , lookup
Magnesium transporter wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Proteolysis wikipedia , lookup
Transcript
© 2016. Published by The Company of Biologists Ltd. Plant mitochondria contain the protein translocase subunits TatB and TatC Chris Carrie1, Stefan Weißenberger1 and Jürgen Soll1,2 1 Department of Biology I, Botany, Ludwig-Maximilians-Universität München, Großhaderner Strasse 2-4, D-82152, Planegg-Martinsried, Germany, 2Munich Center for Integrated Protein Science, CiPSM, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D81377, Munich, Germany To whom correspondence should be addressed: Dr. Chris Carrie, Department of Biology I, Botany, Ludwig-Maximilians-Universität München, Großhaderner Strasse 2-4, D-82152, Planegg-Martinsried, Germany. Phone: +49 89 2180 74761. Fax: +49 89 2180 74752 E-mail: [email protected] Summary statement This paper provides evidence that plant mitochondria contain two subunits of a twin arginine Journal of Cell Science • Advance article translocation pathway indicating the presence of an active Tat pathway. JCS Advance Online Article. Posted on 8 September 2016 Abstract Twin-arginine translocation pathways have been well characterized in bacteria and chloroplasts. However, genes encoding for a TatC protein are found in almost all plant mitochondrial genomes. For the first time it could be demonstrated that this mitochondrial encoded TatC is a functional gene which is translated into a protein in the model plant Arabidopsis thaliana. An inner membrane localized TatB like subunit was also identified, which is nuclear encoded, and is essential for plant growth and development. Indicating that plants potentially require a Tat pathway for mitochondrial biogenesis. Keywords: mitochondria, protein translocation, molecular evolution, protein import, plant Journal of Cell Science • Advance article molecular biology Introduction The twin-arginine translocation (Tat) pathway differs from most other protein translocating systems in that it transports fully folded proteins (Lee et al., 2006). It is named after its targeting signal which contains a pair of adjacent arginine residues (the twin arginines). Identified in all domains of life Tat pathways play essential roles in a number of different cellular processes including: bacterial pathogen virulence, cell separation, phosphate and iron metabolism, photosynthetic and respiratory metabolism (Berks, 2015, Palmer and Berks, 2012). The minimal Tat pathway which is found mostly in archaea and gram-positive bacteria consists of two subunits: TatA (possessing one transmembrane helix (TMH)) and TatC (possessing six TMHs) (Barnett et al., 2008). In general, most Tat systems, including the best studied Escherichia coli Tat system, contain a second functionally distinct member of the TatA family called TatB (Sargent et al., 1999). Outside of prokaryotes a wellestablished Tat pathway has also been identified and characterized inside chloroplasts (Robinson and Klosgen, 1994, Cline and Henry, 1996). It is essential for the assembly of photosystem II and the cytochrome b6f complex in thylakoid membranes (Molik et al., 2001). Genes for Tat pathway components have also been identified in a number of mitochondrial genomes. Including jakobids, whose mitochondria encode for a minimal Tat system of TatA and C (Jacob et al., 2004). TatC like genes are also found in the mitochondrial genomes of higher plants (Unseld et al., 1997). Despite the conservation of Tat pathways across phyla, it was thought to have been completely lost from all opisthokont lineages which includes Fungi, Metazoa, and relatives. So far only two known exceptions have been described, the choanoflagellate Monosiga brevicollis, (Burger et al., 2003) which is interesting as it is homoscleromorph sponges Oscarellidae, (Wang and Lavrov, 2007) both of which have a mitochondrially encoded TatC. However, since most mitochondrial genomes only encode a TatC like protein, no function has ever been attributed to these proteins. Here we present data that demonstrate that in Arabidopsis thaliana the mitochondrial encoded TatC is not a pseudogene. It is also demonstrated that Arabidopsis mitochondria contain a second member of a Tat pathway, a TatB like protein which is an essential gene. This mitochondrial TatB like protein has been long sought after and strengthens the evidence that plant mitochondria contain a Tat pathway in contrast to mitochondria from other eukaryotic lineages. Journal of Cell Science • Advance article thought to be one of the oldest relatives to animals, and the other is a member of the Results Plant mitochondria contain a TatC protein It has been known for more than 20 years that the Arabidopsis thaliana mitochondrial genome contains a gene encoding for a TatC like protein (although it is also known as either orfX or Mttb) (Sunkel et al., 1994, Unseld et al., 1997). However, no functional data has ever been ascribed to it. One of the main reasons for this is that the mtTatC (mitochondrially encoded TatC) has no classical start codon in Arabidopsis and has been thought of as a pseudogene. There are however several pieces of evidence to suggest that mtTatC is a funtional gene that encodes a protein. The mtTatC transcript in Arabidopsis is edited at 36 individual sites (Bentolila et al., 2013). There are also several studies demonstrating that the Arabidopsis mtTatC is an expressed gene (van der Merwe and Dubery, 2007, GutierrezMarcos et al., 2007, Sunkel et al., 1994, Wang et al., 2012). To gather further evidence that mtTatC is a functional gene we searched every plant mitochondrial genome in the NCBI organelle genomes database (http://www.ncbi.nlm.nih.gov/genome/organelle/) for the presence or absence of a TatC like gene. In all we searched 124 plant mitochondrial genomes which are displayed phylogenetically in Figure 1A. Out of 124 plant mitochondrial genomes 102 of them contain a TatC gene (Figure 1A) of which 90 contain classical start codons (Figure 1A). These 102 mitochondrial genomes contained all sequenced higher plant species (Figure 1A). The only major difference was observed in the Chlorophyte lineages (green algae) which is split in two. One half containing mainly the class Chlorophyceae and having no TatC gene and the other half containing mainly the class Trebouxiophyceae which contain a TatC gene (Figure 1A). What can be determined is that the majority of plant mitochondrial genomes contain a TatC gene with a classical start codon. We believe that this conservation like almost all animal, fungi and Chlorophyceae mitochondrial genomes it would have been lost. Therefore, we first aimed to prove that the Arabidopsis mtTatC gene is translated into a protein. To this end we raised an antibody against the peptide VREEGWTSGMRESGIEKKNKSSPPPRTW which corresponds to amino acids 253 to 281 of the Arabidopsis mtTatC. The collected whole serum was then affinity purified against the peptide to obtain a purified antibody. This antibody detected a band of approximately 27 kDa in isolated Arabidopsis mitochondria that was not recognized by the preimmune serum (Figure 1B). To confirm the specificity of the antibody we pre-incubated it with the peptide which abrogated binding of the 27 kDa protein (Figure 1B). While the protein size of 27 kDa Journal of Cell Science • Advance article of a mitochondrial TatC must mean it has some function within plant mitochondria otherwise is slightly lower than the predicted size of 33 kDa it is a well-known phenomenon that TatC and other hydrophobic proteins run at different molecular masses than predicted (Jakob et al., 2009, Mori et al., 2001). From these results it can be concluded that the antibody is specific for the Arabidopsis mtTatC and that it is in fact a functional gene. The Arabidopsis genome encodes a second TatB like protein Since all known Tat pathways contain at least TatC and TatA but the majority contain TatA, TatB and TatC subunits, for plant mitochondria to have a functional Tat pathway other subunits are required. To rectify this situation, we sought to identify any possible TatA or TatB encoding genes in the Arabidopsis genome which may be targeted to mitochondria. We first excluded the chloroplast targeted cpTatA and cpTatB as being dual targeted to mitochondria, by using antibodies to the Pisum sativum (pea) chloroplast TatA (Mori et al., 1999) and TatB (Mori et al., 2001) proteins on isolated chloroplastic and mitochondrial fractions from pea (Figure 1C). As expected cpTatA and cpTatB were only detected in the chloroplastic fractions (Figure 1C). BLAST (Altschul et al., 1990) searches of the Arabidopsis genome using either E. coli TatA or TatB sequences, the Arabidopsis chloroplastic targeted TatA or B sequences, or the mitochondrial encoded sequences of TatA from jakobids were also performed. The only TatA or TatB like sequences recovered by this approach were the known chloroplast Tat subunits. Therefore, we tried another approach and used the program Phyre2 in the back phyre mode (Kelley et al., 2015). This utilizes the known structures of proteins to search genomes for similar proteins. So we used the known structures of the E. coli TatA (PDB: 2NM7) (Zhang et al., 2014b) and TatB (2MI2) (Zhang et al., 2014c) to search the Arabidopsis proteins were identified with high confidence scores (Phyre2 confidence scores are a representation of the probability that the match between the sequence and the template is a true homology) a third unknown protein was also identified with similar confidence scores with the locus At5g43680 (Supplementary Table 1). In fact, At5g43680 came back as the highest ranked protein when using the structure for EcTatB as the search reference. Using the protein sequence of At5g43680 we performed BLAST searches of higher plant genomes. In all cases a protein similar to At5g43680 could be identified. To try and confirm that what we had identified was a TatB or TatA subunit, phylogenetic analysis was utilized using TatA and TatB subunits from a variety of bacterial and jakobid species along with the chloroplastic TatA and TatB subunits from plant species (Figure 2). We observed Journal of Cell Science • Advance article nuclear genome (Supplementary Table 1). While both the known cpTatA and cpTatB that the At5g43680 and related proteins grouped most closely with the TatB proteins from bacteria indicating that At5g43680 is a possible TatB like protein (Figure 2). Although some of the bootstrap values are low we believe the tree is accurate. We also tested Bayesian homology using MrBayes (Ronquist et al., 2012) and an almost identical tree was obtained (data not shown). Secondly when BLAST searches excluding plant species with At5g43680 were carried out the only hits were bacterial TatB proteins (data not shown). Therefore, we believe that At5g43680 is a TatB like protein. This is also supported by basic sequence comparisons as At5g43680 shows a 14% identity and 29% similarity to EcTatB in comparison to 10% identity and 19% similarity to EcTatA. Furthermore, a sequence alignment between EcTatB and At5g43680 showed that it contains the conserved glutamate residue at amino acid position 8 at the start of the transmembrane helix (TMH) and also the invariant glycine between the TMH and the second alpha helix (APH) (Figure 3A). One major difference however is the length, At5g43680 is 61 amino acids longer that EcTatB. This difference is found predominantly in the C-terminus. To try and get a better understanding of the possible function of At5g43680 we built a model of its 3D structure. This was carried out using the Phyre2 in the intensive mode which returned a model fitting the identified structure of EcTatB (Figure 3B) (Zhang et al., 2014c, Kelley et al., 2015). The model is based on the first 104 amino acids of EcTatB and the first 125 amino acids of At5g43680 as the C-termini of both proteins are thought to be unstructured. EcTatB contains four alpha helices which adopt an L-shape conformation (Figure 3B) (Zhang et al., 2014c). The model we obtained for At5g43680 has an almost identical structure containing four alpha helices in an L-shape conformation, further supporting the phylogenetic analysis that it is a TatB like protein. To determine the quality of to EcTatB using Chimera (Pettersen et al., 2004). This indicates that our model for At5g43880 is very close the structure of EcTatB also evidenced by the superimposed structures in Figure 3B. All this data indicate that At5g43680 is most likely a TatB like protein. At5g43680 is an inner mitochondrial membrane protein As yet little or no data is available about the localization of At5g43680. We only know that several peptides of this protein have been identified in whole protein fractions in the pep2pro proteomics database (Baerenfaller et al., 2011), indicating that it is an expressed protein. To determine the subcellular localization of At5g43680 we first performed in vivo Journal of Cell Science • Advance article our model we calculated an overall RMSD of 1.866 and a Q-score of 0.501 when compared GFP targeting analysis. Firstly, we cloned the full length coding sequence for At5g43680 in frame with a C-terminally located GFP tag. Tobacco leaves were infiltrated with agrobacterium carrying the At5g43680-GFP construct. Analysis of the GFP expression of protoplasts isolated from the transformed tobacco leaves displayed a GFP signal which overlapped with mitochondria as visualized using mitotracker (Figure 3C). Since At5g43680 colocalized with mitochondria we decided to rename it as AtmtTatB. To test the AtmtTatB-GFP localization results, in vitro import assays into isolated Arabidopsis mitochondria using radio-labeled precursor proteins were performed. When AtmtTatB was incubated with isolated mitochondria a protease resistant product was observed after incubation with proteinase K of the same size as the precursor protein (Figure 4A, Lanes 1-3), indicating that AtmtTatB is imported into mitochondria but does not contain a cleavable presequence. Pretreatment of the mitochondria with the ionophore valinomycin prior to import abolished the appearance of the protease resistant band of AtmtTatB (Figure 4A, Lanes 4 and 5). Valinomycin dissipates the mitochondrial membrane potential which is required for the import of proteins into or across the inner membrane, indicating that AtmtTatB is imported into the matrix or inner membrane of plant mitochondria. When the outer membrane was removed prior to the addition of proteinase K, AtmtTatB remained protease resistant indicating that if it is located in the inner mitochondrial membrane the majority is facing towards the matrix (Figure 4A, Lanes 6-9). Finally, the addition of 1% Triton X-100 prior to protease treatment demonstrated that when total mitochondria were ruptured AtmtTatB is accessible to the added protease (Figure 4A, Lane 10). The Glycine max AOX (Alternative oxidase 1a) and Arabidopsis Tim23 (Translocase of the inner membrane protein of 23 kDa) proteins were used as controls in the import assays as both are potential dependent manner and processed to a 32 kDa mature protein which was protease resistant even when the outer membrane was ruptured (Figure 4A, Lanes 1-9). Tim23 was also imported in a membrane potential dependent manner and was shown to be protease resistant in intact mitochondria but produced its characteristic smaller membrane protected fragment after outer membrane rupture and protease treatment (Figure 4A). Both proteins were also fully digested by protease K when the mitochondria were ruptured by 1% Triton X100 treatment (Figure 4A, Lane 10). These experiments demonstrated that the AtmtTatB is targeted to and imported into mitochondria and is most likely located either within the mitochondrial inner membrane or mitochondrial matrix. Journal of Cell Science • Advance article associated with the mitochondrial inner membrane. AOX was imported in a membrane To further test the mitochondrial location of AtmtTatB we raised an antibody against the full length protein. Immuno blotting against Arabidopsis mitochondria using the purified AtmtTatB antibody detected a band of 30 kDa that was absent when the preimmune serum was used (Figure 4B). Incubation of the antibody with the antigen prior to western blotting abrogated binding of the antibody to this 30 kDa protein indicating that the antibody is specific (Figure 4B). Using the antibody against mitochondria and ruptured mitochondria treated with proteinase K indicated that AtmtTatB is resistant to protease treatment even when the outer membrane is ruptured (Figure 4C). Supporting the import results that AtmtTatB is located either in the inner mitochondrial membrane or the matrix. To verify AtmtTatB is located in the membrane, mitochondrial membrane fractions were extracted with carbonate. Like the membrane control CoxII, AtmtTatB was located in the pellet membrane fraction as opposed to the soluble fraction which contained subunit H of the glycine decarboxylase (Figure 4D). Putting these results together we could demonstrate that AtmtTatB is located within the inner mitochondrial membrane most likely with its Cterminus facing the matrix. A similar orientation of the EcTatB protein within the cytoplasmic membrane has been described previously (Koch et al., 2012). AtmtTatB is an essential gene Since AtmtTatB is nuclear encoded it is possible to test T-DNA insertion lines for its physiological role to be inferred. We identified two potential insertion lines in CSHL_GT11254 and SALK_003481 and characterized them. The T-DNA insertion for CSHL_GT11254 was found to be directly downstream of the ATG start codon and the TDNA insertion for SALK_003481 was found to be located in exon five (Figure 5). We obtained homozygous plants, only heterozygous and wild-type plants were found. When we examined the siliques of self-fertilized heterozygous plants we observed that one quarter of all embryos were aborted (displayed by the white embryos) (Figure 5). This corroborates the observation that offspring of heterozygous plants produced heterozygous to wild type ratios of 2:1 (Figure 5). To confirm these results, we PCR screened the progeny of a self-fertilized heterozygous plants. After screening 140 plants from the line CSHL_GT11254 we obtained 95 heterozygous plants and 45 wildtype plants which gives a ratio of 2.1:1. After screening 157 plants from the line SALK_003481 we obtained 106 heterozygous plants and 51 wildtype plants which gives a ratio of 2.1:1. These results are almost identical to the predicted results of a heterozygous to wildtype ratio of 2:1 as predicted by Mendelian Journal of Cell Science • Advance article attempted to identify homozygous plants using PCR, however for both lines we never genetics for a self-fertilized embryo lethal gene. We conclude that AtmtTatB is an essential gene which causes the homozygous embryos to abort. Mitochondrial TatC and TatB proteins are located in the same 1500 kDa complex In other organisms the TatB and TatC subunits are normally found in a stable complex termed the TatBC complex (Cline and Mori, 2001, Bolhuis et al., 2001). To determine if the mtTatB and mtTatC proteins are in a stable complex of similar molecular weights we used two dimensional BN-SDS-PAGE followed by immuno blotting. Using the controls of Tom40 from the TOM complex (300 kDa), COXII from complex IV (240 kDa) and Qcr7 from complex III (500 and 1500 kDa), we could determine that mtTatB is located in one complex with the molecular weight of 1500 kDa and mtTatC was located in complexes of molecular weights of 1500 kDa and 150 kDa (Figure 6). As the supercomplex of complexes I + III also runs at 1500 kDa it could be interpreted that mtTatB and C are part of those complexes. However extensive work on identifying the subunits of plant mitochondrial complexes I + III has never identified either mtTatB or C (Meyer et al., 2008, Peters et al., 2013, Klodmann and Braun, 2011, Klodmann et al., 2010), which means it is unlikely they are part of those complexes. The fact that both mtTatB and mtTatC are both found in a high molecular weight complex of the same size is a good indication that they form a stable complex together in a similar manner to that of other organisms. Mitochondrial TatC and TatB proteins are associated with a lack of Bcs1 In yeast and mammalian mitochondria the protein Bcs1 is responsible for the insertion of the Rieske Fe/S (Rip) protein into complex III, (Wagener et al., 2011) which has also been Pett and Lavrov, 2013). By sorting a selection of organisms based on their mitochondrial evolution in the same manner as before by using the protein sequences of COX1 and COB and then over laying this with first the structural arrangement of that organisms Bcs1 and then also including whether or not that organism contains a mitochondrial TatC and a mitochondrial TatB like protein (Figure 7). It can be observed that the majority of organisms which contain a complete Bcs1 like protein which encompasses both the Bcs1 and AAA ATPase domains, do not contain either a mitochondrial encoded TatC gene or mitochondrial TatB like gene. The opposite can then also be observed that the majority of organisms which encode for a mitochondrial TatC and TatB like genes do not contain a protein with a Bcs1 domain but only contain the AAA ATPase domain. This is in agreement with what was Journal of Cell Science • Advance article hypothesized as a substrate for a potential mitochondrial Tat pathway (Hinsley et al., 2001, previously reported (Pett and Lavrov, 2013). The only two exceptions being Naegleria gruberi and Chlamydomonas reinhardtii. This observation indicates that organisms that have evolved a complete Bcs1 protein, i.e. the AAA ATPase and Bcs1 domains have been able to lose their mitochondrial Tat pathway as it is no longer required for Rip insertion. On the other side organisms which do not contain a complete Bcs1 protein have therefore retained their mitochondrial Tat pathway, potentially required for Rip insertion. A similar theory was also proposed by (Pett and Lavrov, 2013). The Arabidopsis Rip protein requires a Tat signal and a ΔpH membrane potential for proper assembly It has been previously demonstrated that some mitochondrial Rip proteins contain Tat-like targeting signals immediately preceding the transmembrane domain (Hinsley et al., 2001, Pett and Lavrov, 2013). To test if the Tat-like targeting signal is functional we analyzed the import and assembly of a mutated Rip protein. We mutated the potential Arabidopsis Rip Tat signal of KR to QQ, glutamines were chosen because substitutions with glutamines had been previously shown to completely abolish Tat pathway export in bacteria (Kreutzenbeck et al., 2007). It was observed using in vitro import assays into isolated Arabidopsis mitochondria that the Rip-KR imports and assembles into complex III and the super complex of I + III very efficiently as seen by the increase in radioactive signal over time (Figure 8A). However, in contrast the Rip-QQ displays a far weaker signal in the complexes indicating that it is not assembled efficiently in Arabidopsis mitochondria (Figure 8A). Interestingly we noticed that in the Rip-QQ samples the signal peaked at 60 mins and subsequently decreased in the further time points while Rip-KR continued to increase (Figure 8A). This suggests that order to test if both the Rip-KR and Rip-QQ proteins are imported and stable within Arabidopsis mitochondria we repeated the experiment but used SDS-PAGE instead of BNPAGE. Both proteins were imported and processed to the mature form in a time dependent manner with similar efficiencies (Figure 8B). Importantly we also checked that both Rip-KR and Rip-QQ were inserted into the inner membrane. This was confirmed by extracting the membranes using 0.1 M Na2CO3 (Supplementary Figure 1). Both Rip-KR and Rip-QQ were located within the membrane fractions (Supplementary Figure 1). From these results we conclude that the Rip-QQ was imported and stable within Arabidopsis mitochondria but cannot be efficiently assembled into complex III, indicating that the insertion of the Rip protein into complex III requires a functional Tat pathway. Journal of Cell Science • Advance article exchanging the Rip KR Tat signal to QQ disrupts the stable assembly into complex III. In To further investigate whether the Arabidopsis mitochondrial Rip protein is inserted by a Tat pathway, we analyzed the assembly in the presence of nigericin. Nigericin is an inonophore which dissipates the ΔpH of the membrane potential but does not effect the electrical component of the membrane potential (Yuan and Cline, 1994). Import and assembly of mitochondrial proteins has been previously demonstrated to rely heavily on the electrical component of the membrane potential and not the ΔpH (Martin et al., 1991, Pfanner and Neupert, 1985). Therefore, we first incubated wiltype Rip-KR with mitochondria under normal import conditions for a pulse time of 7.5 minutes. After the pulse period the mitochondria were re-isolated and washed to remove un-imported protein. The reaction was then split into two, one containing normal import buffer and the other containing in addition to normal import buffer, 2 µM nigericin. Assembly into complex III was analyzed by BNPAGE (Figure 8C). Only the reactions not containing nigericin showed a clear assembly of Rip-KR into complex III and the super complex of I+III indicated by the increase in radioactive signal over time (Figure 8C). The reactions incubated in the presence of nigericin did not show an increase in complex III and super complex I+III intensity (Figure 8C). The fact that Arabidopsis Rip requires the ΔpH is another indication that it is inserted into complex III by a Tat pathway. As originally the Tat pathway was called the ΔpH pathway as many of its substrates in both bacteria and chloroplasts are inhibited by nigericin in a similar manner as what was seen for the Arabidopsis chloroplast Rip protein (Molik et al., 2001) Discussion Since the first plant mitochondrial genome sequences were obtained it has been subunit, TatC was ever identified and was mostly thought of as a pseudogene. Especially in Arabidopsis as the mtTatC did not contain a classical start codon. But even with a functional TatC gene plant mitochondria were still missing other subunits to complete a functional Tat pathway. The identification of a plant mitochondrial TatB like protein opens up a new whole field of research in plant mitochondrial biology. Interestingly mtTatB lacks a cleavable transit peptide but is still correctly targeted to the mitochondria. Most likely this would mean that mtTatB is imported by the carrier import pathway. The carrier import pathway is specialized in the import of inner membrane proteins the majority of which lack cleavable presequences (Sirrenberg et al., 1996). Targeting signals of the carrier import pathway a thought to be Journal of Cell Science • Advance article hypothesized that plant mitochondria contained a Tat pathway. However only ever one mediated by the transmembrane domains of substrate proteins (Endres et al., 1999). Therefore, the mitochondrial targeting signal for mtTatB is most likely its N-terminal transmembrane domain. Several questions however, still require answering such as: where is mtTatA, does the mitochondria Tat pathway require a TatA subunit, why are mtTatB proteins much longer than other TatB proteins, what are the substrates for the mitochondrial Tat pathway and why have plant mitochondria retained a Tat pathway while other eukaryotes have replaced it? To answer the first question, as yet we could not identify any protein within Arabidopsis which is an obvious target for being a mtTatA protein. This may be related to the second question of why mtTatB is much longer than other TatB proteins. It may be possible that mtTatB has gained some sort of dual functionality and performs both the roles of TatA and TatB. This though will require extensive biochemical testing to confirm. So far in our hands attempts to try and complement E. coli Tat mutants with the mitochondrial subunits have not worked. Attempts to also over express full length mtTatC in E. coli have also failed therefore limiting any in vitro reconstitution assays. As for the substrates of the mitochondrial Tat pathway, this study has identified that the Rip protein from complex III as a good candidate. Rip is an interesting candidate as in the mitochondria of yeast and humans it undergoes a unique import and assembly process. Firstly, Rip is synthesized on cytosolic ribosomes and imported in a post translational manner through the outer and inner membrane by the general import pathway utilizing the translocation complexes TOM and the TIM17:23 (Hartl et al., 1986). Secondly, after reaching the matrix Rip has its targeting signal removed in two steps. Thirdly, Rip then has its iron sulfur cluster inserted and the C-terminus is fully folded (Kispal et al., 1997, Kispal et al., 1999). This is all thought to happen in the matrix and chaperoned by the protein Mzm1 to Bcs1 in the inner membrane where Rip is then inserted into the membrane and the fully folded C-terminus is passed back through the inner membrane to the intermembrane space (Cui et al., 2012, Wagener et al., 2011). Fifthly, Rip is the last protein inserted into the so-called pre-Bc1 complex forming the complete and functional complex (Wagener et al., 2011). Up until recently it was also thought that in plant mitochondria a similar pathway existed. However as stated before the closest related protein to human or yeast Bcs1 in plant mitochondria is located in the outer membrane and plays no role in the assembly of the bc1 complex (Zhang et al., 2014a). As for Rip assembly in plant mitochondria it is most probable that of the five steps outlined before the first four are exactly the same. Arabidopsis Rip is a Journal of Cell Science • Advance article at this stage Rip is found as a soluble intermediate (Hartl et al., 1986). Fourthly, Rip is nuclear encoded gene, which its protein product is synthesized in the cytosol and post translationally imported into mitochondria. Similar to yeast AtRip contains a cleavable presequeance and one transmembrane domain therefore most likely also uses the TOM complex and TIM17:23 complexes for translocation through the outer and inner membranes. The plant mitochondrial matrix also contains all the required proteins for iron sulfur biogenesis and also contains a homolog of Mzm1 (Balk and Pilon, 2011). So plant mitochondrial Rip probably also must have a soluble intermediate in the matrix while its iron sulfur cluster is assembled. Therefore, the only divergent part in Rip assembly in plant mitochondria is the use of a Tat pathway for membrane insertion and passing the fully folded C-terminus back into the intermembrane space. This is fully consistent with our observations that mutation of the AtRip Tat targeting signal and also that AtRip requires a ΔpH for assembly. Rip proteins of chloroplasts and bacteria have also been demonstrated previously as requiring a Tat pathway for proper insertion (Molik et al., 2001, Aldridge et al., 2008, Bachmann et al., 2006, De Buck et al., 2007). These observations are strengthened by the correlation of mitochondrial Tat proteins with the absence of a complete Bcs1 proteins in a variety of organisms. It could be argued that AtRip could also be inserted back into the inner membrane by way of the Oxa pathway however this seems unlikely as the Oxa pathway has never been demonstrated to translocate fully folded proteins. This can also be countered by the fact that yeast and humans do not use Oxa for Rip assembly. If this was possible why have Bcs1? It may also be argued that AtRip could be laterally inserted from the TIM17:23 complex into the inner membrane. In this case AtRip would not have a soluble intermediate and the Cterminus would never reach the matrix. The problem here is that all the iron sulfur cluster within the mitochondrial matrix. Therefore, if the C-terminal domain of AtRip never gets to the matrix there is no known mechanism to insert its iron sulfur cluster. Therefore, after excluding all other possibilities and looking at the data presented in this paper it is most likely that AtRip uses a Tat pathway for assembly into complex III. The data presented here demonstrate that plant mitochondria contain the translocation subunits TatB and TatC. Since the TatB subunit is an essential gene it can be extrapolated that this potential Tat pathway is required for plant mitochondrial biogenesis. However, further work is required to determine if this potential mitochondrial Tat pathway requires a TatA subunit or somehow functions with only TatB and TatC subunits. We have also sought to demonstrate that the AtRip protein is a substrate of this potential mitochondrial Tat Journal of Cell Science • Advance article biogenesis machinery and also the Mzm1 chaperone which is specific for Rip are located pathway. AtRip requires the ΔpH and a Tat like targeting signal for proper assembly into complex III. Functional analysis of viable mtTatB mutants (eg: RNAi or antisense knockdowns) will prove invaluable in identifying more possible substrates and also the functional role of the potential plant mitochondrial Tat pathway. As to why plant mitochondria have retained a Tat pathway compared to other eukaryotes is a difficult question to answer. The most obvious explanation is that Bcs1 evolved in eukaryotes later after the split of plants from other eukaryotes. There is some evidence to support this with the base animal Monosiga brevicollis containing both mitochondrial TatC and TatB like proteins and also lacking a complete Bcs1 protein, indicating the mitochondrial Tat pathway was lost rather late in opisthokont evolution. It is also interesting to note that a selection of related green algae also appears to lack both a complete Bcs1 and mitochondrial Tat subunits (eg: Chlamydomonas reinhardtii). How these organisms assemble their mitochondrial Rip is another interesting question. Materials and methods GFP subcellular localization The Agrobacterium tumefaciens strain AGL1 was transformed with the full coding sequence of AtmtTatB (At5g43680) fused to GFP in the vector pK7FWG2 (Karimi et al., 2002) and used to infiltrate 4 - 6 week old Nicotiana benthamiana leaves as described previously (Schweiger et al., 2012). Protoplasts were prepared as outlined in (Koop et al., 1996) except cell walls were digested for 90 mins at 40 rpm in 1% cellulase R10 and 0.3% macerase R10 after vacuum infiltration. Mitotracker (Life Technologies) was added to the protoplast laser scanning microscope at 20°C (Leica, Type: TCS SP5). T-DNA insertion lines The following T-DNA insertion lines were obtained from the Nottingham Arabidopsis Stock Centre (NASC) and Cold Spring Harbor Laboratory (CSHL) respectively: SALK_003481 and CSHL_GT11254 (Alonso et al., 2003, Sundaresan et al., 1995). T-DNA insertions were genotyped by PCR and insertion sites were confirmed by sequencing. Journal of Cell Science • Advance article suspensions to a final concentration of 500 nM. Fluorescence was observed with a confocal Mitochondrial isolation Mitochondria for in vitro import and assays and Western blot analysis were harvested from 14 day old Arabidopsis thaliana seedlings grown in liquid culture as previously described (Lister et al., 2007). Typically between 2 - 4 mg of mitochondrial protein was obtained from each preparation. In vitro import studies For in vitro import studies the full coding sequence of AtmtTatB (AT5G43680) was cloned into the destination vector pDest14 (Invitrogen). The clones for AOX (X68702), Tim23 (At1g72750) and Rip (At5g13430) have been described previously (Murcha et al., 2003, Whelan et al., 1995, Carrie et al., 2015). [35S]Met-labeled precursor proteins were synthesized using the Flexi Rabbit Reticulocyte Lysate (Promega) as previously outlined (Chang et al., 2014). In vitro mitochondrial imports were then performed using isolated mitochondria as previously described in (Whelan et al., 1995, Lister et al., 2007). All in vitro imports were obtained using radiography and images were scanned using a Typhoon scanner (GE Healthcare). Mitochondrial fractionation Outer membrane-ruptured mitochondria were prepared by osmotic shock as previously described (Murcha et al., 2005). Carbonate extraction of mitochondrial membrane proteins was carried out as previously described (Tan et al., 2012). BN-PAGE was performed as in (Eubel and Millar, 2009) using 5% digitonin (Serva). Immunodetection of proteins was performed as described previously (Murcha et al., 2005). Unless specified the equivalent of 50 µg of protein was used in each lane. For the production of the antibody against AtmtTatC the peptide VREEGWTSGMRESGIEKKNKSSPPPRTW which corresponds to amino acids 253 to 281 of the Arabidopsis TatC was produced by Ganaxxon bioscience (Germany) and injected into 2 New Zealand white rabbits as per standard protocols (Cooper and Paterson, 2008). For the antibody against AtmtTatB the full protein sequence of AtmtTatB was expressed in E. coli fused to a six his tag. The protein was purified using denatured IMAC followed by electro-elution. The final protein was concentrated to approximately 2 mg/ml and then sent to Pineda Antikörper-service Journal of Cell Science • Advance article BN-PAGE and immunoblotting (Germany) for antibody production. Both antibodies were affinity purified using their respective antigens. The antibodies CoxII (A S04 052) and GDC-H (A S05 074) were purchased from Agrisera (Sweden). Antibodies against Tom40 and Qcr7 have been previously published (Kuhn et al., 2011, Carrie et al., 2009). Phylogenetic analysis For the phylogenetic tree in Figure 1 the protein sequences of Cytochrome c 1 (COX1) and Cytochrome b (COB) were concatenated prior to analysis. Protein sequences were obtained from the NCBI organelle genomes database. The protein sequences were first aligned using MEGA5 (Tamura et al., 2011) with the Muscle algorithm (Edgar, 2004). The evolutionary history was then inferred using Maximum Likelihood method based on the Whelan and Goldman model (Whelan and Goldman, 2001). The bootstrap consensus tree was inferred from 1000 replicates with the percentage of replicate trees in which the associated proteins clustered together in the bootstrap test shown next to branches. For the phylogenetic tree of the TatA and B sequences in Figure 2 the process was exactly the same. For the phylogenetic tree in Figure 6 the procedure and use of COX1 and COB sequences was exactly the same. Journal of Cell Science • Advance article For species and sequence information see Supplementary Table 2 for all phylogenetic trees. Acknowledgements Part of this work was started while CC was supported by a Humboldt Research Fellowship from the Alexander von Humboldt Foundation. The Tom20 antibody was a kind gift from Dr. Serena Schwenkert. The pea cpTatA and cpTatB antibodies were a kind gift from Prof. Kenneth C. Cline. Competing interests None Author contributions CC carried out all experimental work. SW carried out the modeling of mtTatB. Both CC and Journal of Cell Science • Advance article JS planned and designed all experiments and all authors co-wrote the manuscript. ALDRIDGE, C., SPENCE, E., KIRKILIONIS, M. A., FRIGERIO, L. & ROBINSON, C. 2008. Tat-dependent targeting of Rieske iron-sulphur proteins to both the plasma and thylakoid membranes in the cyanobacterium Synechocystis PCC6803. Mol Microbiol, 70, 140-50. ALONSO, J. M., STEPANOVA, A. N., LEISSE, T. J., KIM, C. J., CHEN, H., SHINN, P., STEVENSON, D. K., ZIMMERMAN, J., BARAJAS, P., CHEUK, R., GADRINAB, C., HELLER, C., JESKE, A., KOESEMA, E., MEYERS, C. C., PARKER, H., PREDNIS, L., ANSARI, Y., CHOY, N., DEEN, H., GERALT, M., HAZARI, N., HOM, E., KARNES, M., MULHOLLAND, C., NDUBAKU, R., SCHMIDT, I., GUZMAN, P., AGUILAR-HENONIN, L., SCHMID, M., WEIGEL, D., CARTER, D. E., MARCHAND, T., RISSEEUW, E., BROGDEN, D., ZEKO, A., CROSBY, W. L., BERRY, C. C. & ECKER, J. R. 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science, 301, 653-7. ALTSCHUL, S. F., GISH, W., MILLER, W., MYERS, E. W. & LIPMAN, D. J. 1990. Basic local alignment search tool. J Mol Biol, 215, 403-10. BACHMANN, J., BAUER, B., ZWICKER, K., LUDWIG, B. & ANDERKA, O. 2006. The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase. FEBS J, 273, 4817-30. BAERENFALLER, K., HIRSCH-HOFFMANN, M., SVOZIL, J., HULL, R., RUSSENBERGER, D., BISCHOF, S., LU, Q., GRUISSEM, W. & BAGINSKY, S. 2011. pep2pro: a new tool for comprehensive proteome data analysis to reveal information about organ-specific proteomes in Arabidopsis thaliana. Integr Biol (Camb), 3, 225-37. BALK, J. & PILON, M. 2011. Ancient and essential: the assembly of iron-sulfur clusters in plants. Trends Plant Sci, 16, 218-26. BARNETT, J. P., EIJLANDER, R. T., KUIPERS, O. P. & ROBINSON, C. 2008. A minimal Tat system from a gram-positive organism: a bifunctional TatA subunit participates in discrete TatAC and TatA complexes. J Biol Chem, 283, 2534-42. BENTOLILA, S., OH, J., HANSON, M. R. & BUKOWSKI, R. 2013. Comprehensive highresolution analysis of the role of an Arabidopsis gene family in RNA editing. PLoS Genet, 9, e1003584. BERKS, B. C. 2015. The twin-arginine protein translocation pathway. Annu Rev Biochem, 84, 843-64. BOLHUIS, A., MATHERS, J. E., THOMAS, J. D., BARRETT, C. M. & ROBINSON, C. 2001. TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli. J Biol Chem, 276, 20213-9. BURGER, G., FORGET, L., ZHU, Y., GRAY, M. W. & LANG, B. F. 2003. Unique mitochondrial genome architecture in unicellular relatives of animals. Proc Natl Acad Sci U S A, 100, 892-7. CARRIE, C., KUHN, K., MURCHA, M. W., DUNCAN, O., SMALL, I. D., O'TOOLE, N. & WHELAN, J. 2009. Approaches to defining dual-targeted proteins in Arabidopsis. Plant J, 57, 1128-39. CARRIE, C., VENNE, A. S., ZAHEDI, R. P. & SOLL, J. 2015. Identification of cleavage sites and substrate proteins for two mitochondrial intermediate peptidases in Arabidopsis thaliana. J Exp Bot, 66, 2691-708. Journal of Cell Science • Advance article References Journal of Cell Science • Advance article CHANG, W., SOLL, J. & BOLTER, B. 2014. A new member of the psToc159 family contributes to distinct protein targeting pathways in pea chloroplasts. Front Plant Sci, 5, 239. CLINE, K. & HENRY, R. 1996. Import and routing of nucleus-encoded chloroplast proteins. Annu Rev Cell Dev Biol, 12, 1-26. CLINE, K. & MORI, H. 2001. Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport. J Cell Biol, 154, 719-29. COOPER, H. M. & PATERSON, Y. 2008. Production of polyclonal antisera. Curr Protoc Mol Biol, Chapter 11, Unit 11 12. CUI, T. Z., SMITH, P. M., FOX, J. L., KHALIMONCHUK, O. & WINGE, D. R. 2012. Late-stage maturation of the Rieske Fe/S protein: Mzm1 stabilizes Rip1 but does not facilitate its translocation by the AAA ATPase Bcs1. Mol Cell Biol, 32, 4400-9. DE BUCK, E., VRANCKX, L., MEYEN, E., MAES, L., VANDERSMISSEN, L., ANNE, J. & LAMMERTYN, E. 2007. The twin-arginine translocation pathway is necessary for correct membrane insertion of the Rieske Fe/S protein in Legionella pneumophila. FEBS Lett, 581, 259-64. EDGAR, R. C. 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics, 5, 113. ENDRES, M., NEUPERT, W. & BRUNNER, M. 1999. Transport of the ADP/ATP carrier of mitochondria from the TOM complex to the TIM22.54 complex. EMBO J, 18, 321421. EUBEL, H. & MILLAR, A. H. 2009. Systematic monitoring of protein complex composition and abundance by blue-native PAGE. Cold Spring Harb Protoc, 2009, pdb prot5221. GUTIERREZ-MARCOS, J. F., DAL PRA, M., GIULINI, A., COSTA, L. M., GAVAZZI, G., CORDELIER, S., SELLAM, O., TATOUT, C., PAUL, W., PEREZ, P., DICKINSON, H. G. & CONSONNI, G. 2007. empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant growth in maize. Plant Cell, 19, 196-210. HARTL, F. U., SCHMIDT, B., WACHTER, E., WEISS, H. & NEUPERT, W. 1986. Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase. Cell, 47, 939-51. HINSLEY, A. P., STANLEY, N. R., PALMER, T. & BERKS, B. C. 2001. A naturally occurring bacterial Tat signal peptide lacking one of the 'invariant' arginine residues of the consensus targeting motif. FEBS Lett, 497, 45-9. JACOB, Y., SEIF, E., PAQUET, P. O. & LANG, B. F. 2004. Loss of the mRNA-like region in mitochondrial tmRNAs of jakobids. RNA, 10, 605-14. JAKOB, M., KAISER, S., GUTENSOHN, M., HANNER, P. & KLOSGEN, R. B. 2009. Tat subunit stoichiometry in Arabidopsis thaliana challenges the proposed function of TatA as the translocation pore. Biochim Biophys Acta, 1793, 388-94. KARIMI, M., INZE, D. & DEPICKER, A. 2002. GATEWAY vectors for Agrobacteriummediated plant transformation. Trends Plant Sci, 7, 193-5. KELLEY, L. A., MEZULIS, S., YATES, C. M., WASS, M. N. & STERNBERG, M. J. 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc, 10, 845-58. KISPAL, G., CSERE, P., GUIARD, B. & LILL, R. 1997. The ABC transporter Atm1p is required for mitochondrial iron homeostasis. FEBS Lett, 418, 346-50. KISPAL, G., CSERE, P., PROHL, C. & LILL, R. 1999. The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins. EMBO J, 18, 39819. Journal of Cell Science • Advance article KLODMANN, J. & BRAUN, H. P. 2011. Proteomic approach to characterize mitochondrial complex I from plants. Phytochemistry, 72, 1071-80. KLODMANN, J., SUNDERHAUS, S., NIMTZ, M., JANSCH, L. & BRAUN, H. P. 2010. Internal architecture of mitochondrial complex I from Arabidopsis thaliana. Plant Cell, 22, 797-810. KOCH, S., FRITSCH, M. J., BUCHANAN, G. & PALMER, T. 2012. Escherichia coli TatA and TatB proteins have N-out, C-in topology in intact cells. J Biol Chem, 287, 1442031. KOOP, H. U., STEINMULLER, K., WAGNER, H., ROSSLER, C., EIBL, C. & SACHER, L. 1996. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta, 199, 193-201. KREUTZENBECK, P., KROGER, C., LAUSBERG, F., BLAUDECK, N., SPRENGER, G. A. & FREUDL, R. 2007. Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities. J Biol Chem, 282, 790311. KUHN, K., CARRIE, C., GIRAUD, E., WANG, Y., MEYER, E. H., NARSAI, R., DES FRANCS-SMALL, C. C., ZHANG, B., MURCHA, M. W. & WHELAN, J. 2011. The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana. Plant J, 67, 1067-80. LEE, P. A., TULLMAN-ERCEK, D. & GEORGIOU, G. 2006. The bacterial twin-arginine translocation pathway. Annu Rev Microbiol, 60, 373-95. LISTER, R., CARRIE, C., DUNCAN, O., HO, L. H., HOWELL, K. A., MURCHA, M. W. & WHELAN, J. 2007. Functional definition of outer membrane proteins involved in preprotein import into mitochondria. Plant Cell, 19, 3739-59. MARTIN, J., MAHLKE, K. & PFANNER, N. 1991. Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences. J Biol Chem, 266, 18051-7. MEYER, E. H., TAYLOR, N. L. & MILLAR, A. H. 2008. Resolving and identifying protein components of plant mitochondrial respiratory complexes using three dimensions of gel electrophoresis. J Proteome Res, 7, 786-94. MOLIK, S., KARNAUCHOV, I., WEIDLICH, C., HERRMANN, R. G. & KLOSGEN, R. B. 2001. The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts? J Biol Chem, 276, 42761-6. MORI, H., SUMMER, E. J. & CLINE, K. 2001. Chloroplast TatC plays a direct role in thylakoid (Delta)pH-dependent protein transport. FEBS Lett, 501, 65-8. MORI, H., SUMMER, E. J., MA, X. & CLINE, K. 1999. Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways. J Cell Biol, 146, 45-56. MURCHA, M. W., LISTER, R., HO, A. Y. & WHELAN, J. 2003. Identification, expression, and import of components 17 and 23 of the inner mitochondrial membrane translocase from Arabidopsis. Plant Physiol, 131, 1737-47. MURCHA, M. W., MILLAR, A. H. & WHELAN, J. 2005. The N-terminal cleavable extension of plant carrier proteins is responsible for efficient insertion into the inner mitochondrial membrane. J Mol Biol, 351, 16-25. PALMER, T. & BERKS, B. C. 2012. The twin-arginine translocation (Tat) protein export pathway. Nat Rev Microbiol, 10, 483-96. PETERS, K., BELT, K. & BRAUN, H. P. 2013. 3D Gel Map of Arabidopsis Complex I. Front Plant Sci, 4, 153. Journal of Cell Science • Advance article PETT, W. & LAVROV, D. V. 2013. The twin-arginine subunit C in Oscarella: origin, evolution, and potential functional significance. Integr Comp Biol, 53, 495-502. PETTERSEN, E. F., GODDARD, T. D., HUANG, C. C., COUCH, G. S., GREENBLATT, D. M., MENG, E. C. & FERRIN, T. E. 2004. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem, 25, 1605-12. PFANNER, N. & NEUPERT, W. 1985. Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J, 4, 281925. ROBINSON, C. & KLOSGEN, R. B. 1994. Targeting of proteins into and across the thylakoid membrane--a multitude of mechanisms. Plant Mol Biol, 26, 15-24. RONQUIST, F., TESLENKO, M., VAN DER MARK, P., AYRES, D. L., DARLING, A., HOHNA, S., LARGET, B., LIU, L., SUCHARD, M. A. & HUELSENBECK, J. P. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol, 61, 539-42. SARGENT, F., STANLEY, N. R., BERKS, B. C. & PALMER, T. 1999. Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein. J Biol Chem, 274, 36073-82. SCHWEIGER, R., MULLER, N. C., SCHMITT, M. J., SOLL, J. & SCHWENKERT, S. 2012. AtTPR7 is a chaperone-docking protein of the Sec translocon in Arabidopsis. J Cell Sci, 125, 5196-207. SIRRENBERG, C., BAUER, M. F., GUIARD, B., NEUPERT, W. & BRUNNER, M. 1996. Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22. Nature, 384, 582-5. SUNDARESAN, V., SPRINGER, P., VOLPE, T., HAWARD, S., JONES, J. D., DEAN, C., MA, H. & MARTIENSSEN, R. 1995. Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev, 9, 1797810. SUNKEL, S., BRENNICKE, A. & KNOOP, V. 1994. RNA editing of a conserved reading frame in plant mitochondria increases its similarity to two overlapping reading frames in Escherichia coli. Mol Gen Genet, 242, 65-72. TAMURA, K., PETERSON, D., PETERSON, N., STECHER, G., NEI, M. & KUMAR, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 28, 2731-9. TAN, Y. F., MILLAR, A. H. & TAYLOR, N. L. 2012. Components of mitochondrial oxidative phosphorylation vary in abundance following exposure to cold and chemical stresses. J Proteome Res, 11, 3860-79. UNSELD, M., MARIENFELD, J. R., BRANDT, P. & BRENNICKE, A. 1997. The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nat Genet, 15, 57-61. VAN DER MERWE, J. A. & DUBERY, I. A. 2007. Expression of mitochondrial tatC in Nicotiana tabacum is responsive to benzothiadiazole and salicylic acid. J Plant Physiol, 164, 1231-4. WAGENER, N., ACKERMANN, M., FUNES, S. & NEUPERT, W. 2011. A pathway of protein translocation in mitochondria mediated by the AAA-ATPase Bcs1. Mol Cell, 44, 191-202. WANG, X. & LAVROV, D. V. 2007. Mitochondrial genome of the homoscleromorph Oscarella carmela (Porifera, Demospongiae) reveals unexpected complexity in the common ancestor of sponges and other animals. Mol Biol Evol, 24, 363-73. WANG, Y., CARRIE, C., GIRAUD, E., ELHAFEZ, D., NARSAI, R., DUNCAN, O., WHELAN, J. & MURCHA, M. W. 2012. Dual location of the mitochondrial Journal of Cell Science • Advance article preprotein transporters B14.7 and Tim23-2 in complex I and the TIM17:23 complex in Arabidopsis links mitochondrial activity and biogenesis. Plant Cell, 24, 2675-95. WHELAN, J., HUGOSSON, M., GLASER, E. & DAY, D. A. 1995. Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria. Plant Mol Biol, 27, 769-78. WHELAN, S. & GOLDMAN, N. 2001. A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol, 18, 691-9. YUAN, J. & CLINE, K. 1994. Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity. J Biol Chem, 269, 18463-7. ZHANG, B., VAN AKEN, O., THATCHER, L., DE CLERCQ, I., DUNCAN, O., LAW, S. R., MURCHA, M. W., VAN DER MERWE, M., SEIFI, H. S., CARRIE, C., CAZZONELLI, C., RADOMILJAC, J., HOFTE, M., SINGH, K. B., VAN BREUSEGEM, F. & WHELAN, J. 2014a. The mitochondrial outer membrane AAA ATPase AtOM66 affects cell death and pathogen resistance in Arabidopsis thaliana. Plant J, 80, 709-27. ZHANG, Y., HU, Y., LI, H. & JIN, C. 2014b. Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure. PLoS One, 9, e103157. ZHANG, Y., WANG, L., HU, Y. & JIN, C. 2014c. Solution structure of the TatB component of the twin-arginine translocation system. Biochim Biophys Acta, 1838, 1881-8. Journal of Cell Science • Advance article Figures Figure 1. Presence or absence of TatC like genes in plant mitochondrial genomes and confirmation in Arabidopsis thaliana that TatC is an expressed protein. A) The evolutionary history of plant mitochondria was inferred using the Maximum Likelihood method on concatenated protein sequences of Cytochrome c oxidase 1 (Cox1) and Cytochrome b (COB). Displayed is the consensus tree and bootstrap vales after 1000 replicates. Blue dots represent TatC genes with a classical start codons and red dots represent TatC genes without a classical start codon. B) Immuno blotting of Arabidopsis mitochondria separated by SDS-PAGE labeled with either pre-immune or purified antibody raised against AtmtTatC. Labeling of the TatC protein was abrogated by pre-incubation of the antibody with the TatC peptide. C) Western blot analysis of mitochondrial and chloroplast fractions from pea. Antibodies to CpTatA and cpTatB were tested for mitochondrial localization. Controls for mitochondria were the Voltage dependent anion channel protein, VDAC, the Rieske iron sulfur cluster protein from complex III, Rip and the translocase of the outer membrane protein of 40 kDa, Tom40. To control for chloroplasts, the light harvesting II Journal of Cell Science • Advance article complex protein was used, LHCII. At5g43680 like proteins was inferred using the maximum Likelihood method. Displayed is a bootstrap consensus tree inferred from 100 replicates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. Red text indicate the proteins identified by using At5g43680 in BLAST searches and are annotated as TatAB as it is unknown whether they are TatA or TatB proteins. Purple text indicate bacterial TatB proteins. Brown text indicates bacterial TatA proteins. Light blue text indicates TatA proteins from the mitochondrial genomes of jakobids. Green text indicates TatA and TatB proteins which are located in chloroplasts or are from cyanobacteria. Journal of Cell Science • Advance article Figure 2. Phylogenetic analysis of At5g43680. The evolutionary history of TatA, TatB and See Supplementary Table 2 for accession numbers of protein sequences used and also for Journal of Cell Science • Advance article species names abbreviations. Figure 3. Multiple sequence alignment between At5g43680 and EcTatB and a structural model for At5g43680 and GFP localization. A) Sequence alignment of the EcoTatB and At5g43680. Indicated are the transmembrane domains and aliphatic alpha helices along with the conserved Glutamate at residue 8 and the conserved Glycine at the end of the identical and grey amino acids are similar. B) Comparison of the structure of the E coli TatB protein (EcoTatB) and the model for At5g43680 protein from Arabidopsis. Indicated are the transmembrane domains (THM) and the three aliphatic alpha helices (APH, α3 and α4). C) Protoplasts of transformed tobacco leaves expressing At5g43680 tagged to the N-terminus of GFP monitored by confocal laser scanning microscopy. Also shown are mitotracker false colored purple for mitochondrial localization and the chlorophyll auto-fluorescence (red channel) indicating the location of the chloroplasts. Journal of Cell Science • Advance article transmembrane domain. Numbers indicate the amino acid number. Black amino acids are Figure 4. At5g43680 is an essential inner mitochondrial membrane localized TatB protein. A) In vitro import of radio labeled AtmtTatB into isolated mitochondria. The control proteins AOX and Tim23 are also shown. Lanes contain: 1 precursor protein alone, 2 precursor protein incubated with mitochondria under conditions that support import into mitochondria, 3 as lane 2 but with Proteinase K added after incubation of the precursor with mitochondria, 4 and 5 as lanes 2 and 3 but with valinomycin added to the import assay before membrane was ruptured after the incubation period with precursor protein but before the addition of Proteinase K, 10 is the same as lane 3 except mitochondria were first treated with Triton X-100 before Proteinase K treatment. Mit: mitochondria, Mit*OM: mitochondria with the outer membrane ruptured, Pk: Proteinase K, Val: valinomycin, Tx100: Triton X100, p: precursor protein band, m: mature protein band, m*: inner membrane protected fragment of Tim23.B) Immuno blotting of Arabidopsis mitochondria separated by SDS-PAGE labeled with either pre-immune serum or the antibody raised against AtmtTatB. Labeling of the AtmtTatB protein was abrogated by pre-incubation of the antibody with the AtmtTatB antigen used in antibody production. C) Sub-mitochondrial localization of AtmtTatB analyzed by protease protection. Proteinase K (Pk) was applied to mitochondria (Mit) or Journal of Cell Science • Advance article the addition of precursor protein, lanes 6-9 as lanes 2-5 except that the mitochondrial outer mitoplasts (Mit*OM, hypertonically swollen mitochondria). Antibodies to the translocase of the outer membrane of 20 kDa protein (Tom20), Cytochrome c oxidase subunit II (CoxII) and the H protein of glycine decarboxylase (GDC-H) were used as controls. D) Membrane association of AtmtTatB. Mitochondria were subjected to carbonate extraction using Na2CO3 (pH 11) and separated into membrane (M) and soluble (S) fractions. Again CoxII and GDC- Journal of Cell Science • Advance article H were used as membrane and soluble controls respectively. Figure 5. mtTatB is an essential gene. Gene structure of AtmtTatB displaying the locations of the T-DNA insertions and pictures of open siliques displaying aborted seeds from heterozygous plants. Siliques from wild type plants from both lines are shown for Journal of Cell Science • Advance article comparison. +: viable seed, -: aborted seed, Ls - Landsberg erecta, Col-0 - Columbia 0. SDS-PAGE stained with coomassie. B) BN-SDS-PAGE analyzed by immuno blotting using antibodies against the indicated proteins. Tom40 – translocase of the outer membrane of 40 kDa, CoxII – cytochrome c oxidase subunit II, Qcr7 – ubiquinol:cytochrome c reductase Journal of Cell Science • Advance article Figure 6. Mitochondrial TatB and TatC are located in a complex of 1500 kDa. A) BN- subunit 7, mtTatB and mtTatC. The molecular weights in kDa is given for the known Journal of Cell Science • Advance article complexes. Figure 7. Mitochondrial TatC and TatB proteins are associated with a lack of Bcs1. The evolutionary history of mitochondria from a variety of organisms was inferred using the Maximum Likelihood method on concatenated protein sequences of Cytochrome c oxidase 1 1000 replicates. The arrangement of the structural domains of the closest related protein to Bcs1 is shown and was determined by the SMART database. Also indicated is the presence or absence of mitochondrial TatC and TatB genes. Journal of Cell Science • Advance article (Cox1) and Cytochrome b (COB). Displayed is the consensus tree and bootstrap vales after Journal of Cell Science • Advance article Figure 8. Arabidopsis mitochondrial Rieske Fe/S protein requires a Tat signal and a ΔpH for assembly. A) [35S]Met-labeled Arabidopsis mitochondrial Rieske Fe/S proteins with Rip-KR or with Rip-QQ signals were incubated with isolated mitochondria in conditions that support import. After certain time points the mitochondria were analyzed by BN-PAGE and analyzed by autoradiography. The location of complex III and the super complex formed between complexes I and III are indicated. B) Displays the same import experiment as B however the proteins were separated by SDS-PAGE to display import and not assembly. p: precursor protein band, m: mature protein band. C) Wildtype AtRip-KR was incubated with isolated mitochondria in normal import conditions for a pulse period of 7.5 minutes. After the pulse period the mitochondria were re-isolated and washed of un-imported protein and the reaction was split in two. One half of the mitochondria were incubated in normal import buffer while the second half contained 2 μM nigericin (Nig). Samples were then collected at Journal of Cell Science • Advance article the indicated time points and analyzed by BN-PAGE. J. Cell Sci. 129: doi:10.1242/jcs.190975: Supplementary information Supplementary figure and tables Soluble Membrane Soluble Membrane Precursor Rip-KR Rip-KK pRip mRip Supplementary Figure 1. Membrane insertion of AtRip-KR and AtRip-QQ. [35S]Met-labeled Arabidopsis mitochondrial Rieske Fe/S proteins with Rip-KR or with Rip-QQ signals were incubated with isolated mitochondria in conditions that support import. After soluble fractions were then separated by SDS-PAGE. Journal of Cell Science • Supplementary information import mitochondrial membranes were extracted using 0.1 M Na2CO3. Both membrane and J. Cell Sci. 129: doi:10.1242/jcs.190975: Supplementary information Supplementary table 1. BackPhyre results of Arabidopsis genome searching with the structures of TatA and TatB. Results are displayed in ranking order according the Phyre program. Displayed are the GI accession numbers and the NCBI description, the confidence of the prediction, as well as the sequence percentage identity to the bait. Also shown are the respective AT accession numbers as well as the TAIR10 description. Supplementary table 2. Species and accession numbers for all proteins used in phylogenetic analysis for Figures 1,2 and 6. Click here to Download Table S2 Journal of Cell Science • Supplementary information Click here to Download Table S1 J. Cell Sci. 129: doi:10.1242/jcs.190975: Supplementary information Soluble Membrane Soluble Membrane Precursor Rip-KR Rip-KK pRip mRip Fig. S1. Membrane insertion of AtRip-KR and AtRip-QQ. [35S]Met-labeled Arabidopsis mitochondrial Rieske Fe/S proteins with Rip-KR or with Rip-QQ signals were incubated with isolated mitochondria in conditions that support import. After import mitochondrial membranes were extracted using 0.1 M Na2CO3. Both membrane and Journal of Cell Science • Supplementary information soluble fractions were then separated by SDS-PAGE. J. Cell Sci. 129: doi:10.1242/jcs.190975: Supplementary information Table S1. BackPhyre results of Arabidopsis genome searching with the structures of TatA and TatB. Results are displayed in ranking order according the Phyre program. Displayed are the GI accession numbers and the NCBI description, the confidence of the prediction, as well as the sequence percentage identity to the bait. Also shown are the respective AT accession numbers as well as the TAIR10 description. Journal of Cell Science • Supplementary information Click here to Download Table S1 J. Cell Sci. 129: doi:10.1242/jcs.190975: Supplementary information Table S2. BackPhyre results of Arabidopsis genome searching with the structures of TatA and TatB. Results are displayed in ranking order according the Phyre program. Displayed are the GI accession numbers and the NCBI description, the confidence of the prediction, as well as the sequence percentage identity to the bait. Also shown are the respective AT accession numbers as well as the EcoTatA PDB: 2NM7 Rank Description Confidence %i.d. TAIR accession Tair description 1 gi|15241912|ref|NP_198227.1| 99.8 38 AT5G28750 cpTatA 2 gi|15237156|ref|NP_200057.1| 99.8 27 AT5G52440 cpTatB 3 gi|22327551|ref|NP_199181.2| 99.7 26 AT5G43680 4 gi|22328976|ref|NP_194480.2| 23.4 17 AT4G27500 unknown protein Proton pump interactor 1 Confidence %i.d. TAIR accession TAIR description EcoTatB PDB: 2MI2 Rank Description 1 gi|22327551|ref|NP_199181.2| 99.9 17 AT5G43680 unknown protein 2 gi|15237156|ref|NP_200057.1| 99.9 22 AT5G52440 cpTatB 3 gi|15241912|ref|NP_198227.1| 99.9 19 AT5G28750 cpTatA 4 gi|18378920|ref|NP_563645.1| 61.7 17 AT1G02090 COP9 5 gi|42571297|ref|NP_973739.1| 60.9 16 AT1G02090 COP9 6 gi|15217502|ref|NP_174992.1| 56.6 16 AT1G42700 unknown protein 7 gi|30678240|ref|NP_849576.1| 53.3 16 AT1G02090 COP9 8 gi|30689895|ref|NP_850429.1| 49.3 15 AT2G45000 Nuclear pore protein 9 gi|79325107|ref|NP_001031638.1| 45.5 21 AT4G14315 unknown protein 10 gi|18407236|ref|NP_564778.1| 32.9 29 AT1G61563 11 gi|15240395|ref|NP_198039.1| 32 11 AT5G26880 12 gi|15232376|ref|NP_188721.1| 28.7 33 AT3G20850 RALFL8 AGL26 Transcription factor Proline rich family protein 13 gi|22330102|ref|NP_175258.2| 22.1 17 AT1G48240 14 gi|42572273|ref|NP_974232.1| 20.9 18 AT3G05860 SNARE 12 MADS-box transcription factor family protein Journal of Cell Science • Supplementary information TAIR10 description.