Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F₁-ATPase -subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and -glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial -presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.     

Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency

We describe a novel method for enhancing protein import into mitochondria, by tandemly duplicating the N-terminal cleavable leader peptide using a gene manipulation strategy. The import into isolated yeast mitochondria of passenger proteins (yeast mitochondrial ATP synthase subunits 8 and 9 and some mutagenised derivatives) that show little or no import when endowed with one such leader (that of Neurospora crassa mitochondrial ATP synthase subunit 9) is remarkably improved when the leader is tandemly duplicated. The import of these chimaeric proteins bearing a double leader is so rapid that a series of partially processed precursor intermediates accumulates inside the mitochondria before the final proteolytic release of leader sequences from the passenger proteins. It is considered that the duplicated leader greatly accelerates delivery of the import precursors to outer membrane receptor elements and the associated translocation systems, thereby enhancing precursor uptake into mitochondria.     

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.     

A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.

Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Chloroplast β-Barrel Proteins Are Assembled into the Mitochondrial Outer Membrane in a Process That Depends on the TOM and TOB Complexes

Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.     

Functional analysis of a recently originating,atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast

A gene family of at least five members encodes the tobacco mitochondrial Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichia coli -glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS proteins were localized by cell fractionation. The aminoterminal 75 and 71 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-terminal sequence of RISP4, which contains an atypical mitochondrial presequence, can translocate the GUS protein specifically into tobacco mitochondria with apparently low efficiency.Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the tobacco RISP3 and RISP4 presequences can direct import and processing of a GUS fusion protein in yeast mitochondria. Plant presequences, however, direct mitochondrial import in yeast less efficiently than the yeast presequence, indicating subtle differences between the plant and yeast mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an amphipathic secondary structure; however, these structural properties may improve the efficiency of mitochondrial import.     

Processing, targeting and import into the mitochondria and peroxisomes of proteins coded by nuclear genes]

I have herein discussed two gene-enzyme families in maize whose protein products participate to purge toxic oxidants from cells, and are thus of importance to all aerobic organisms. We have demonstrated that plant mitochondria import precursor proteins (i.e., preSOD-3) in a manner analogous to other eukaryotic cells. The "transit peptide" (TP) of preSOD-3 is 31 amino acid long and has similar properties to other reported TPs for mitochondrial and chloroplastic proteins. Import to peroxisomes is uniquely different from that for mitochondria and chloroplasts in that no consensus presequence seems to be involved. Instead, targeting signals seem to be integral parts of peroxisomal proteins.     

Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts

The precursors of the F₁-ATPase -subunits fromNicotiana plumbaginifolia andNeurospora crassa were imported into isolated spinach (Spinacia oleracea L.) leaf mitochondria. Both F₁ precursors were imported and processed to mature size products. No import of the mitochondrial precursor proteins into isolated intact spinach chloroplasts was seen. Moreover, the precursor of the 33 kDa protein of photosynthetic water-splitting enzyme was not imported into the leaf mitochondria. This study provides the first experimental report ofin vitro import of precursor proteins into plant mitochondria isolated from photosynthetic tissue and enables studies of protein sorting between mitochondria and chloroplasts in a system which is homologous with respect to organelles. The results suggest a high organellar specificity in the plant cell for the cytoplasmically synthesized precursor proteins.     

Aberrant mitochondrial processing of chimaeric import precursors containing subunits 8 and 9 of yeast mitochondrial ATP synthase

A set of chimaeric precursors which contain the same leader sequences but different passenger proteins has been analyzed for the site of protease cleavage following import into yeast mitochondria. Each precursor comprises the leader of Neurospora crassa subunit 9 of mitochondrial ATP synthase fused to subunit 8 or 9 of the corresponding yeast enzyme. Precursors containing the first five residues of mature N. crassa subunit 9 interposed between the leader and the yeast passenger protein were cleaved at the natural site of the N. crassa subunit 9 precursor. Direct fusions without interposed sequences were cleaved at novel sites. Cleavage occurred between the 3rd and 4th residues of yeast subunit 8, but for yeast subunit 9, cleavage occurred within the leader, 8 residues upstream of the passenger protein.     

Protein import into mitochondria in a homologous yeast in vitro system

To study the import of proteins into mitochondria we developed a homologous in vitro system in which mitochondria and cell-free translation extract are both derived from the yeast Saccharomyces cerevisiae. This system allows the synthesis of precursor proteins in the presence of isolated mitochondria and offers a means of analyzing yeast mutants defective in mitochondrial protein import. The in vitro import of an artificial precursor protein into yeast mitochondria in the presence of its substrate analog was analyzed subsequent to synthesis in either a yeast or rabbit reticulocyte cell-free translation reaction. Results suggest that a component(s) present in the yeast cytosolic extract may interact with the precursor protein.     

Plant mitochondrial rhomboid, AtRBL12, has different substrate specificity from its yeast counterpart

Rhomboid proteins comprise a class of serine proteases that are conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. This paper provides experimental evidence for the existence of rhomboid proteases in plant mitochondria and chloroplasts. Among 15 putative rhomboid-like proteins in Arabidopsis thaliana, we selected five predicted as mitochondrially targeted. For these proteins we performed the GFP transient assay, and identified two homologues, AtRBL11 (At5g25752) and AtRBL12 (At1g18600) to be targeted into plastids and mitochondria, respectively. Phylogenetic analysis reveals that AtRBL12 or AtRBL11 have only one clear orthologue in plant species with completely sequenced genomes. Complementation of the yeast lacking a functional copy of mitochondrial rhomboid with AtRBL12 indicates that this plant protease, in contrast to the human orthologue, does not recognize the yeast substrates, cytochrome c peroxidase (Ccp1) or dynamin-like GTPase (Mgm1). In agreement with this, we did not observe processing of Mgm1 when labeled precursor of this protein was incubated in vitro with Arabidopsis mitochondrial extract. Our results imply that plant mitochondrial rhomboids function in a specific manner and thus differ from their yeast and mammal counterparts.     

Functional analysis of mitochondrial protein import in yeast

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.     

Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.     

Mitochondrial import and processing of mutant human ornithine transcarbamylase precursors in cultured cells.

We have investigated mitochondrial import and processing of the precursor for human ornithine transcarbamylase (OTC; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) in HeLa cells stably transformed with cDNA sequences encoding OTC precursors carrying mutations in their leader peptides. The mutant precursors studied included two with amino acid substitutions in the 32-amino-acid leader peptide (glycine for arginine at position 23, designated gly23; glycines for arginines at positions 15, 23, and 26, designated gly15,23,26) and two with deletions (deletion of residues 8 to 22, designated d8-22; deletion of residues 17 to 32, designated N16). Specific immunoprecipitation with anti-OTC antiserum of extracts of L-[35S]methionine-labeled cells expressing these mutations yielded only precursor species; neither mature nor intermediate-size OTC subunits were observed. Fractionation of radiolabeled cells, however, revealed important differences among the various mutants: the gly23 precursor was associated with mitochondria and was not detected in the cytosol; the d8-22 and N16 precursors were found with both the mitochondrial fraction and the cytosol; only the gly15,23,26 precursor was detected exclusively in the cytosol. A large fraction of each of the mitochondrially associated OTC species was in a trypsin-protected compartment. In particular, the gly23 precursor behaved in trypsin protection and mitochondrial fractionation studies in a manner consistent with its translocation into the mitochondrial matrix. On the other hand, the lack of binding of the gly23 protein to a delta-N-phosphonoacetyl-L-ornithine affinity column, which specifically recognizes active OTC enzyme, indicated that, despite its intramitochondrial location, the mutant protein did not assemble into the normal, active trimer. Further, the gly23 mutant precursor was unstable within the mitochondria and was degraded with a t1/2 of less further than 4 h. Thus, we have shown that, in intact HeLa cells, cleavage of the OTC leader peptide is not required for translocation into mitochondria, but is required for assembly into active enzyme.     

Different legumin protein domains act as vacuolar targeting signals.

Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons. We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles. To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively. In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole. Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole. A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles. With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain. We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences.     

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.     

A novel ubiquitous protein 'chaperonin' supports the endosymbiotic origin of mitochondrion and plant chloroplast

The deduced amino acid sequences for a major mitochondrial protein (designated P1, related to the 'chaperonin' family of proteins) from human and Chinese hamster cells show extensive similarity (greater than 60% identity observed over the entire length) with a related protein present in evolutionarily as divergent organisms as Escherichia coli, Coxiella burnetii, Mycobacterium species, cyanobacteria as well as in yeast mitochondria and higher plant chloroplasts. Of the different groups of bacteria for which sequence data is available, maximum similarity of the mammalian/yeast P1 protein is observed with the corresponding protein from purple bacteria (especially C. burnetii) while the protein from plant chloroplasts exhibited highest similarity with the corresponding protein from cyanobacteria. The sequence data for this protein thus support the contention that the endosymbiont that gave rise to mitochondrion was a member of purple bacteria, while plant chloroplast originated from a member of the cyanobacterial lineage.     

Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.     

Protein transport into mitochondria is conserved between plant and yeast species

Protein targeting into plant mitochondria was investigated by in vitro translocation experiments. The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria. Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species. beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria. This translocation used protease-sensitive components of the outer membrane. Conversely, the N. plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria. However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria. We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms. These results are discussed in the context of a poly- or monophyletic origin of mitochondria.