Genomic organization, expression, and subcellular localization of mouse mitochondrial seryl-tRNA synthetase

We report here the identification and characterization of the mouse mitochondrial seryl-tRNA synthetase (mtSerRS). The genomic organization of mouse mtSerRS has been elucidated. The mouse mtSerRS gene containing 16 exons encodes a 519 residue protein with a strong homology to the mitochondria-like seryl-tRNA synthetase of bacteria, yeast, and other homologs. The mouse mtSerRS is ubiquitously expressed in various tissues, but more abundantly in tissues with high metabolic rates including heart and liver. Surprisingly, this gene, unlike other nuclear genes encoding mitochondrial proteins, exhibited a low expression in skeletal muscle and brain. Furthermore, immunofluorescence analysis of NIH3T3 cells expressing the mtSerRS-GFP fusion protein demonstrated that the mouse mtSerRS localizes in mitochondrion. These observations suggest that the mouse mtSerRS is an evolutionarily conserved protein involved in aminoacylation. Thus, it may play a role in the fidelity in mitochondrial translation and pathogenesis of deafness-associated mutations in the mitochondrial tRNA(Ser(UCN)).     

Dual targeting to mitochondria and chloroplasts.

Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Their endosymbiotic origin explains why these organelles contain their own DNA, nonetheless only a few dozens of genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the nuclear genome of the host, the product of their expression being targeted back to the corresponding organelle. Although targeting of proteins to mitochondria and chloroplasts is generally highly specific, an increasing number of examples have been discovered where the same protein is imported into both organelles. The object of this review is to compare and discuss these examples in order to try and identify common features of dual-targeted proteins. The study helps throw some light on the factors determining organelle targeting specificity, and suggests that dual-targeted proteins may well be far more common than once thought.     

Superposition of a tRNASer acceptor stem microhelix into the seryl-tRNA synthetase complex

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNAs. Seryl-tRNA synthetase is a class II synthetase, which depends on rather few and simple identity elements in tRNA(Ser) to determine the amino acid specificity. tRNA(Ser) acceptor stem microhelices can be aminoacylated with serine, which makes this part of the tRNA a valuable tool for investigating the structural motifs in a tRNA(Ser)-seryl-tRNA synthetase complex. A 1.8A-resolution tRNA(Ser) acceptor stem crystal structure was superimposed to a 2.9A-resolution crystal structure of a tRNA(Ser)-seryl-tRNA synthetase complex for a visualization of the binding environment of the tRNA(Ser) microhelix.     

The proximal region of a noncatalytic eukaryotic seryl-tRNA synthetase extension is required for protein stability in vitro and in vivo

Eukaryotic cytosolic seryl-tRNA synthetases (SerRS) have idiosyncratic C-terminal extensions not present in prokaryotic counterparts. The extensions of two eukaryotic SerRSs were subjected to mutagenesis and partial truncation. Only minor parts of the yeast or maize SerRS extensions, adjacent to the catalytic core (7 of 20 and 8 of 26 amino acids, respectively), were found to be indispensable for protein stability. Truncated proteins with substantially shortened extensions displayed unaltered catalytic properties and could complement a Saccharomyces cerevisiae strain with a disrupted SerRS gene, if these proximal regions were left intact. Although the yeast C-terminal SerRS extension is required for Pex21p binding, the maize counterpart with an appended yeast SerRS extension remained incapable of Pex21p binding, implying that additional regions of yeast SerRS may also contribute to the interaction with the peroxin. The proximal region of the eukaryotic SerRS C-terminal extension is indispensable for protein stability, while the remaining part of the extension remains available for other functions, such as species-specific protein:protein interactions.     

Inactivation of organellar glutamyl- and seryl-tRNA synthetases leads to developmental arrest of chloroplasts and mitochondria in higher plants

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.     

Dual targeting of Myxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxyfluorfen resistance

Much attention has been paid to the signal sequences of eukaryotic protoporphyrinogen oxidases (protoxes); both the organelles targeted by protoxes and the role of protoxes in conferring resistance against protox‐inhibiting herbicides, such as oxyfluorfen, have been examined. However, there have been no reports on the translocation of prokaryotic protoxes. This study investigated the targeting ability of Myxococcus xanthus protox in vitro and in vivo. In an in vitro translocation assay using a dual import system, M. xanthus protein was detected in chloroplasts and mitochondria, suggesting that the M. xanthus protox protein was targeted into both organelles. In order to confirm the in vitro dual targeting ability of M. xanthus, we used a stable transgenic strategy to investigate dual targeting in vivo. In transgenic rice plants overexpressing M. xanthus protox, M. xanthus protox antibody cross‐reacted with proteins with predicted molecular masses of 50 kDa from both chloroplasts and mitochondria, and this in vivo transgene expression corresponded to a prominent increase in chloroplastic and mitochondrial protox activity. Seeds from the transgenic lines M4 and M7 germinated in solid Murashige and Skoog media of up to 500 µm of oxyfluorfen, whereas wild‐type seeds did not germinate in 1 µm . After 4‐week‐old‐rice plants were treated with oxyfluorfen for 3 d, lines M4 and M7 exhibited normal growth, whereas the wild‐type line was severely bleached and necrotized. The herbicidal resistance is attributed to the insignificant accumulation of photodynamic protoporphyrin IX in cytosol because the high chloroplastic and mitochondrial protox activity in oxyfluorfen‐treated transgenic lines, compared with that in oxyfluorfen‐treated and untreated wild‐type plants, metabolizes protoporphyrinogen IX to chlorophyll and heme. A practical application of the dual targeting of M. xanthus protox for obtaining outstanding resistance to peroxidizing herbicides is discussed.     

The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting

We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.     

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.     

Targeting of glutamine synthetase to the mitochondria of transgenic tabacco

Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia -F₁ ATPase to target the GS polypeptide to the mitochondrion. The other (CYT-4) contained a single copy of a cytosolic GS construct. Leaves of in vitro plantlets expressed the constructs and contained a novel GS polypeptide, which assembled into active GS isoenzymes constituting about 25% of the total GS activity. In in vitro plantlets of MIT-1, but not CYT-4, the novel polypeptide was found to be associated with the mitochondria. Moreover in MIT-1, the size of the novel polypeptide was not that predicted of the precursor (44.9 kDa) but was about 39 kDa, the same size as the authentic GS polypeptide in CYT-4. These results are consistent with the precursor being imported into the mitochondria and cleaved near the fusion junction between the two sequences. These experiments have therefore shown that the presequence of the -F₁ ATPase has successfully targeted the GS polypeptide to the mitochondria of transgenic tobacco where it has assembled into an active isoenzyme. However, in fully regenerated plants growing photoautotrophically in growth-room conditions, although the constructs were still expressed, the polypeptide did not accumulate to the same levels as in in vitro plantlets and new isoenzyme activities were now barely detectable. Moreover in leaves of the mature MIT-1 plants, the polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.     

Dual targeting and function of a protease in mitochondria and chloroplasts

Here we show, using the green fluorescent protein (GFP) fusion system, that an Arabidopsis thaliana zinc-metalloprotease (AtZn-MP) is targeted to both mitochondria and chloroplasts. A deletion mutant lacking the amino-terminal 28 residues, with translation initiation at the second methionine residue, was imported into chloroplasts only. However, a mutated form of the full-length targeting peptide, in which the second methionine residue is changed to leucine, was imported to both organelles. No GFP fluorescence was detected when a frame-shift mutation was introduced between the first and second ATG codons of the Zn-MP–GFP construct, suggesting no alternative translational initiation. Our results show that the dual targeting of the Zn-MP is due to an ambiguous targeting peptide. Furthermore, we show that the recombinant AtZn-MP degrades mitochondrial and chloroplastic targeting peptides, indicating its function as a signal peptide degrading protease in both mitochondria and chloroplasts.     

Development of ultrastructural damage to chloroplasts in a plastoquinone-deficient mutant of maize

The ultrastructure of a recently discovered mutant of maize (mutant hcf103-114) that is completely lacking plastoquinone (Cook, W.B., Miles, D., 1992. Nuclear mutations affecting plastoquinone accumulation in maize. Photosyn. Res. 31, 99–111) was investigated. This mutant fails to green and dies at an early age. Tissues along a developmental gradient (from base to tip of a maize leaf) were fixed and prepared for examination via electron microscopy. Initial development was normal in both mesophyll cell (MC) and bundle sheath cell (BSC) chloroplasts. Starch, which was abundant in BSC chloroplasts of wild type maize, did not accumulate in the mutant. As tissue aging progressed, both plastid types exhibited symptoms typical of photooxidative injury. Injury, seen as chloroplast swelling, lipid accumulation and envelope disruption, appeared sooner in BSC chloroplasts than in MC chloroplasts. Chloroplasts in guard cells possessed starch granules and only showed ultrastructural injury after the starch granules disappeared. Stomata developed normally in the hcf103-114 mutant. The results are discussed in terms of the known roles of plastoquinone in chloroplast metabolism.     

Differential transmission of the Cucumis organellar genomes

 Although plants generally show maternal transmission of the organellar genomes, previous research has demonstrated that the mitochondrial (mt) genome of cucumber is paternally transmitted. In this study, we identified RFLPs in the organellar genomes of melon, squash, and watermelon to establish organellar DNA transmission. Serial dilutions of DNA demonstrated that our hybridizations revealed the presence of a polymorphic cytoplasm when it represented at least 1% of the DNA sample. At this level of sensitivity, the chloroplast genomes of melon, squash, and watermelon were maternally transmitted. The mitochondrial genomes of squash and watermelon were maternally transmitted; however, melon, like cucumber, showed paternal transmission of the mitochondrial genome. Because most angiosperms and the related genera Cucurbita and Citrullus show maternal transmission of the mtDNA, paternal transmission in Cucumis is likely the derived state. The Cucumis mitochondrial genomes are several-fold larger than those of other cucurbits. Based on 55 probe-enzyme combinations, mtDNA size differences could not be explained by duplication of the entire genome or partial duplication of regions hybridizing with the mitochondrial probes. Because the chloroplast, mitochondrial, and nuclear genomes of Cucumis are differentially transmitted, this genus is an excellent system to study the role of intergenomic transfer in the evolution of extremely large mitochondrial genomes. Received: 20 November 1997 / Accepted: 30 December 1997     

Hydroquinone peroxidase activity of maize root mitochondria

Summary. The oxidation of hydroquinone with H₂O₂ in the presence of mitochondria isolated from maize (Zea mays L.) roots was studied. The results indicate that a reduced form of quinone may be a substrate of mitochondrial peroxidases. Specific activities in different mitochondrial isolates, the apparent K _m for hydrogen peroxide and hydroquinone, and the influence of some known peroxidase inhibitors or effectors are presented. Zymographic assays revealed that all mitochondrial peroxidases, which were stained with 4-chloro-1-naphthol, were capable of oxidizing hydroquinone. A possible antioxidative role of hydroquinone peroxidase in H₂O₂ scavenging within the mitochondria, in cooperation with ascorbate or coupled with mitochondrial NAD(P)H dehydrogenases, is proposed. Correspondence: M. Vuletić, Laboratory of Plant Physiology, Maize Research Zemun Polje, P.O. Box 89, 11185 Belgrade, Serbia.     

An integrated physiological and genetic approach to the dynamics of FtsZ targeting and organisation in a moss, Physcomitrella patens

Plant FtsZ (filamentous temperature-sensitive Z) proteins are regarded as descendants of prokaryotic cell division proteins. We could show previously that four FtsZ isoforms of the moss Physcomitrella patens assemble into, and interact in, distinct structures inside the chloroplasts and in the cytosol. Their organisation and localisation patterns indicate an involvement in chloroplast and cell division and in the maintenance of chloroplast shape and integrity. The cellular processes of chloroplast division and maintenance of chloroplast shape were disturbed either by application of the beta-lactam antibiotic ampicillin or by a mutation that presumably affects signal transduction of the plant hormone cytokinin. When cells of these plants were analysed microscopically, there was no indication that cytosolic functions of FtsZ proteins were affected. Furthermore, FtsZ proteins continued to build three-dimensional plastoskeleton networks, even in considerably enlarged or malformed chloroplasts. On the other hand, macrochloroplast formation promoted the localisation of FtsZ proteins in filaments that emanate from the plastids and, therefore, most likely represent stromules. Annular FtsZ structures that are regarded as essential components of the division apparatus were absent from macrochloroplasts of ampicillin-treated cells. Thus, the distribution of FtsZ proteins after inhibition of chloroplast division further strengthens our hypothesis on the functions of distinct isoforms. In addition, the results provide further insight into the regulation of protein targeting and dynamics of plastoskeletal elements.     

Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.     

Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria

Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery. However, the situation is confusing for transmembrane domain (TMD)-containing signal-anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs: a basic residue and an arginine-rich region flanking the N- and C-termini of TMDs, respectively, and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co-translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.     

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.     

Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons

Protoporphyrinogen oxidase (Protox) is the final enzyme in the common pathway of chlorophyll and heme biosynthesis. Two Protox isoenzymes have been described in tobacco, a plastidic and a mitochondrial form. We isolated and sequenced spinach Protox cDNA, which encodes a homolog of tobacco mitochondrial Protox (Protox II). Alignment of the deduced amino acid sequence between Protox II and other tobacco mitochondrial Protox homologs revealed a 26-amino acid N-terminal extension unique to the spinach enzyme. Immunoblot analysis of spinach leaf extract detected two proteins with apparent molecular masses of 57 and 55 kDa in chloroplasts and mitochondria, respectively. In vitro translation experiments indicated that two translation products (59 and 55 kDa) are produced from Protox II mRNA, using two in-frame initiation codons. Transport experiments using green fluorescent protein-fused Protox II suggested that the larger and smaller translation products (Protox IIL and IIS) target exclusively to chloroplasts and mitochondria, respectively.