Gamma carbonic anhydrases in plant mitochondria

Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (γCA), a Zn containing enzyme from Methanosarcina thermophila(CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of γCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from α and γ proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain γCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.     

线粒体蛋白质运输机制

：线粒体的大多数蛋白质是由核基因编码、细胞质合成,而最终运输到线粒体。在此过程中,需要线粒体外膜和内膜的蛋白质运输机器(至少三种主要的移位酶复合物)来保证前体蛋白质的正确运输。     

The role of plant mitochondria in the biosynthesis of coenzymes

This last decade, many efforts were undertaken to understand how coenzymes, including vitamins, are synthesized in plants. Surprisingly, these metabolic pathways were often “quartered” between different compartments of the plant cell. Among these compartments, mitochondria often appear to have a key role, catalyzing one or several steps in these pathways. In the present review we will illustrate these new and important biosynthetic functions found in plant mitochondria by describing the most recent findings about the synthesis of two vitamins (folate and biotin) and one non-vitamin coenzyme (lipoate). The complexity of these metabolic routes raise intriguing questions, such as how the intermediate metabolites and the end-product coenzymes are exchanged between the various cellular territories, or what are the physiological reasons, if any, for such compartmentalization.     

Post-Golgi protein traffic in the plant secretory pathway

The Golgi apparatus in plants is organized as a multitude of individual stacks that are motile in the cytoplasm and in close association with the endoplasmic reticulum (ER) (Boevink et al. in Plant J 15:441–447, 1998). These stacks operate as a sorting centre for cargo molecules, providing modification and redirection to other organelles as appropriate. In the post-Golgi direction, these include vacuole and plasma membrane, and specialized transport routes to each are required to prevent mislocalization. Recent evidence in plant cells points to the existence of post-Golgi organelles that function as intermediate stations for efficient protein traffic, as well as to the influence of small GTPases such as Rabs and ARFs on post-Golgi trafficking. This review focuses on the latest findings on post-Golgi trafficking routes and on the involvement of GTPases and their effectors on the trafficking of proteins in the plant secretory pathway. Sally L. Hanton and Loren A. Matheson have contributed equally to this work.     

RNA editing sites in plant mitochondria can share cis-elements

RNA editing in flowering plant mitochondria alters numerous C nucleotides in a given mRNA molecule to U residues. To investigate whether neighbouring editing sites can influence each other we analyzed in vitro RNA editing of two sites spaced 30 nt apart. Deletion and competition experiments show that these two sites carry independent essential specificity determinants in the respective upstream 20-30 nucleotides. However, deletion of a an upstream sequence region promoting editing of the upstream site concomitantly decreases RNA editing of the second site 50-70 nucleotides downstream. This result suggests that supporting cis-/trans-interactions can be effective over larger distances and can affect more than one editing event.     

Sorting out glycosylation enzymes in the Golgi apparatus

The study of glycosylation and glycosylation enzymes has been instrumental for the advancement of Cell Biology. After Neutra and Leblond showed that the Golgi apparatus is the main site of glycosylation, elucidation of oligosaccharide structures by Baenziger and Kornfeld and subsequent mapping of glycosylation enzymes followed. This enabled development of an in vitro transport assay by Rothman and co-workers using glycosylation to monitor intra Golgi transport which, complemented by yeast genetics by Schekman and co-workers, provided much of the fundamental insights and key components of the secretory pathway that we today take for granted. Glycobiology continues to play a key role in Cell Biology and here, we look at the use of glycosylation enzymes to elucidate intra Golgi transport.     

Phosphoenolpyruvate efflux from kidney cortex mitochondria of rabbit

(1) The relationship between phosphoenolpyruvate formation and its accumulation in kidney cortex mitochondria of rabbit was studied in the presence of glutamate as substrate. (2) In mitochondria incubated in either State 4 or under uncoupled conditions, both 1,2,3-benzenetricarboxylate and atractyloside resulted in a marked elevation of the intramitochondrial phosphoenolpyruvate accompanied by a 2–4-fold decline in production of this compound. The same effect was induced by n-butylmalonate in uncoupled mitochondria, while both phosphoenolpyruvate efflux and its production were inhibited to a smaller extent in mitochondria incubated with 1,2,3-benzenetricarboxylate in State 3. (3) Citrate, malate or 2-phosphoglycerate caused a fast displacement of phosphoenolpyruvate from atractyloside-inhibited mitochondria to the reaction medium. In contrast, on the addition of ATP to mitochondria incubated with 1,2,3-benzenetricarboxylate, the rate of phosphoenolpyruvate efflux was lower than that induced by either malate or citrate. (4) Despite the presence of both 1,2,3-benzenetricarboxylate and atractyloside, arsenite and rotenone plus antimycin resulted in a leakage of phosphoenolpyruvate from the mitochondria, probably via a carrier-independent mechanism. (5) Based on the present results it seems that depending on the metabolic condition, the tricarboxylate carrier and the adenine nucleotide translocase are functioning to different extents in the efflux of phosphoenolpyruvate from rabbit renal mitochondria to the surrounding medium.     

Role of autolysins in the EDTA-induced lysis of Pseudomonas aeruginosa

Abstract A DNA fragment containing the genes secE, nusG and rplK of Staphylococcus carnosus was cloned using the Escherichia coli rplK gene as a probe. The S. carnosus secE homologue encodes a protein of 65 amino acid residues which is homologous to the carboxyl-terminal region of the E. coli SecE protein. The S. carnosus SecE polypeptide which, in contrast to the E. coli SecE protein, contains only one putative transmembrane segment, could fully replace the E. coli SecE protein in two different secE mutants. These results strongly suggest that the identified secE gene encodes an important component of the S. carnosus protein export apparatus.     

Facilitated glucose and dehydroascorbate transport in plant mitochondria

Ascorbate, dehydroascorbate, and glucose transport was investigated in plant mitochondria and mitoplasts prepared from cultured BY2 tobacco cells. Using a rapid filtration method with radiolabeled ligands, we observed a specific glucose and dehydroascorbate transport, which was temperature and time dependent and saturable. Inhibition of mitochondrial respiration by KCN and the uncoupler 2,4-dinitrophenol did not influence the transport of the investigated compounds. Dehydroascorbate transport was inhibited by glucose and genistein, while glucose uptake was decreased upon 3-O-methyl-glucose, D-mannose, cytochalasin B or genistein addition. On the other hand, a low affinity low capacity ascorbate transport was found. Oxidizing agents (potassium ferricyanide or ascorbate oxidase) increased ascorbate uptake. The results demonstrate the presence of dehydroascorbate and glucose transport in plant mitochondria and suggest that it is mediated by the same or closely related transporter(s).     

A tripartite group II intron in mitochondria of an angiosperm plant

In mitochondria of flowering plants the nad5 open reading frame is assembled from five exons via two conventional cis-splicing and two trans-splicing events. Trans-splicing between exons c and d in wheat, petunia and Arabidopsis involves a bipartite group II intron structure, while in Oenothera a large portion of intron domains I–IV is missing from the major genomic locus. This intron region has been lost downstream of exon c and is now found in a distant genomic region. Intragenomic recombination across an 11 nucleotide sequence has separated these intron parts, which now have to be reassembled from three independent RNA precursors. This organisation coexists with highly substoichiometric copy numbers of the bipartite intron arrangement, consistent with an evolutionary origin of the tripartite intron by genomic disruption. Received: 28 August 1996 / Accepted: 11 December 1996     

The 23-kDa light-stress-regulated heat-shock protein of Chenopodium rubrum L. is located in the mitochondria

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et␣al. (1995, Biochem J 311:805–813) and LaFayette et␣al. (1996, Plant Mol Biol 30:159–169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions. Received: 11 July 1996 / Accepted: 24 August 1996     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

The plant translational apparatus

Protein synthesis in both eukaryotic and prokaryotic cells is a complex process requiring a large number of macromolecules: initiation factors, elongation factors, termination factors, ribosomes, mRNA, amino-acylsynthetases and tRNAs. This review focuses on our current knowledge of protein synthesis in higher plants.Abbreviations eIF eukaryotic initiation factor - eEF eukaryotic elongation factor - EST expressed sequence tag - eRF eukaryotic release factor - GUS -glucoronidase - HCR heme-controlled repressor - PKR double-stranded - RNA activated protein kinase - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Modulation of matrix Ca2+ content by the ADP/ATP carrier in brown adipose tissue mitochondria. Influence of membrane lipid composition

The role of the adenine nucleotide translocase on Ca²⁺ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 M Ca²⁺, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca²⁺ release, even though it inhibits the ADP exchange reaction. However, when the Ca²⁺ concentration was increased to 150 M, carboxyatractyloside did induce Ca²⁺ release, and ADP favored Ca²⁺ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It is suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.     

A matrix-located processing peptidase of plant mitochondria

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc₁ complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the F_Ad subunit of the ATP synthetase and the tobacco F₁ subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bc₁ complex of the respiratory chain.     

The electron transport systems of Fasciola hepatica mitochondria.

The electron transport systems of Fasciola hepatica mitochondria were investigated spectrophotometrically at room temperature and at −196°. The mitochondria were found to contain substrate reducible a-, b- and c-type cytochromes. All of the cytochrome components of the classical mammalian type of respiratory chain were present, although the concentration of cytochromes aa₃ was low. In addition to the mammalian type of respiratory chain, the Fasciola mitochondria contained a substrate reducible b-type cytochrome component (557 nm) which included a CO reactive o-type cytochrome. The results suggest that F. hepatica mitochondria contain a branched electron transport system including a mammalian type of chain and involving two terminal oxidases and at least two b-type cytochromes.     

In vitro RNA editing in plant mitochondria does not require added energy

RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.     

The experimental herbicide UKJ72J is an inhibitor of succinate oxidation in plant mitochondria

Fragments derived from human plasma fibronectin by enzymatic degradation were tested in the Boyden chamber for chemotactic activity towards various fibroblast strains. The results provide clear evidence that the chemotactic activity is restricted to a defined region of the fibronectin molecule which is the same for various fibroblast strains. The active domain is localized between the collagen binding site and the major heparin binding site, about 170 kDa apart from the N-terminal and about 70 kDa from the C-terminal ends of the two subunit peptide chains.     

Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.