Putative Mitochondrial Targeting Sequence Research Articles

Computational and mitochondrial functional studies of novel compound heterozygous variants in SPATA5 gene support a causal link with epileptogenic encephalopathy

The SPATA5 gene encodes a 892 amino-acids long protein that has a putative mitochondrial targeting sequence and has been proposed to function in maintenance of mitochondrial function and integrity during mouse spermatogenesis. Several studies have associated homozygous or compound heterozygous mutations in SPATA5 gene to microcephaly, intellectual disability, seizures and hearing loss. This suggests a role of the SPATA5 gene also in neuronal development. Recently, our group presented results validating the use of blood cells for the assessment of mitochondrial function for diagnosis and follow-up of mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsy. In this study, we were able to diagnose a patient with epileptogenic encephalopathy using next generation sequencing. We found two novel compound heterozygous variants in SPATA5 that are most likely causative. To analyze the impact of SPATA5 mutations on mitochondrial functional studies directly on the patients' mononuclear cells and platelets were undertaken. Oxygen consumption rates in platelets and PBMCs were impaired in the patient when compared to a healthy control. Also, a decrease in mitochondrial mass was observed in the patient monocytes with respect to the control. This suggests a true pathogenic effect of the mutations in mitochondrial function, especially in energy production and possibly biogenesis, leading to the observed phenotype.

Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

ObjectiveCholesterol plays a pivotal role in mitochondrial steroidogenesis, membrane structure, and respiration. Mitochondrial membranes are intrinsically low in cholesterol content and therefore must be replenished with cholesterol from other subcellular membranes. However, the molecular mechanisms underlying mitochondrial cholesterol transport remains poorly understood. The Aster-B gene encodes a cholesterol binding protein recently implicated in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER). In this study, we investigated the function and underlying mechanism of Aster-B in mediating mitochondrial cholesterol transport. MethodsCRISPR/Cas9 gene editing was carried out to generate cell lines deficient in Aster-B expression. The effect of Aster-B deficiency on mitochondrial cholesterol transport was examined by both confocal imaging analysis and biochemical assays. Deletion mutational analysis was also carried out to identify the function of a putative mitochondrial targeting sequence (MTS) at the N-terminus of Aster-B for its role in targeting Aster-B to mitochondria and in mediating mitochondrial cholesterol trafficking. ResultsAblation of Aster-B impaired cholesterol transport from the ER to mitochondria, leading to a significant decrease in mitochondrial cholesterol content. Aster-B is also required for mitochondrial transport of fatty acids derived from hydrolysis of cholesterol esters. A putative MTS at the N-terminus of Aster-B mediates the mitochondrial cholesterol uptake. Deletion of the MTS or ablation of Arf1 GTPase which is required for mitochondrial translocation of ER proteins prevented mitochondrial cholesterol transport, leading to mitochondrial dysfunction. ConclusionsWe identified Aster-B as a key regulator of cholesterol transport from the ER to mitochondria. Aster-B also coordinates mitochondrial cholesterol trafficking with uptake of fatty acids derived from cholesterol esters, implicating the Aster-B protein as a novel regulator of steroidogenesis.

A conserved mammalian mitochondrial isoform of acetyl-CoA carboxylase ACC1 provides the malonyl-CoA essential for mitochondrial biogenesis in tandem with ACSF3.

Mitochondrial fatty acid synthesis (mtFAS) is a highly conserved pathway essential for mitochondrial biogenesis. The mtFAS process is required for mitochondrial respiratory chain assembly and function, synthesis of the lipoic acid cofactor indispensable for the function of several mitochondrial enzyme complexes and essential for embryonic development in mice. Mutations in human mtFAS have been reported to lead to neurodegenerative disease. The source of malonyl-CoA for mtFAS in mammals has remained unclear. We report the identification of a conserved vertebrate mitochondrial isoform of ACC1 expressed from an ACACA transcript splicing variant. A specific knockdown (KD) of the corresponding transcript in mouse cells, or CRISPR/Cas9-mediated inactivation of the putative mitochondrial targeting sequence in human cells, leads to decreased lipoylation and mitochondrial fragmentation. Simultaneous KD of ACSF3, encoding a mitochondrial malonyl-CoA synthetase previously implicated in the mtFAS process, resulted in almost complete ablation of protein lipoylation, indicating that these enzymes have a redundant function in mtFAS. The discovery of a mitochondrial isoform of ACC1 required for lipoic acid synthesis has intriguing consequences for our understanding of mitochondrial disorders, metabolic regulation of mitochondrial biogenesis and cancer.

Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking.

A potential antioxidant enzyme belonging to the atypical 2-Cys peroxiredoxin subfamily characterized from rock bream, Oplegnathus fasciatus.

Peroxiredoxins (Prxs), a diverse family of antioxidant enzymes, exert their antioxidant function through which different peroxide species are detoxified. This study describes both structural and functional characterization of a mitochondrial Prx identified in rock bream, Oplegnathus fasciatus (RbPrx5). The ORF (573 bp) of RbPrx5 encoded a protein of 190 amino acids (20 kDa) containing a putative mitochondrial targeting sequence (residues 1-20) and a thioredoxin-2 motif (residues 31-190) and three conserved Cys residues. Homology assessment and phylogenetic analysis clearly disclosed relatively higher amino acid sequence similarities and a closer evolutionary position of RbPrx5 with those of other teleost homologs. The ORF of RbPrx5 was distributed among six exons as found in other vertebrates, but it possessed an additional exon in its 5'-UTR. In silico examination of RbPrx5 gene's putative promoter region revealed the presence of several cis-elements which may be important in its transcriptional regulation. Constitutive expression of RbPrx5 was detected in eleven tissues with the highest level in the heart. Modulation of RbPrx5 transcription was evidenced from varying mRNA levels in head kidney post in vivo LPS-, poly I:C-, Edwardsiella tarda bacterial- and rock bream iridoviral-challenges. The antioxidant function of RbPrx5 was investigated using recombinant RbPrx5 protein. Results of an in vitro mixed-function oxidase assay demonstrated a dose-dependent inhibition of DNA damage by rRbPrx5. A H2O2 tolerance assay showed that in vivo overexpression of rRbPrx5 increased the bacterial survival under H2O2-mediated oxidative stress condition. These findings provide an overall insight into the structural, expressional and functional aspects of RbPrx5.

Citrate synthase from the liver fluke Fasciola hepatica

Citrate synthase catalyses the first step of the Krebs' tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K m for oxaloacetate was found to be 0.22mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.

GFP-tagging of Arabidopsis acyl-activating enzymes raises the issue of peroxisome-chloroplast import competition versus dual localization

Protein sequence analysis of a subfamily of 18 Arabidopsis acyl-activating enzymes (AAE) for organelle targeting signals revealed that eight of them possessed putative peroxisomal targeting signals (PTS1), five of which belonged to Clade VI of the AAE superfamily. Peroxisomal localization was confirmed by confocal microscopy of green fluorescent protein (GFP)-AAE fusion proteins co-localizing with peroxisomal RFP. The sequence analysis also revealed that all enzymes of Clade VI possess N-terminal regions indicative of chloroplast transit peptides (cTP). Among the five Clade VI peroxisomal enzymes tested, masking the PTS1 signal with GFP redirected three to plastids. In addition, three other peroxisomal AAEs appeared to be redirected to plastids in AAE-GFP fusion constructs. Due to the lack of evidence supporting plastid localization, we propose that competition dictates the exclusive localization to peroxisomes. AAE2 of Clade VI was the only enzyme with a putative mitochondrial targeting sequence, and it appeared to be targeted to mitochondria. The remainder of the AAEs appeared to be localized to plastids or cytosol. The AAE9-GFP fusion protein appeared to be located within discreet structures within plastids that may be plastoglobules. AAE15-GFP, but not AAE16-GFP appeared to be located in the chloroplast envelope. The number of examples is increasing whereby proteins located within other compartments contribute to plastid function. We provide an example of this through the light-sensitive phenotype of mutants of AAE2.

Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn(2+) ions for phosphatase activity, whereas Mg(2+) and Ca(2+) ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nm and 10 μm for Mn(2+) and Mg(2+) ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca(2+)-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K(m) for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.

Hydrogenosome-localization of arginine deiminase in Trichomonas vaginalis

The arginine dihydrolase (ADH) pathway has an analogous function to the urea cycle in mitochondria-containing cells, by removing nitrogen from amino acids and generating ATP. Subcellular localization of the ADH pathway enzymes in Trichomonas vaginalis revealed that arginine deiminase (ADI) localizes to the hydrogenosome, a mitochondrion-like organelle of anaerobic protists. However the other enzymes of the ADH pathway, ornithine carbamyltransferase and carbamate kinase localize to the cytosol. Three gene sequences of T. vaginalis ADI (ADI 1–3) were identified in the T. vaginalis genome, all having putative mitochondrial targeting sequences. The ADI sequences were cloned and used to probe T. vaginalis using a carboxyterminal di-hemogglutinin epitope tag which demonstrated co-localization with malic enzyme confirming the hydrogenosome localization of this enzyme.

Schistosoma mansoni Stomatin Like Protein-2 Is Located in the Tegument and Induces Partial Protection against Challenge Infection

BackgroundSchistosomiasis affects more than 200 million individuals worldwide, with a further 650 million living at risk of infection, constituting a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection, and the development of an effective vaccine still remains the most desirable means of control for this disease.Methodology/Principal FindingsHerein, we report the cloning and characterization of a S. mansoni Stomatin-like protein 2 (SmStoLP-2). In silico analysis predicts three putative sites for palmitoylation (Cys11, Cys61 and Cys330), which could contribute to protein membrane association; and a putative mitochondrial targeting sequence, similar to that described for human Stomatin-like protein 2 (HuSLP-2). The protein was detected by Western blot with comparable levels in all stages across the parasite life cycle. Fractionation by differential centrifugation of schistosome tegument suggested that SmStoLP-2 displays a dual targeting to the tegument membranes and mitochondria; additionally, immunolocalization experiments confirm its localization in the tegument of the adult worms and, more importantly, in 7-day-old schistosomula. Analysis of the antibody isotype profile to rSmStoLP-2 in the sera of patients living in endemic areas for schistosomiasis revealed that IgG1, IgG2, IgG3 and IgA antibodies were predominant in sera of individuals resistant to reinfection as compared to those susceptible. Next, immunization of mice with rSmStoLP-2 engendered a 30%–32% reduction in adult worm burden. Protective immunity in mice was associated with specific anti-rSmStoLP-2 IgG1 and IgG2a antibodies and elevated production of IFN-γ and TNF-α, while no IL-4 production was detected, suggesting a Th1-predominant immune response.Conclusions/SignificanceData presented here demonstrate that SmStoLP-2 is a novel tegument protein located in the host-parasite interface. It is recognized by different subclasses of antibodies in patients resistant and susceptible to reinfection and, based on the data from murine studies, shows protective potential against schistosomiasis. These results indicate that SmStoLP-2 could be useful in a combination vaccine.

Genetic Mapping of Glutaric Aciduria, Type 3, to Chromosome 7 and Identification of Mutations in C7orf10

While screening Old Order Amish children for glutaric aciduria type 1 (GA1) between 1989 and 1993, we found three healthy children who excreted abnormal quantities of glutaric acid but low 3-hydroxyglutaric acid, a pattern consistent with glutaric aciduria type 3 (GA3). None of these children had the GCDH c.1262C-->T mutation that causes GA1 among the Amish. Using single-nucleotide polymorphism (SNP) genotypes, we identified a shared homozygous 4.7 Mb region on chromosome 7. This region contained 25 genes including C7orf10, an open reading frame with a putative mitochondrial targeting sequence and coenzyme-A transferase domain. Direct sequencing of C7orf10 revealed that the three Amish individuals were homozygous for a nonsynonymous sequence variant (c.895C-->T, Arg299Trp). We then sequenced three non-Amish children with GA3 and discovered two nonsense mutations (c.322C-->T, Arg108Ter, and c.424C-->T, Arg142Ter) in addition to the Amish mutation. Two pathogenic alleles were identified in each of the six patients. There was no consistent clinical phenotype associated with GA3. In affected individuals, urine molar ratios of glutarate to its derivatives (3-hydroxyglutarate, glutarylcarnitine, and glutarylglycine) were elevated, suggesting impaired formation of glutaryl-CoA. These observations refine our understanding of the lysine-tryptophan degradation pathway and have important implications for the pathophysiology of GA1.

AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.

CDNA cloning and characterization of the novel genes related to aldehyde dehydrogenase from wild Chinese grape (Vitis pseudoreticulataW. T. Wang)

mRNA differential display was employed to study the gene differential expression of wild Chinese grape (Vitis pseudoreticulata W. T. Wang) infected by Uncinula necator in different periods, a cDNA fragment of T11AC/B0319-456 coded by aldehyde dehydrogenase (ALDH) gene has been obtained. 5' RACE and 3' RACE have been used to clone the whole cDNA sequences of ALDH which consists of three cDNA sequences, whose sizes are 1887, 1956 and 1961 bp, and they encoded a polypeptide size of 537, 524 and 477 designated as VpALDH2a, VpALDH2b and VpALDH1a, respectively. The deduced amino acid sequence shared highly identity with other plants and Human ALDH. Both VpALDH2a and VpALDH2b protein contain putative mitochondrial targeting sequence except VpALDH1a, it indicates that VpALDH2a and VpALDH2b are mitochondrial enzymes, and VpALDH1a is cytosolic enzyme. The VpALDH2a was subcloned into the expression vector pGEX-4T-1, transformed into E.coli BL 21-coden plus induced by IPTG, and about Mr. 85 kD of GST-ALDH fusion protein displayed in SDS-PAGE gel.

Characterization of mitochondrial ferritin in Drosophila.

Mitochondrial function depends on iron-containing enzymes and proteins, whose maturation requires available iron for biosynthesis of iron-sulfur clusters and heme. Little is known about how mitochondrial iron homeostasis is maintained, although the recent discovery of a mitochondrial ferritin in mammals and plants has uncovered a potential key player in the process. Here, we show that Drosophila melanogaster expresses mitochondrial ferritin from an intron-containing gene. It has high similarity to the mouse and human mitochondrial ferritin sequences and, as in mammals, is expressed mainly in testis. This ferritin contains a putative mitochondrial targeting sequence and an epitope-tagged version localizes to mitochondria in transfected cells. Overexpression of mitochondrial ferritin fails to alter both total-body iron levels and iron that is bound to secretory ferritins. However, the viability of iron-deficient flies is compromised by overexpression of mitochondrial ferritin, suggesting that it may sequester iron at the expense of other important cellular functions. The conservation of mitochondrial ferritin in an insect species underscores the importance of this iron-storage molecule.

Wild-type PINK1 Prevents Basal and Induced Neuronal Apoptosis, a Protective Effect Abrogated by Parkinson Disease-related Mutations

Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations.

Expression, crystallization and preliminary X-ray crystallographic analysis of human agmatinase

Agmatine, which results from the decarboxylation of L-arginine by arginine decarboxylase, is a metabolic intermediate in the biosynthesis of putresine and higher polyamines (spermidine and spermine). Recent studies indicate that agmatine can have several important biochemical effects in humans, ranging from effects on the central nervous system to cell proliferation in cancer and viral replication. Agmatinase catalyses the hydrolysis of agmatine to putresine and urea and is a major target for drug action and development. The human agmatinase gene encodes a 352-residue protein with a putative mitochondrial targeting sequence at the N-terminus. Human agmatinase (residues Ala36-Val352) has been overexpressed as a fusion with both N- and C-terminal purification tags in Escherichia coli and crystallized in the presence of Mn2+ and 1,6-diaminohexane at 297 K using polyethylene glycol 4000 as a precipitant. X-ray diffraction data were collected at 100 K to 2.49 A from a flash-frozen crystal. The crystals are tetragonal, belonging to space group P4(2), with unit-cell parameters a = b = 114.54, c = 125.65 A, alpha = beta = gamma = 90 degrees. Three monomers are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.66 A3 Da(-1) and a solvent content of 66.4%.

Molecular basis for mitochondrial localization of viral particles during beet necrotic yellow vein virus infection.

During infection, Beet necrotic yellow vein virus (BNYVV) particles localize transiently to the cytosolic surfaces of mitochondria. To understand the molecular basis and significance of this localization, we analyzed the targeting and membrane insertion properties of the viral proteins. ORF1 of BNYVV RNA-2 encodes the 21-kDa major coat protein, while ORF2 codes for a 75-kDa minor coat protein (P75) by readthrough of the ORF1 stop codon. Bioinformatic analysis highlighted a putative mitochondrial targeting sequence (MTS) as well as a major (TM1) and two minor (TM3 and TM4) transmembrane regions in the N-terminal part of the P75 readthrough domain. Deletion and gain-of-function analyses based on the localization of green fluorescent protein (GFP) fusions showed that the MTS was able to direct a reporter protein to mitochondria but that the protein was not persistently anchored to the organelles. GFP fused either to MTS and TM1 or to MTS and TM3-TM4 efficiently and specifically associated with mitochondria in vivo. The actual role of the individual domains in the interaction with the mitochondria seemed to be determined by the folding of P75. Anchoring assays to the outer membranes of isolated mitochondria, together with in vivo data, suggest that the TM3-TM4 domain is the membrane anchor in the context of full-length P75. All of the domains involved in mitochondrial targeting and anchoring were also indispensable for encapsidation, suggesting that the assembly of BNYVV particles occurs on mitochondria. Further data show that virions are subsequently released from mitochondria and accumulate in the cytosol.

A novel senescence‐associated gene encoding γ‐aminobutyric acid (GABA):pyruvate transaminase is upregulated during rice leaf senescence

The gene Osl2, which is specifically upregulated during leaf senescence in rice (Oryza sativa L. cv. Tainong 67), was cloned and functionally characterized. The protein coding region of the gene consists of 19 exons encoding 516 amino acids, with a putative mitochondrial targeting sequence and conserved sequence of the pyridoxal 5′‐phosphate‐binding domain. The recombinant Osl2 fusion protein over‐expressed in Escherichia coli displays pyruvate‐dependent γ‐aminobutyric acid (GABA) transaminase (EC 2.6.1.19) activity. Examination of the expression patterns of the Osl2 gene in rice reveals that Osl2‐specific transcripts are induced in the senescing leaves. The temporal profile of Osl2 protein accumulation is correlated with that of pyruvate‐dependent GABA transaminase activity in rice leaves, with the highest expression level at the S3 senescent stage. The potential role for GABA transaminase during rice leaf senescence is discussed.

Mitochondrial phosphatidylserine decarboxylase from higher plants. Functional complementation in yeast, localization in plants, and overexpression in Arabidopsis.

Plants are known to synthesize ethanolamine (Etn) moieties by decarboxylation of free serine (Ser), but there is also some evidence for phosphatidyl-Ser (Ptd-Ser) decarboxylation. Database searches identified diverse plant cDNAs and an Arabidopsis gene encoding 50-kD proteins homologous to yeast (Saccharomyces cerevisiae) and mammalian mitochondrial Ptd-Ser decarboxylases (PSDs). Like the latter, the plant proteins have putative mitochondrial targeting and inner membrane sorting sequences and contain near the C terminus a Glycine-Serine-Threonine motif corresponding to the site of proteolysis and catalytic pyruvoyl residue formation. A truncated tomato (Lycopersicon esculentum) cDNA lacking the targeting sequence and a chimeric construct in which the targeting and sorting sequences were replaced by those from yeast PSD1 both complemented the Etn requirement of a yeast psd1 psd2 mutant, and PSD activity was detected in the mitochondria of the complemented cells. Immunoblot analysis of potato (Solanum tuberosum) mitochondria demonstrated that PSD is located in mitochondrial membranes, and mRNA analysis in Arabidopsis showed that the mitochondrial PSD gene is expressed at low levels throughout the plant. An Arabidopsis knockup mutant grew normally but had 6- to 13-fold more mitochondrial PSD mRNA and 9-fold more mitochondrial PSD activity. Total membrane PSD activity was, however, unchanged in the mutant, showing mitochondrial activity to be a minor part of the total. These results establish that plants can synthesize Etn moieties via a phospholipid pathway and have both mitochondrial and extramitochondrial PSDs. They also indicate that mitochondrial PSD is an important housekeeping enzyme whose expression is strongly regulated at the transcriptional level.

Isocitrate dehydrogenase of Plasmodium falciparum.

Erythrocytic stages of the malaria parasite Plasmodium falciparum rely on glycolysis for their energy supply and it is unclear whether they obtain energy via mitochondrial respiration albeit enzymes of the tricarboxylic acid (TCA) cycle appear to be expressed in these parasite stages. Isocitrate dehydrogenase (ICDH) is either an integral part of the mitochondrial TCA cycle or is involved in providing NADPH for reductive reactions in the cell. The gene encoding P. falciparum ICDH was cloned and analysis of the deduced amino-acid sequence revealed that it possesses a putative mitochondrial targeting sequence. The protein is very similar to NADP+-dependent mitochondrial counterparts of higher eukaryotes but not Escherichia coli. Expression of full-length ICDH generated recombinant protein exclusively expressed in inclusion bodies but the removal of 27 N-terminal amino acids yielded appreciable amounts of soluble ICDH consistent with the prediction that these residues confer targeting of the native protein to the parasites' mitochondrion. Recombinant ICDH forms homodimers of 90 kDa and its activity is dependent on the bivalent metal ions Mg2+ or Mn2+ with apparent Km values of 13 micro m and 22 micro m, respectively. Plasmodium ICDH requires NADP+ as cofactor and no activity with NAD+ was detectable; the for NADP+ was found to be 90 micro m and that of d-isocitrate was determined to be 40 micro m. Incubation of P. falciparum under exogenous oxidative stress resulted in an up-regulation of ICDH mRNA and protein levels indicating that the enzyme is involved in mitochondrial redox control rather than energy metabolism of the parasites.