A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:proto¬chlorophyllide oxidoreductase (POR) A

NADPH: protochlorophyllide oxido reductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Amongthem are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radio labeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of35S-Oep16/Ptc16 is stimulated by GTP.35S-Oep16/Ptc16forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photo oxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.

Influence of denitrifiers abundance on N2O emissions in long term tillage system under a rainfed legume crop

Current studies about nitrous oxide (N2O) emissions from legume crops have raised considerable doubt, observing a high variability between sites (0.03-7.09 kg N2O–N ha−1 y -1) [1]. This high variability has been associated to climate and soil conditions, legume species and soil management practices (e.g. conservation or conventional tillage). Conservation tillage (i.e. no tillage (NT) and minimum tillage (MT)) has spread during the last decades because promotes several positive effects (increase of soil organic content, reduction of soil erosion and enhancement of carbon (C) sequestration). However, these benefits could be partly counterbalanced by negative effects on the release of N2O emissions. Among processes responsible for N2O production and consumption in soils, denitrification plays an importantrole both in tilled and no-tilled ropping systems [2]. Recently, amplification of functional bacterial genes involved in denitrification is being used to examine denitrifiers abundance and evaluate their influence on N2O emissions. NirK and nirS are functional genes encoding the cytochrome cd1 and copper nitrite reductase, which is the key enzyme regulating the denitrification process.

Regulation of chloroplast protein import through a protochlorophyllide-responsive transit peptide

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme of chlorophyll biosynthesis in angiosperms. In barley, two POR enzymes, termed PORA and PORB, exist. Both are nucleus-encoded plastid proteins that must be imported posttranslationally from the cytosol. Whereas the import of the precursor of PORA, pPORA, previously has been shown to depend on Pchlide, the import of pPORB occurred constitutively. To study this striking difference, chimeric precursor proteins were constructed in which the transit sequences of the pPORA and pPORB were exchanged and fused to either their cognate polypeptides or to a cytosolic dihydrofolate reductase (DHFR) reporter protein of mouse. As shown here, the transit peptide of the pPORA (transA) conferred the Pchlide requirement of import onto both the mature PORB and the DHFR. By contrast, the transit peptide of the pPORB directed the reporter protein into both chloroplasts that contained or lacked translocation-active Pchlide. In vitro binding studies further demonstrated that the transit peptide of the pPORA, but not of the pPORB, is able to bind Pchlide. We conclude that the import of the authentic pPORA and that of the transA-PORB and transA-DHFR fusion proteins is regulated by a direct transit peptide-Pchlide interaction, which is likely to occur in the plastid envelope, a major site of porphyrin biosynthesis.

The heme oxygenase gene (pbsA) in the red alga Rhodella violacea is discontinuous and transcriptionally activated during iron limitation

Heme oxygenase (HO) catalyzes the opening of the heme ring with the release of iron in both plants and animals. In cyanobacteria, red algae, and cryptophyceae, HO is a key enzyme in the synthesis of the chromophoric part of the photosynthetic antennae. In an attempt to study the regulation of this key metabolic step, we cloned and sequenced the pbsA gene encoding this enzyme from the red alga Rhodella violacea. The gene is located on the chloroplast genome, split into three distant exons, and is presumably expressed by a trans-splicing mechanism. The deduced polypeptide sequence is homologous to other reported HOs from organisms containing phycobilisomes (Porphyra purpurea and Synechocystis sp. strain PCC 6803) and, to a lesser extent, to vertebrate enzymes. The expression is transcriptionally activated under iron deprivation, a stress condition frequently encountered by algae, suggesting a second role for HO as an iron-mobilizing agent in photosynthetic organisms.

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

Degradation of Hepatic Stearyl CoA Δ9-Desaturase

Δ9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt–washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.

Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle

The glyoxylate cycle is regarded as essential for postgerminative growth and seedling establishment in oilseed plants. We have identified two allelic Arabidopsis mutants, icl-1 and icl-2, which lack the glyoxylate cycle because of the absence of the key enzyme isocitrate lyase. These mutants demonstrate that the glyoxylate cycle is not essential for germination. Furthermore, photosynthesis can compensate for the absence of the glyoxylate cycle during postgerminative growth, and only when light intensity or day length is decreased does seedling establishment become compromised. The provision of exogenous sugars can overcome this growth deficiency. The icl mutants also demonstrate that the glyoxylate cycle is important for seedling survival and recovery after prolonged dark conditions that approximate growth in nature. Surprisingly, despite their inability to catalyze the net conversion of acetate to carbohydrate, mutant seedlings are able to break down storage lipids. Results suggest that lipids can be used as a source of carbon for respiration in germinating oilseeds and that products of fatty acid catabolism can pass from the peroxisome to the mitochondrion independently of the glyoxylate cycle. However, an additional anaplerotic source of carbon is required for lipid breakdown and seedling establishment. This source can be provided by the glyoxylate cycle or, in its absence, by exogenous sucrose or photosynthesis.

Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.

Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas’ disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of ∼1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

Oxidative Turnover of Soybean Root Glutamine Synthetase. In Vitro and in Vivo Studies1

Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. GS in plants is an octameric enzyme. Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J. Temple, T.J. Knight, P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Gen Genet 236: 315–325; S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996] Plant Physiol 112: 1723–1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Taylor & Francis, London, pp 155–177). Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the ·OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level. This would suggest either translational or posttranslational control of GS levels.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Genetic Regulation and Cellular Expression1

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric β-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.