Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells.
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Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a approximately 4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II. (+info)
In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes.
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In Salmonella typhimurium, the CobU, CobS, CobT, and CobC proteins have been proposed to catalyze the late steps in adenosylcobalamin biosynthesis, which define the nucleotide loop assembly pathway. This paper reports the in vitro assembly of the nucleotide loop of adenosylcobalamin from its precursors adenosylcobinamide, 5, 6-dimethylbenzimidazole, nicotinate mononucleotide, and GTP. Incubation of these precursors with the CobU, CobS, and CobT proteins resulted in the synthesis of adenosylcobalamin-5'-phosphate. This cobamide was isolated by HPLC, identified by UV-visible spectroscopy and mass spectrometry, and shown to support growth of a cobalamin auxotroph. Adenosylcobalamin-5'-phosphate was also isolated from reaction mixtures containing adenosylcobinamide-GDP (the product of the CobU reaction) and alpha-ribazole-5'-phosphate (the product of the CobT reaction) as substrates and CobS. These results allowed us to conclude that CobS is the cobalamin(-5'-phosphate) synthase enzyme in S. typhimurium. The CobC enzyme, previously shown to dephosphorylate alpha-ribazole-5'-phosphate to alpha-ribazole, was shown to dephosphorylate adenosylcobalamin-5'-phosphate to adenosylcobalamin. Adenosylcobinamide was converted to adenosylcobalamin in reactions where all four enzymes were present in the reaction mixture. This in vitro system offers a unique opportunity for the rapid synthesis and isolation of cobamides with structurally different lower-ligand bases that can be used to investigate the contributions of the lower-ligand base to cobalamin-dependent reactions. (+info)
A Bacillus subtilis operon containing genes of unknown function senses tRNATrp charging and regulates expression of the genes of tryptophan biosynthesis.
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Strains of Bacillus subtilis containing a temperature-sensitive tryptophanyl-tRNA synthetase produce elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess tryptophan. This increase is because of reduced availability of the tryptophan-activated trp RNA-binding attenuation protein (TRAP). To test the hypothesis that this elevated trp gene expression was caused by the overproduction of a transcript capable of binding and sequestering TRAP, a computer program was designed to search the B. subtilis genome sequence for additional potential TRAP binding sites. A region containing a stretch of (G/A)AG trinucleotide repeats, characteristic of a TRAP binding site, was identified in the yczA-ycbK operon. We show that transcriptional regulation of the yczA-ycbK operon is controlled by the T-box antitermination mechanism in response to the level of uncharged tRNA(Trp), and that the presence of a trpS1 mutant allele increases production of the yczA-ycbK transcript. Elevated yczA-ycbK expression was shown to activate transcription of the trp operon. Deletion of the yczA-ycbK operon abolishes the trpS1 effect on trp gene expression. The purpose of increasing expression of the genes of tryptophan biosynthesis in the trpS mutant would be to provide additional tryptophan to overcome the charged tRNA(Trp) deficiency. Therefore, in B. subtilis, as in Escherichia coli, transcription of the tryptophan biosynthetic genes is regulated in response to changes in the extent of charging of tRNA(Trp) as well as the availability of tryptophan. (+info)
A Bacillus subtilis gene of previously unknown function, yhaG, is translationally regulated by tryptophan-activated TRAP and appears to be involved in tryptophan transport.
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Computer analysis of the Bacillus subtilis genome sequence revealed a gene with no previously attributed function, yhaG, specifying a transcript containing a presumptive binding site for the tryptophan-activated regulatory protein, TRAP. The presumptive TRAP binding site overlaps the yhaG Shine-Dalgarno sequence and translation initiation region. TRAP was shown to regulate expression of yhaG translationally. Production of the yhaG transcript in vivo was found to compete for the binding of TRAP to other known TRAP binding sites. YhaG is likely to be a transmembrane protein involved in tryptophan transport. (+info)
Phosphatidylinositol/phosphatidylcholine transfer proteins in yeast.
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Phosphatidylinositol transfer proteins (PITPs) are now becoming widely recognized as intriguing proteins that participate in the coordination and coupling of phospholipid metabolism with vesicle trafficking, and in the regulation of important signaling cascades. Yet, only in one case is there a large body of evidence that speaks to the precise identities of PITP-dependent cellular reactions, and to the mechanisms by which PITPs execute function in eukaryotic cells. At present, yeast provide the most powerful system for analysis of the physiology of PITP function in vivo, and the mechanism by which this function is carried out. Here, we review the recent progress and remaining questions in the area of PITP function in yeast. (+info)
The heterotrimeric Thermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln).
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Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn). Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated. (+info)
Phosphatidylserine synthase-1 and -2 are localized to mitochondria-associated membranes.
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We report the subcellular localization of enzymes involved in phosphatidylserine biosynthesis in mammalian cells. Several lines of evidence suggest that phosphatidylserine synthase-1 (PSS1) is highly enriched in mitochondria-associated membranes (MAM) and is largely excluded from the bulk of the endoplasmic reticulum (ER). Taking advantage of the substrate specificity of PSS1, we showed that (i) MAM contain choline exchange activity, whereas this activity is very low in the bulk of the ER, (ii) serine exchange activity is inhibited by choline to a much greater extent in MAM than in ER, and (iii) MAM use phosphatidylcholine and phosphatidylethanolamine as substrates for phosphatidylserine biosynthesis, whereas the ER utilizes only phosphatidylethanolamine. According to immunoblotting of proteins from both CHO-K1 cells and murine liver, PSS1 is localized to MAM, and in hepatoma cells stably expressing PSS1 this protein is highly enriched in MAM. Since the ER contains serine and ethanolamine exchange activities, we had predicted that PSS2 would account for the serine exchange activity in the ER. Unexpectedly, using immunoblotting experiments, we found that (i) PSS2 of CHO-K1 cells is present only in MAM and (ii) PSS2 is restricted to MAM of McArdle cells expressing recombinant PSS2. These data leave open the question of which enzyme imparts PSS activity to the ER and suggest that a third isoform of PSS might be located in the ER. (+info)
Decay of mutualistic potential in aphid endosymbionts through silencing of biosynthetic loci: Buchnera of Diuraphis.
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Buchnera, the primary bacterial endosymbiont of aphids, is known to provision essential amino acids lacking in the hosts' diet of plant sap. The recent discovery of silenced copies of genes for tryptophan biosynthesis (trpEG) in certain Buchnera lineages suggests a decay in symbiotic functions in some aphid species. However, neither the distribution of pseudogenes among lineages nor the impact of this gene silencing on amino-acid availability in hosts has been assessed. In Buchnera of the aphid Diuraphis noxia, tandem repeats of these pseudogenes have persisted in diverse lineages, and thpEG pseudogenes have originated at least twice within this aphid genus. Measures of amino-acid concentrations in Diuraphis species have shown that the presence of the pseudogene is associated with a decreased availability of tryptophan, indicating that gene silencing decreases nutrient provisioning by symbionts. In Buchnera of Diuraphis, rates of nonsynonymous substitutions are elevated in functional trpE copies, supporting the hypothesis that pseudogene origin and persistence reflect a reduced selection for symbiont biosynthetic contributions. The parallel evolution of trpEG pseudogenes in Buchnera of Diuraphis and certain other aphid hosts suggests that either selection at the host level is not effective or that fitness in these aphids is not limited by tryptophan availability. (+info)