scholarly journals A lysine accumulation phenotype of ScIpk2Δ mutant yeast is rescued by Solanum tuberosum inositol phosphate multikinase

2007 ◽  
Vol 403 (3) ◽  
pp. 381-389 ◽  
Author(s):  
Samuel E. K. Caddick ◽  
Christopher J. Harrison ◽  
Ioanna Stavridou ◽  
Sue Johnson ◽  
Charles A. Brearley
Keyword(s):  
Solanum Tuberosum ◽  
Amino Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Amino Acid Content ◽  
Sequence Divergence ◽  
Direct Interaction ◽  
Nitrogen Sources ◽  
Inositol Polyphosphate ◽  
Wild Type

Inositol phosphates and the enzymes that interconvert them are key regulators of diverse cellular processes including the transcriptional machinery of arginine synthesis [York (2006) Biochim. Biophys. Acta 1761, 552–559]. Despite considerable interest and debate surrounding the role of Saccharomyces cerevisiae inositol polyphosphate kinase (ScIPK2, ARG82, ARGRIII) and its inositol polyphosphate products in these processes, there is an absence of data describing how the transcripts of the arginine synthetic pathway, and the amino acid content of ScIpk2Δ, are altered under different nutrient regimes. We have cloned an IPMK (inositol phosphate multikinase) from Solanum tuberosum, StIPMK (GenBank® accession number EF362785), that despite considerable sequence divergence from ScIPK2, restores the arginine biosynthesis pathway transcripts ARG8, acetylornithine aminotransferase, and ARG3, ornithine carbamoyltransferase of ScIpk2Δ yeast to wild-type profiles. StIPMK also restores the amino acid profiles of mutant yeast to wild-type, and does so with ornithine or arginine as the sole nitrogen sources. Our data reveal a lysine accumulation phenotype in ScIpk2Δ yeast that is restored to a wild-type profile by expression of StIPMK, including restoration of the transcript profiles of lysine biosynthetic genes. The StIPMK protein shows only 18.6% identity with ScIPK2p which probably indicates that the rescue of transcript and diverse amino acid phenotypes is not mediated through a direct interaction of StIPMK with the ArgR–Mcm1 transcription factor complex that is a molecular partner of ScIPK2p.

scholarly journals Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

1987 ◽  
Vol 7 (6) ◽  
pp. 2231-2242 ◽  
Author(s):  
J E Rudolph ◽  
M Kimble ◽  
H D Hoyle ◽  
M A Subler ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Sequence Divergence ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Structural Constraints ◽  
Wild Type ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Single Amino Acid Residue ◽  
Beta 2

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.

scholarly journals Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation

1989 ◽  
Vol 260 (1) ◽  
pp. 237-241 ◽  
Author(s):  
I H Batty ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Muscarinic Receptor ◽  
Metabolic Flux ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Inositol Polyphosphate ◽  
Receptor Stimulation ◽  
Cortex Slices ◽  
Rapid Kinetics

The rapid kinetics of [3H]inositol phosphate accumulation and turnover were examined in rat cerebral-cortex slices after muscarinic-receptor stimulation. Markedly increased [3H]inositol polyphosphate concentrations were observed to precede significant stimulated accumulation of [3H]inositol monophosphate. New steady-state accumulations of several 3H-labelled products were achieved after 5-10 min of continued agonist stimulation, but were rapidly and effectively reversed by subsequent receptor blockade. The results show that muscarinic-receptor activation involves phosphoinositidase C-catalysed hydrolysis initially of polyphosphoinositides rather than of phosphatidylinositol. Furthermore, prolonged carbachol stimulation is shown not to cause receptor desensitization, but to allow persistent hydrolysis of [3H]phosphatidylinositol bisphosphate and permit sustained metabolic flux through the inositol tris-/tetrakis-phosphate pathway.

scholarly journals Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of anl-Arginine/γ-Aminobutyric Acid Permease inMycobacterium bovis BCG

2000 ◽  
Vol 182 (4) ◽  
pp. 919-927 ◽  
Author(s):  
Anjali Seth ◽  
Nancy D. Connell
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Nitrogen Sources ◽  
Single Copy ◽  
Aminobutyric Acid ◽  
Cosmid Library ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Genes Encoding ◽  
Type Gene

ABSTRACT Genes encoding l-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter fromMycobacterium bovis BCG and the effects of its deletion onl-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to therocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using 14C-labeled substrates. Greatly reduced uptake of l-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),l-[14C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenousl-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. l-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.

scholarly journals Inositol polyphosphate multikinase regulation ofTrypanosoma bruceilife stage development

2018 ◽  
Vol 29 (9) ◽  
pp. 1137-1152 ◽  
Author(s):  
Igor Cestari ◽  
Atashi Anupama ◽  
Kenneth Stuart
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Tsetse Fly ◽  
Developmental Changes ◽  
Mammalian Host ◽  
Surface Coat ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphate Multikinase

Many cellular processes change during the Trypanosoma brucei life cycle as this parasite alternates between the mammalian host and tsetse fly vector. We show that the inositol phosphate pathway helps regulate these developmental changes. Knockdown of inositol polyphosphate multikinase (IPMK), which phosphorylates Ins(1,4,5)P3 and Ins(1,3,4,5)P4, resulted in changes in bloodstream forms that are characteristic of insect stage procyclic forms. These changes include expression of the procyclic surface coat, up-regulation of RNA-binding proteins that we show to regulate stage-specific transcripts, and activation of oxidative phosphorylation with increased ATP production in bloodstream forms. These changes were accompanied by development of procyclic morphology, which also occurred by the expression of a catalytically inactive IPMK, implying that regulation of these processes entails IPMK activity. Proteins involved in signaling, protein synthesis and turnover, and metabolism were affinity-enriched with the IPMK substrate or product. Developmental changes associated with IPMK knockdown or catalytic inactivation reflected processes that are enriched with inositol phosphates, and chemical and genetic perturbation of these processes affected T. brucei development. Hence, IPMK helps regulate T. brucei development, perhaps by affecting inositol phosphate interactions with proteins of the regulatory network that controls energy metabolism and development.

Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function

1987 ◽  
Vol 7 (6) ◽  
pp. 2231-2242
Author(s):  
J E Rudolph ◽  
M Kimble ◽  
H D Hoyle ◽  
M A Subler ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Sequence Divergence ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Structural Constraints ◽  
Wild Type ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Single Amino Acid Residue ◽  
Beta 2

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.

Mutant ras gene induces elevated levels of inositol tris- and hexakisphosphates in Dictyostelium

1988 ◽  
Vol 89 (1) ◽  
pp. 13-20
Author(s):  
G.N. Europe-Finner ◽  
M.E. Luderus ◽  
N.V. Small ◽  
R. Van Driel ◽  
C.D. Reymond ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cyclic Amp ◽  
Gene Product ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Regulatory Protein ◽  
Signal Transduction Pathway ◽  
Mutant Form ◽  
Wild Type ◽  
Ras Gene

Previous studies of Europe-Finner & Newell indicated that in amoebae of Dictyostelium discoideum, signal transduction used for chemotaxis to cyclic AMP involved transient formation of inositol tris- and polyphosphates. Evidence was also presented for the involvement of a GTP-binding G-protein. Here we report evidence for the involvement of a ras gene product in the D. discoideum inositol phosphate pathway. Use was made of strains of Dictyostelium transformed with a wild-type D. discoideum ras gene (ras-Gly12) or a mutant form of the gene (ras-Thr12). Experiments using separation of soluble inositol phosphates by Dowex anion-exchange resin chromatography indicated that cells transformed with the wild-type ras-Gly12 gene were unaffected in their basal levels of inositol polyphosphates and in the inositol phosphates formed in response to stimulation with the chemotactic agent cyclic AMP. In contrast, cells transformed with the mutant ras-Thr12 gene showed a basal level of inositol polyphosphate that was several-fold elevated over the controls and stimulation of these cells with cyclic AMP produced only a small further elevation. When the inositol phosphates were analysed by h.p.l.c. it was found that the basal level of inositol 1,4,5-trisphosphate was raised three- to fivefold in the ras-Thr12 strain compared to the strain transformed with ras-Gly12, and that inositol hexakisphosphate (which was found to be present in large amounts relative to other inositol phosphates in D. discoideum cells) was also raised to a similar extent in the ras-Thr12-transformed cells. We propose that the Dictyostelium ras gene product codes for a regulatory protein involved in the inositol phosphate chemotactic signal-transduction pathway.

scholarly journals The inositol phosphates in WRK1 rat mammary tumour cells

1992 ◽  
Vol 286 (2) ◽  
pp. 459-468 ◽  
Author(s):  
N S Wong ◽  
C J Barker ◽  
A J Morris ◽  
A Craxton ◽  
C J Kirk ◽  
...  
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Periodate Oxidation ◽  
Tumour Cells ◽  
Mammary Tumour ◽  
Inositol Polyphosphate ◽  
Inositol Hexakisphosphate ◽  
Site Specific

1. A detailed structural survey has been made of the inositol phosphates of unstimulated and vasopressin-stimulated WRK-1 rat mammary tumour cells. Inositol phosphate peaks were separated by h.p.l.c., and structural assignments were made for more than 20 compounds by combinations of: (a) co-chromatography with labelled standards; (b) site-specific enzymic dephosphorylation; (c) complete and partial periodate oxidation, followed by h.p.l.c. of polyols and their stereospecific oxidation by dehydrogenases; and (d) ammoniacal hydrolysis. 2. The ‘inositol monophosphates’ fraction from unstimulated cells included an uncharacterized peak, probably containing some glycerophosphoinositol, and Ins(1:2-cyclic)P. Stimulation provoked accumulation of both Ins1P and Ins3P, of Ins2P, and of Ins5P and/or the enantiomers Ins4P and Ins6P. The proportions of Ins1P and Ins3P were determined by partial periodate oxidation and enantiomeric identification of the resulting glucitols. 3. Three inositol bisphosphate peaks were detected in unstimulated cells: Ins(1,4)P2 [this was distinguished chemically from its enantiomer Ins(3,6)P2], Ins(3,4)P2 and/or Ins(1,6)P2, and Ins(4,5)P2 and/or Ins(5,6)P2. On stimulation, Ins(1,4)P2 and Ins(3,4)P2 [and/or Ins(1,6)P2] levels increased, and Ins(1:2-cyclic,4)P2 and Ins(1,3)P2 were also formed. 4. Three inositol trisphosphate peaks were obtained from unstimulated cells: all increased during stimulation. These were Ins(1,3,4)P3 [with some Ins(1:2-cyclic,4,5)P3], Ins(1,4,5)P3 and Ins(3,4,5)P3 [and/or Ins(1,5,6)P3]. During stimulation, another compound, probably Ins(1,4,6)P3, appeared in the ‘Ins(1,4,5)P3 peak’. The ‘Ins(3,4,5)P3 peak’ contained a second trisphosphate, probably Ins(2,4,5)P3. 5. Three inositol tetrakisphosphates, namely Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, were present in unstimulated cells, and all accumulated during stimulation. 6. Ins(1,3,4,5,6)P5, which is the most abundant inositol polyphosphate in these cells, a less abundant inositol pentakisphosphate and inositol hexakisphosphate were all unresponsive to stimulation.

scholarly journals Functional Analysis of Fructosyl-Amino Acid Oxidases of Aspergillus oryzae

2004 ◽  
Vol 70 (10) ◽  
pp. 5882-5890 ◽  
Author(s):  
Shin-ichi Akazawa ◽  
Tetsuya Karino ◽  
Nobuyuki Yoshida ◽  
Tohoru Katsuragi ◽  
Yoshiki Tani
Keyword(s):  
Amino Acid ◽  
Aspergillus Oryzae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Nitrogen Sources ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Carbon And Nitrogen Sources ◽  
Carbon And Nitrogen

ABSTRACT Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N ε-fructosyl N α-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain ΔfaoAo2 did not grow. Addition of glucose or (NH4)2SO4 to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO2 as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.

Defective intestinal amino acid absorption in Ace2 null mice

2012 ◽  
Vol 303 (6) ◽  
pp. G686-G695 ◽  
Author(s):  
Dustin Singer ◽  
Simone M. R. Camargo ◽  
Tamara Ramadan ◽  
Matthias Schäfer ◽  
Luca Mariotta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Weight Gain ◽  
Amino Acid ◽  
Amino Acid Content ◽  
Wild Type ◽  
Neutral Amino Acids ◽  
Low Protein ◽  
Dependent Transport ◽  
Niacin Deficiency ◽  
The Impact

Mutations in the main intestinal and kidney luminal neutral amino acid transporter B0AT1 (Slc6a19) lead to Hartnup disorder, a condition that is characterized by neutral aminoaciduria and in some cases pellagra-like symptoms. These latter symptoms caused by low-niacin are thought to result from defective intestinal absorption of its precursor l-tryptophan. Since Ace2 is necessary for intestinal B0AT1 expression, we tested the impact of intestinal B0AT1 absence in ace2 null mice. Their weight gain following weaning was decreased, and Na+-dependent uptake of B0AT1 substrates measured in everted intestinal rings was defective. Additionally, high-affinity Na+-dependent transport of l-proline, presumably via SIT1 (Slc6a20), was absent, whereas glucose uptake via SGLT1 (Slc5a1) was not affected. Measurements of small intestine luminal amino acid content following gavage showed that more l-tryptophan than other B0AT1 substrates reach the ileum in wild-type mice, which is in line with its known lower apparent affinity. In ace2 null mice, the absorption defect was confirmed by a severalfold increase of l-tryptophan and of other neutral amino acids reaching the ileum lumen. Furthermore, plasma and muscle levels of glycine and l-tryptophan were significantly decreased in ace2 null mice, with other neutral amino acids displaying a similar trend. A low-protein/low-niacin diet challenge led to differential changes in plasma amino acid levels in both wild-type and ace2 null mice, but only in ace2 null mice to a stop in weight gain. Despite the combination of low-niacin with a low-protein diet, plasma niacin concentrations remained normal in ace2 null mice and no pellagra symptoms, such as photosensitive skin rash or ataxia, were observed. In summary, mice lacking Ace2-dependent intestinal amino acid transport display no total niacin deficiency nor clear pellagra symptoms, even under a low-protein and low-niacin diet, despite gross amino acid homeostasis alterations.

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