The catabolic function of the α-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine–oxoglutarate reductase, but not saccharopine dehydrogenase

2000 ◽  
Vol 351 (1) ◽  
pp. 215-220
Author(s):  
Xiaohong ZHU ◽  
Guiliang TANG ◽  
Gad GALILI
Keyword(s):  
Adipic Acid ◽  
Expression System ◽  
Yeast Cells ◽  
Deletion Mutants ◽  
Null Mutant ◽  
Anabolic Activity ◽  
Saccharopine Dehydrogenase ◽  
Intermediate Metabolites ◽  
Acid Pathway ◽  
Transcriptional Controls

Whereas plants and animals use the α-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine–oxoglutarate reductase (lysine–ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the α-amino adipic acid pathway in plants.

Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers

2000 ◽  
Vol 351 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Catherine P. DARLEY ◽  
Olivier C. M. VAN WUYTSWINKEL ◽  
Karel VAN DER WOUDE ◽  
Willem H. MAGER ◽  
Albertus H. DE BOER
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Fusion ◽  
Direct Evidence ◽  
Protein Targeting ◽  
Expression System ◽  
Yeast Cells ◽  
Null Mutant ◽  
Heterologous Expression System ◽  
Na Ions ◽  
Convenient Model

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole is one mechanism to confer Na+-tolerance on these organisms. In the present study we provide direct evidence that the ArabidopsisthalianaAt-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na+/H+ exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na+ accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na+/H+ exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20–40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na+/H+ antiporters.

scholarly journals Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520

1994 ◽  
Vol 14 (1) ◽  
pp. 277-286
Author(s):  
M Raymond ◽  
S Ruetz ◽  
D Y Thomas ◽  
P Gros
Keyword(s):  
Multidrug Resistance ◽  
Mammalian Cells ◽  
Drug Transport ◽  
Function Analysis ◽  
Expression System ◽  
Functional Expression ◽  
Yeast Cells ◽  
Mutant Form ◽  
Cellular Resistance ◽  
P Glycoprotein

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

scholarly journals Decreased Phosphorylation and Increased Methionine Oxidation of α-Synuclein in the Methionine Sulfoxide Reductase A Knockout Mouse

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Derek B. Oien ◽  
Gonzalo A. Carrasco ◽  
Jackob Moskovitz
Keyword(s):  
Knockout Mouse ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Yeast Cells ◽  
Mammalian Brain ◽  
Methionine Oxidation ◽  
Null Mutant ◽  
Supportive Evidence ◽  
Methionine Sulfoxide Reductase A ◽  
Protein Fibrillation

Previously, we have showed that overexpression of methionine-oxidized α-synuclein in methionine sulfoxide reductase A (MsrA) null mutant yeast cells inhibits α-synuclein phosphorylation and increases protein fibrillation. The current studies show that ablation of mouse MsrA gene caused enhanced methionine oxidation of α-synuclein while reducing its own phophorylation levels, especially in the hydrophobic cell-extracted fraction. These data provide supportive evidence that a compromised MsrA function in mammalian brain may cause enhanced pathologies associated with altered α-synuclein oxidation and phosphorylation levels.

scholarly journals Small heat-shock protein Hsp12 contributes to yeast tolerance to freezing stress

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 2021-2028 ◽  
Author(s):  
A. Pacheco ◽  
C. Pereira ◽  
M. J. Almeida ◽  
M. J. Sousa
Keyword(s):  
Heat Shock ◽  
Freezing Tolerance ◽  
Small Heat Shock Protein ◽  
Yeast Cells ◽  
Freezing Stress ◽  
Prolonged Storage ◽  
Null Mutant ◽  
Wild Type ◽  
Shock Proteins

The HSP12 gene encodes one of the two major small heat-shock proteins of Saccharomyces cerevisiae and is induced under different conditions, such as low and high temperatures, osmotic or oxidative stress and high sugar or ethanol concentrations. However, few studies could demonstrate any correlation between HSP12 deletion or overexpression and a phenotype of sensitivity/resistance, making it difficult to attribute a role for Hsp12p under several of these stress conditions. We investigated the possible role of Hsp12p in yeast freezing tolerance. Contrary to what would be expected, the hsp12 null mutant when subjected to prolonged storage at −20 °C showed an increased resistance to freezing when compared with the isogenic wild-type strain. Because the mutant strain displayed a higher intracellular trehalose concentration than the wild-type, which could mask the effect of manipulating HSP12, we overexpressed the HSP12 gene in a trehalose-6-phosphate synthase (TPS1) null mutant. The tps1Δ strain overexpressing HSP12 showed an increase in resistance to freezing storage, indicating that Hsp12p plays a role in freezing tolerance in a way that seems to be interchangeable with trehalose. In addition, we show that overexpression of HSP12 in this tps1Δ strain also increased resistance to heat shock and that absence of HSP12 compromises the ability of yeast cells to accumulate high levels of trehalose in response to a mild heat stress.

Expression of actin mutants to study their roles in cardiomyopathyThis paper is one of a selection of papers published this Special Issue, entitled Young Investigator's Forum.

2006 ◽  
Vol 84 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Lori A. Rutkevich ◽  
David J. Teal ◽  
John F. Dawson
Keyword(s):  
Cell Biology ◽  
Insect Cells ◽  
Expression System ◽  
Yeast Cells ◽  
Mutant Proteins ◽  
Advantages And Disadvantages ◽  
Cardiac Actin ◽  
Important Characterization ◽  
Mutant Forms

Mutations in the human cardiac actin gene (ACTC) have been implicated in the development of hypertrophic or dilated cardiomyopathy in humans. To determine the molecular mechanism for the disease development, a system for the expression of mutant cardiac actin proteins that may be lethal to eukaryotic cells must be developed. Here, we explore some of the advantages and disadvantages of human ACTC expression in yeast and insect cells. We show that human ACTC is incapable of rescuing a yeast endogenous actin (ACT1) - knockout in yeast cells and that coexpression of human ACTC in yeast results in slower growth, making yeast an unsuitable expression system. However, we show that it is possible for yeast cells to express a polymerization-deficient ACT1 mutant, thereby allowing us to examine the cell biology of this mutation in the future. Finally, mutant forms of human cardiac actin can be expressed in and purified from insect cells in a properly folded and functional form, permitting important characterization of the biochemical mechanisms responsible for cardiomyopathy development in humans. These studies allow for further research into the biochemical characteristics of previously untenable actin mutant proteins.

scholarly journals Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

2004 ◽  
Vol 85 (10) ◽  
pp. 3115-3122 ◽  
Author(s):  
Luisa Rubino ◽  
Vitantonio Pantaleo ◽  
Beatriz Navarro ◽  
Marcello Russo
Keyword(s):  
Plant Cells ◽  
Open Reading Frames ◽  
Ringspot Virus ◽  
Yeast Cells ◽  
Deletion Mutants ◽  
Replication Proteins ◽  
Satellite Rna ◽  
Replicase Proteins ◽  
Replication Strategy ◽  
Reading Frames

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.

scholarly journals Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae

1996 ◽  
Vol 315 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Toshiko KASAHARA ◽  
Michihiro KASAHARA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Specific Activity ◽  
Expression System ◽  
Yeast Cells ◽  
Transport Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crude Membrane

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze–thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4±0.2 mM (mean±S.E.M.) and was inhibited by cytochalasin B (IC50 = 0.44±0.03 μM), HgCl2 (IC50 = 3.5±0.5 μM), phloretin (IC50 = 49±12 μM) and phloridzin (IC50 = 355±67 μM). To compare these properties with native GLUT1, we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.

Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae

1991 ◽  
Vol 37 (5) ◽  
pp. 662-666 ◽  
Author(s):  
C Angele ◽  
T Oster ◽  
A Visvikis ◽  
J M Michels ◽  
M Wellman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Clinical Chemistry ◽  
Rat Kidney ◽  
Expression System ◽  
Yeast Cells ◽  
Single Chain ◽  
Hep G2 ◽  
Gamma Glutamyltransferase ◽  
Activity Measurements

Abstract To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.

scholarly journals RA92A recombinant protein as immunogen to protect cattle against tick challenge in Brazil and Uganda

2021 ◽  
Vol 37 ◽  
pp. e37068
Author(s):  
Rodrigo Casquero Cunha ◽  
Margaret Saimo-Kahwa ◽  
Marcos Valério Garcia ◽  
Francisco Denis Souza Santos ◽  
Emukule Samuel ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Expression System ◽  
Clinical Signs ◽  
Experimental Period ◽  
Adult Tick ◽  
Blood Parameters ◽  
Rhipicephalus Appendiculatus ◽  
Yeast Cells ◽  
Normal Ranges ◽  
Pichia Pastoris Yeast

In this study, the recombinant gut protein rRa92A produced in Pichia pastoris yeast cells was used to immunize cattle in two experiments, one in Brazil and the other in Uganda. In both experiments, the animals were intramuscularly (IM) injected with 200 µg of recombinant protein in Brazil on days 0, 30 and 51 and in Uganda on days 0, 30. Blood samples for sera separation were collected from different days in both experiments. These samples were analyzed by ELISAs. In Brazil, ticks collected from the animals during the experimental period were analyzed for biological parameters. At Uganda, blood was collected to assess blood parameters, clinical signs were recorded and adult tick (Rhipicephalus appendiculatus) counts were performed. All animals of the vaccinated groups were shown to produce antibodies, and it was not possible to detect an effect on Rhipicephalus microplus. All the clinical parameters were considered within the normal ranges for both the experimental and control groups in Uganda. Antibody absorbance was elevated after each immunization and remained high until the end of the experiments, remaining low in the control animals. The results of stall test carried out in Brazil using R. microplus tick showed efficacy of 21.95%. The rRa92A immunization trial experiments in Uganda showing a decrease of 55.2% in the number of engorged adult ticks, which was statistically significant (p<0.05). Assessment of the immunogenicity of Ra92A produced in the P. pastoris expression system in bovines is reported for the first time, and the protein acted as a concealed antigen.

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