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.

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.

scholarly journals Global Phenotype Screening and Transcript Analysis Outlines the Inhibitory Mode(s) of Action of Two Amphibian-Derived, α-Helical, Cationic Peptides on Saccharomyces cerevisiae

2007 ◽  
Vol 51 (11) ◽  
pp. 3948-3959 ◽  
Author(s):  
C. Oliver Morton ◽  
Andrew Hayes ◽  
Michael Wilson ◽  
Bharat M. Rash ◽  
Stephen G. Oliver ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Inhibitory Action ◽  
Protein Targeting ◽  
Telomere Maintenance ◽  
Yeast Cells ◽  
Membrane Disruption ◽  
Dna Integrity ◽  
Cationic Peptides

ABSTRACT Dermaseptin S3(1-16) [DsS3(1-16)] and magainin 2 (Mag 2) are two unrelated, amphibian-derived cationic peptides that adopt an α-helical structure within microbial membranes and have been proposed to kill target organisms via membrane disruption. Using a combination of global deletion mutant library phenotypic screening, expression profiling, and physical techniques, we have carried out a comprehensive in vitro analysis of the inhibitory action of these two peptides on the model fungus Saccharomyces cerevisiae. Gene ontology profiling (of biological processes) was used to identify both common and unique effects of each peptide. Resistance to both peptides was conferred by genes involved in telomere maintenance, chromosome organization, and double-strand break repair, implicating a common inhibitory action of DNA damage. Crucially, each peptide also required unique genes for maintaining resistance; for example, DsS3(1-16) required genes involved in protein targeting to the vacuole, and Mag 2 required genes involved in DNA-dependent DNA replication and DNA repair. Thus, DsS3(1-16) and Mag 2 have both common and unique antifungal actions that are not simply due to membrane disruption. Physical techniques revealed that both peptides interacted with DNA in vitro but in subtly different ways, and this observation was supported by the functional genomics experiments that provided evidence that both peptides also interfered with DNA integrity differently in vivo. This implies that both peptides are able to pass through the cytoplasmic membrane of yeast cells and damage DNA, an inhibitory action that has not been previously attributed to either of these peptides.

scholarly journals Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

1992 ◽  
Vol 3 (8) ◽  
pp. 895-911 ◽  
Author(s):  
S C Ogg ◽  
M A Poritz ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Er Membrane

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.

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 Heterologous expression of the human ZIP4 zinc transporter in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Yuting Liu ◽  
Elizabeth M. Bafaro ◽  
Robert E. Dempski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Expression System ◽  
Protein A ◽  
Brain Dysfunction ◽  
Acrodermatitis Enteropathica ◽  
Wild Type ◽  
Membrane Domain ◽  
Heterologous Expression System ◽  
Gfp Fusion Protein

The human (h) transporter, hZIP4 is the primary zinc importer in the intestine and is also expressed in a variety of organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the zinc deficiency disease acrodermatitis enteropathica (AE), which disrupts digestive and immune system homeostasis. Structure-function studies of hZIP4 have been greatly hindered by the absence of a robust heterologous expression system. Here, we report the heterologous expression of hZIP4 in Saccharomyces cerevisiae. Both a wild type and a mutant S. cerevisiae strain, in which the endogenous zinc transporters are deleted, were used to test the expression and localization of an hZIP4-GFP fusion protein. A full-length hZIP4-GFP and a truncated membrane domain only (mhZIP4-GFP) protein were successfully produced and targeted to the plasma membrane in yeast.

Yeast Cells Allow High-Level Expression and Formation of Polyomavirus-Like Particles

1999 ◽  
Vol 380 (3) ◽  
pp. 381-386 ◽  
Author(s):  
K. Sasnauskas ◽  
O. Buzaite ◽  
F. Vogel ◽  
B. Jandrig ◽  
R. Razanskas ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Expression System ◽  
Yeast Cells ◽  
Human Polyomavirus ◽  
Yeast Expression System ◽  
E Coli ◽  
Human Use ◽  
Capsid Protein Vp1 ◽  
High Level

AbstractPolyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated inE. colior insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeastSaccharomyces cerevisiaefor expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1- coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels inSaccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.

scholarly journals Eukaryotic translation factor eIF5A contributes to acetic acid tolerance in Saccharomyces cerevisiae via transcriptional factor Ume6p

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yanfei Cheng ◽  
Hui Zhu ◽  
Zhengda Du ◽  
Xuena Guo ◽  
Chenyao Zhou ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Adaptive Response ◽  
Acid Tolerance ◽  
Peptide Bond ◽  
Transcriptional Factor ◽  
Yeast Cells ◽  
Acetic Acid Tolerance ◽  
Translation Factor ◽  
Industrial Strains

Abstract Background Saccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown. Results In this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance. Conclusion Translation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.

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