scholarly journals Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline

2021 ◽  
Vol 9 (9) ◽  
pp. 1902
Author(s):  
Akira Nishimura ◽  
Yurie Takasaki ◽  
Shota Isogai ◽  
Yoichi Toyokawa ◽  
Ryoya Tanahashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Alcoholic Beverage ◽  
Binding Pocket ◽  
Yeast Cells ◽  
Water Retention Capacity ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Retention Capacity ◽  
Distillation Residues

Awamori, the traditional distilled alcoholic beverage of Okinawa, Japan, is brewed with the yeast Saccharomyces cerevisiae. During the distillation process after the fermentation, enormous quantities of distillation residues containing yeast cells must be disposed of, and this has recently been recognized as a major problem both environmentally and economically. Proline, a multifunctional amino acid, has the highest water retention capacity among amino acids. Therefore, distillation residues with large amounts of proline could be useful in cosmetics. Here, we isolated a yeast mutant with high levels of intracellular proline and found a missense mutation (Gln79His) on the PRO1 gene encoding the γ-glutamyl kinase Pro1, a limiting enzyme in proline biosynthesis. The amino acid change of Gln79 to His in Pro1 resulted in desensitization to the proline-mediated feedback inhibition of GK activity, leading to the accumulation of proline in cells. Biochemical and in silico analyses showed that the amino acid residue at position 79 is involved in the stabilization of the proline binding pocket in Pro1 via a hydrogen-bonding network, which plays an important role in feedback inhibition. Our current study, therefore, proposed a possible mechanism underlying the feedback inhibition of γ-glutamyl kinase activity. This mechanism can be applied to construct proline-accumulating yeast strains to effectively utilize distillation residues.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

scholarly journals The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding.

1992 ◽  
Vol 12 (12) ◽  
pp. 5683-5689 ◽  
Author(s):  
M R Parthun ◽  
D A Mangus ◽  
J A Jaehning
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Yeast Cells ◽  
Stable Complex ◽  
Affinity Column ◽  
Gene Encoding ◽  
Heat Stable ◽  
Gel Retardation ◽  
Final Purification Step

A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.

The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein

1988 ◽  
Vol 8 (3) ◽  
pp. 1282-1289
Author(s):  
W Haggren ◽  
D Kolodrubetz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Mobility ◽  
Hmg Proteins ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Library ◽  
Associated Proteins ◽  
Genomic Dna Library

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function.

Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin

2002 ◽  
Vol 363 (3) ◽  
pp. 609-617 ◽  
Author(s):  
Olli H. LAITINEN ◽  
Vesa P. HYTÖNEN ◽  
Mervi K. AHLROTH ◽  
Olli T. PENTIKÄINEN ◽  
Ciara GALLAGHER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Modelling ◽  
Chemical Properties ◽  
Binding Pocket ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Subunit Interface ◽  
Physico Chemical ◽  
Biotin Binding ◽  
Biochemical Analyses

Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin—biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys111→isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin—biotin technological applications.

scholarly journals The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

Substitution of histone H3 arginine 72 to alanine leads to deregulation of isoleucine biosynthesis in budding yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Pilendra Kumar Thakre ◽  
Rakesh Kumar Sahu ◽  
Raghuvir Singh Tomar
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Crucial Role ◽  
Minimal Medium ◽  
Budding Yeast ◽  
Histone H3 ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isoleucine Biosynthesis ◽  
Minimal Media

Histone residues play an essential role in the regulation of various biological processes. In the present study, we have utilized the H3/H4 histone mutant library to probe functional aspects of histone residues in amino acid biosynthesis. We found that histone residue H3R72 plays a crucial role in the regulation of isoleucine biosynthesis. Substitution of arginine residue (H3R72) of histone H3 to alanine (H3R72A) renders yeast cells unable to grow in the minimal media. Histone mutant H3R72A requires the external supplementation of either isoleucine, serine, or threonine for the growth in minimal media. We also observed that H3R72 residue and leucine amino acid in synthetic complete media might play a crucial role in determining the intake of isoleucine and threonine in yeast. Further, gene deletion analysis of ILV1 and CHA1 in H3R72A mutant confirmed that isoleucine is the sole requirement for growth in minimal medium. Altogether, we have identified that histone H3R72 residue may be crucial for yeast growth in the minimal medium by regulating isoleucine biosynthesis through the Ilv1 enzyme in budding yeast Saccharomyces cerevisiae.

Cloning and characterisation of ZmZLP1, a gene encoding an endoplasmic reticulum-localised zinc transporter in Zea mays

2010 ◽  
Vol 37 (3) ◽  
pp. 194 ◽  
Author(s):  
Yao-Guang Xu ◽  
Bao-Sheng Wang ◽  
Jing-Juan Yu ◽  
Guang-Ming Ao ◽  
Qian Zhao
Keyword(s):  
Endoplasmic Reticulum ◽  
Zea Mays ◽  
Metal Ion ◽  
Yeast Cells ◽  
Growth Defect ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Maize Pollen ◽  
Unfolded Protein ◽  
The Unfolded Protein Response

The ZmZLP1 (ZmZIP-like protein) gene was isolated from a cDNA library of Zea mays L. (maize) pollen. Bioinformatics analysis indicated that ZmZLP1 shares many characteristics of the ZIP (ZRT/IRT-like protein) family of metal ion transporters. Under general nutrient conditions, the expression of ZmZLP1 was detected in both mature pollen and, less strongly, in male inflorescences, whereas an induction of the ZmZLP1 transcript was observed in roots after 12 h of zinc deprivation. The visualisation of GFP showed that ZmZLP1 was targeted to the endoplasmic reticulum (ER). To investigate the gene’s functions, we fused ZmZLP1 with the signal peptide of the plasma membrane-localised protein AtIRT1 and transformed this fusion protein into the zinc uptake-deficient yeast (Saccharomyces cerevisiae) strain ZHY3 and the wild-type strain DEY1457. The IRT1-ZmZLP1 transformants grew poorly on zinc-limited medium, and this growth defect was rescued by zinc supplementation, suggesting that ZmZLP1 is responsible for transporting zinc from the ER to the cytoplasm. Further research indicated that ZmZLP1 is involved in the unfolded protein response (UPR) pathway and enhances the heat resistance of yeast cells.

scholarly journals Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (2) ◽  
pp. 390-395 ◽  
Author(s):  
S Powers ◽  
K O'Neill ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Yeast Cells ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Genes ◽  
Evolutionarily Conserved

Two mutant alleles of RAS2 were discovered that dominantly interfere with wild-type RAS function in the yeast Saccharomyces cerevisiae. An amino acid substitution which caused the dominant interference was an alanine for glycine at position 22 or a proline for alanine at position 25. Analogous mutations in human H-ras also dominantly inhibited RAS function when expressed in yeast cells. The inhibitory effects of the mutant RAS2 or H-ras genes could be overcome by overexpression of CDC25, but only in the presence of wild-type RAS. These results suggest that these mutant RAS genes interfere with the normal interaction of RAS and CDC25 proteins and suggest that this interaction is direct and has evolutionarily conserved features.

The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding

1992 ◽  
Vol 12 (12) ◽  
pp. 5683-5689
Author(s):  
M R Parthun ◽  
D A Mangus ◽  
J A Jaehning
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Yeast Cells ◽  
Stable Complex ◽  
Affinity Column ◽  
Gene Encoding ◽  
Heat Stable ◽  
Gel Retardation ◽  
Final Purification Step

A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.

scholarly journals Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 390-395
Author(s):  
S Powers ◽  
K O'Neill ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Yeast Cells ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Genes ◽  
Evolutionarily Conserved

Two mutant alleles of RAS2 were discovered that dominantly interfere with wild-type RAS function in the yeast Saccharomyces cerevisiae. An amino acid substitution which caused the dominant interference was an alanine for glycine at position 22 or a proline for alanine at position 25. Analogous mutations in human H-ras also dominantly inhibited RAS function when expressed in yeast cells. The inhibitory effects of the mutant RAS2 or H-ras genes could be overcome by overexpression of CDC25, but only in the presence of wild-type RAS. These results suggest that these mutant RAS genes interfere with the normal interaction of RAS and CDC25 proteins and suggest that this interaction is direct and has evolutionarily conserved features.

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