Inositol regulates phosphatidylglycerolphosphate synthase expression in Saccharomyces cerevisiae

The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the synthesis of cardiolipin, a phospholipid found predominantly in the mitochondrial inner membrane. To determine whether PGPS is regulated by cross-pathway control, we analyzed PGPS expression under conditions in which the regulation of general phospholipid synthesis could be examined. The addition of inositol resulted in a three- to fivefold reduction in PGPS expression in wild-type cells in the presence or absence of exogenous choline. The reduction in enzyme activity in response to inositol was seen in minutes, suggesting that inactivation or degradation of the enzyme plays an important role in inositol-mediated repression of PGPS. In cho2 and opi3 mutants, which are blocked in phosphatidylcholine synthesis, inositol-mediated repression of PGPS did not occur unless choline was added to the media. Three previously identified genes that regulate general phospholipid synthesis, INO2, INO4, and OP11, did not affect PGPS expression. Thus, ino2 and ino4 mutants, which are unable to derepress biosynthetic enzymes involved in general phospholipid synthesis, expressed wild-type levels of PGPS activity under derepressing conditions. PGPS expression in the opi1 mutant, which exhibits constitutive synthesis of general phospholipid biosynthetic enzymes, was fully repressed in the presence of inositol and partially repressed even in the absence of inositol. These results demonstrate for the first time that an enzymatic step in cardiolipin synthesis is coordinately controlled with general phospholipid synthesis but that this control is not mediated by the same genetic regulatory circuit.

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Prohibitin Family Members Interact Genetically with Mitochondrial Inheritance Components in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.4043 ◽

1998 ◽

Vol 18 (7) ◽

pp. 4043-4052 ◽

Cited By ~ 131

Author(s):

Karen H. Berger ◽

Michael P. Yaffe

Keyword(s):

Saccharomyces Cerevisiae ◽

Outer Membrane ◽

Integral Membrane Proteins ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Morphology ◽

Mitochondrial Inheritance ◽

Loss Of Function ◽

Wild Type ◽

Mitochondrial Inner Membrane ◽

Synthetically Lethal

ABSTRACT Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae. Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability. In otherwise wild-type genetic backgrounds, null mutations in PHB1 andPHB2 did not confer any obvious phenotypes. However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p. These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology.

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Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3320-3328.1986 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3320-3328

Author(s):

J P Hirsch ◽

S A Henry

Keyword(s):

Saccharomyces Cerevisiae ◽

Blot Hybridization ◽

Constitutive Expression ◽

Genomic Clone ◽

Phospholipid Synthesis ◽

Wild Type ◽

Inositol 1 ◽

Regulatory Link ◽

In Cells ◽

Phosphate Synthase

The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression.

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Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1

Biochemical Journal ◽

10.1042/bj20031880 ◽

2004 ◽

Vol 380 (1) ◽

pp. 131-137 ◽

Cited By ~ 22

Author(s):

Christopher J. ENDRES ◽

Dhruba J. SENGUPTA ◽

Jashvant D. UNADKAT

Keyword(s):

Amino Acid ◽

Yeast Cells ◽

Nucleoside Transporter ◽

Wild Type ◽

Equilibrative Nucleoside Transporter ◽

Ic50 Values ◽

Km Value ◽

Adenosine Transport ◽

The Media ◽

First Time

We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 {e.g. NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside], dilazep or dipyridamole} were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92→Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260±16 µM) when compared with the wild-type transporter (65±7.8 µM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.

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Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3320 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3320-3328 ◽

Cited By ~ 150

Author(s):

J P Hirsch ◽

S A Henry

Keyword(s):

Saccharomyces Cerevisiae ◽

Blot Hybridization ◽

Constitutive Expression ◽

Genomic Clone ◽

Phospholipid Synthesis ◽

Wild Type ◽

Inositol 1 ◽

Regulatory Link ◽

In Cells ◽

Phosphate Synthase

The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression.

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Gene Dosage Effect of l-Proline Biosynthetic Enzymes on l-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6527-6532.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6527-6532 ◽

Cited By ~ 56

Author(s):

Yukiyasu Terao ◽

Shigeru Nakamori ◽

Hiroshi Takagi

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Dosage ◽

Freeze Tolerance ◽

Proline Accumulation ◽

Dosage Effect ◽

Yeast Cells ◽

Biosynthetic Enzymes ◽

Wild Type ◽

Gene Dosage Effect ◽

Carboxylate Reductase

ABSTRACT We have previously reported that l-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with l-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding γ-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of l-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant γ-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three l-proline biosynthetic enzymes, including Δ1-pyrroline-5-carboxylate reductase, which converts Δ1-pyrroline-5-carboxylate into l-proline, on l-proline accumulation and freeze tolerance in a non-l-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on l-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant γ-glutamyl kinase and the wild-type γ-glutamyl phosphate reductase was effective for l-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Δ1-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of l-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular l-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.

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Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.2.383 ◽

1996 ◽

Vol 142 (2) ◽

pp. 383-391 ◽

Cited By ~ 2

Author(s):

Yasumasa Tsukamoto ◽

Jun-ichi Kato ◽

Hideo Ikeda

Keyword(s):

Saccharomyces Cerevisiae ◽

Quantitative Analysis ◽

Structural Analysis ◽

Recombination Rate ◽

Type Strain ◽

Negative Selection ◽

Wild Type Strain ◽

Illegitimate Recombination ◽

Wild Type ◽

In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽

10.1093/genetics/123.1.81 ◽

1989 ◽

Vol 123 (1) ◽

pp. 81-95 ◽

Cited By ~ 1

Author(s):

E J Louis ◽

J E Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Chromosome ◽

Stop Codon ◽

Fold Increase ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Nonsense Mutations ◽

Chromosome Iii ◽

Meiosis I ◽

Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

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Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽

10.3390/pathogens10010054 ◽

2021 ◽

Vol 10 (1) ◽

pp. 54

Author(s):

Christine Landlinger ◽

Lenka Tisakova ◽

Vera Oberbauer ◽

Timo Schwebs ◽

Abbas Muhammad ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Bactericidal Activity ◽

High Selectivity ◽

Ex Vivo ◽

Vaginal Microbiome ◽

Wild Type ◽

Wild Type Enzyme ◽

Promising Alternative ◽

First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽

10.1093/genetics/149.1.45 ◽

1998 ◽

Vol 149 (1) ◽

pp. 45-56

Author(s):

Luther Davis ◽

JoAnne Engebrecht

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Heterologous Expression ◽

Protein Translation ◽

Developmental Pathways ◽

G1 Phase ◽

Growth Defect ◽

Wild Type ◽

Drosophila Homolog ◽

Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

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