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A D Panek

Publications and source records attributed to A D Panek.

At least 55 records · Page 3Linked to original sources
PubMed1993

Role of the trehalose carrier in dehydration resistance of Saccharomyces cerevisiae.

Yeast cells are well known for their ability to survive complete dehydration, a phenomenon that is directly linked to the presence of the sugar trehalose in these cells. This sugar apparently endows the cells with the capacity to survive dehydration. Previous studies on in vitro models showed that trehalose must be present on both sides of the bilayer to stabilize dry membranes. The present report demonstrates that a specific trehalose carrier seems to enable the sugar to protect the yeast cell membrane by translocating trehalose from the cytosol to the extracellular environment. Saccharomyces cerevisiae mutant strains which lack the trehalose carrier did not survive after dehydration although they accumulated endogenous trehalose. Furthermore, when carrier mutants were dehydrated in the presence of exogenous trehalose the cells became more resistant showing increased survival.

Carrier Proteins↗
PubMed1993

A dependable method for the synthesis of [14C]trehalose.

A new method for the preparation of [14C]trehalose was developed, based on the ability of yeast cells to accumulate trehalose under stress. The method is simple and reliable. It utilizes a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted. Thus, exogenously supplied glucose is not metabolized, but is instead converted to trehalose. The [14C]-trehalose obtained is pure, it is hydrolyzed by trehalase, and it is not susceptible to the action of alpha-glucosidase. The yield of this method is in the order of 35% of the [14C]glucose supplied.

Carbon Radioisotopes↗
PubMed1993

Protective role of trehalose during heat stress in Saccharomyces cerevisiae.

Yeast cells accumulate the disaccharide trehalose in response to certain stress conditions. In an attempt to verify the role that trehalose plays when yeast cells are faced with heat stress, yeast mutant strains with specific lesions in trehalose metabolism were used. Cultures growing exponentially on glucose were shifted from 28 to 40 degrees C for 1 h. Accumulation of trehalose was correlated with heat tolerance, measured as resistance to 50.5 degrees C. Additionally, it was observed that the trehalose carrier was not involved in the mechanism of thermotolerance acquisition. Mutants that lack the carrier were also able to acquire thermotolerance as long as synthesis of the disaccharide took place.

Adaptation, Physiological↗
PubMed1993

Trehalose metabolism during sporulation in Saccharomyces cerevisiae.

S. cerevisiae has at least two enzymes for trehalose synthesis. In this paper we report some characteristics of the ADPG-dependent trehalose-6-phosphate synthase. As opposed to the UDPG-dependent enzyme, the ADPG-dependent activity did not respond to a glucose signal nor to a heat shock. Mutant strains, deficient in the UDPG-dependent enzyme but possessing the ADPG-dependent activity, did not accumulate trehalose either during growth or during sporulation. A diploid, homozygous for the deficiency in the UDPG-dependent synthase activity, was constructed. During sporulation the ADPG-dependent activity remained constant. However, when mutant spores were subjected to a glucose signal, trehalose was accumulated to the same extent as in control spores.

Adenosine Diphosphate Glucose↗
PubMed1993

Yeast--100 years of contribution to biochemistry.

For centuries mankind has benefited from yeast for its survival as well as for joy and even solace in moments of pain. Today the yeast cell is considered a universal model yielding answers to important and intriguing questions about cell division, differentiation, transduction of signals and cell disorders. Classical and molecular genetic methods have been successfully applied to understand the association of genes with proteins and functions-a knowledge which has also led to the development of new biotechnologies.

Biochemistry↗
PubMed1992

Characterization of trehalase in Rhodotorula rubra.

Trehalase activity in Rhodotorula rubra was found to be bound to the particulate fraction of a cell-free extract in contrast with the soluble trehalase activity of Saccharomyces cerevisiae. The enzyme was strongly repressed by glucose and derepressed during growth on maltose, trehalose and glycerol. This increase in activity was due to a "de novo" synthesis as seen by inhibition with cycloheximide, a mechanism not described for Saccharomyces cerevisiae. Catabolite inactivation by addition of glucose was also demonstrated. This particulate enzyme does not respond to activation by the cAMP-dependent protein kinase.

Cycloheximide↗
PubMed1992

Trehalase activity and its regulation during growth of Saccharomyces cerevisiae.

Trehalase activity decreased in 95% at the onset of the transition phase of growth of S. cerevisiae. The question which we raised was whether this phenomenon was due to proteolysis or to conversion of the enzyme to a less active form (dephosphorylation). Immunological methods allowed to identify the presence of the trehalase protein during cell growth. At the same stage of growth, an increase in the non-phosphorylated enzyme was detected "in vitro". Results utilizing mutant strains also indicated that regulation occurred by interconversion of forms. The same mechanism also seems to control trehalase activity in non proliferating conditions.

Blotting, Western↗
PubMed1991

Activation of yeast trehalase by heat shock.

1. Activation of Saccharomyces cerevisiae trehalase by heat shock was shown in all strains tested, including mutants in which the response to a glucose signal was absent. A low concentration of cAMP favored the response as seen in 2nd log cells or in ras2 and cyr1ts mutant strains. The heat shock effect upon trehalase activity was not observed under conditions of catabolite repression. 2. Neither hexokinase PII nor the heat shock protein hsp26 seemed to be involved in the activation of trehalase by heat shock. However, mutant strains deleted in the polyubiquitin gene showed only a 2-fold activation of the enzyme while in control strains a 5- to 7-fold irreversible activation was observed. 3. An alternative mechanism of trehalase activation by removal of an inhibitor through ligation with ubiquitin is discussed. Activation by cAMP-independent phosphorylation is also considered.

Culture Media↗
PubMed1991

Trehalose transport in yeast cells.

Saccharomyces cerevisiae possesses a specific transporter for trehalose. Transport is low until after glucose exhaustion in stationary phase, and addition of glucose in stationary phase results in loss of transport, an event that is reversible when the glucose is removed. The effect of glucose is not competitive inhibition: the increase in trehalose transport in stationary phase requires a lag time after glucose is depleted; and recovery of transport after glucose inhibition requires protein synthesis. Finally, we provide evidence that the trehalose transporter is distinct from that for maltose.

Binding, Competitive↗
PubMed1991

Trehalose-transporting membrane vesicles from yeasts.

We have isolated and characterized a membrane vesicle fraction from yeasts that is capable trehalose transport. The kinetics of the transport system were similar to those seen in the intact cells. The transport depends on a transmembrane pH gradient. If the gradient is collapsed, trehalose accumulated inside the vesicles is leaked into the medium. After aging for several days the ability of the vesicles to transport was lost. However, transport was partially restored by elevating internal pH in the vesicles.

Biological Transport↗
PubMed1990

Isolation and characterization of a new mutant of Saccharomyces cerevisiae with altered synthesis of 5-aminolevulinic acid.

A new gene, RHM1, required for normal production of 5-aminolevulinic acid by Saccharomyces cerevisiae, was identified by a novel screening method. Ethyl methanesulfonate treatment of a fluorescent porphyric strain bearing the pop3-1 mutation produced nonfluorescent or weakly fluorescent mutants with defects in early stages of tetrapyrrole biosynthesis. Class I mutants defective in synthesis of 5-aminolevulinate regained fluorescence when grown on medium supplemented with 5-aminolevulinate, whereas class II mutants altered in later biosynthetic steps did not. Among six recessive class I mutants, at least three complementation groups were found. One mutant contained an allele of HEM1, the structural gene for 5-aminolevulinate synthase, and two mutants contained alleles of the regulatory gene CYC4. The remaining mutants contained genes complementary to both hem1 and cyc4. Mutant strain DA3-RS3/68 contained mutant gene rhm1, which segregated independently of hem1 and cyc4 during meiosis. 5-Aminolevulinate synthase activity of the rhm1 mutant was 35 to 40% of that of the parental pop3-1 strain, whereas intracellular 5-aminolevulinate concentration was only 3 to 4% of the parental value. Transformation of an rhm1 strain with a multicopy plasmid containing the cloned HEM1 gene restored normal levels of 5-aminolevulinate synthase activity, but intracellular 5-aminolevulinate was increased to only 9 to 10% of normal. We concluded that RHM1 could control either targeting of 5-aminolevulinate synthase to the mitochondrial matrix or the activity of the enzyme in vivo.

5-Aminolevulinate Synthetase↗
PubMed1990

On the determination of trehalose-6-phosphate synthase in Saccharomyces.

Trehalose-6-phosphate synthase activity was determined by colorimetric, spectrophotometric and trehalose specific assays. All methods gave comparable results thus confirming our previous findings (1) and those reported by Elander (2). Different strains and mutants of Saccharomyces were carefully re-investigated in relation with the recent claim made by Vandercammen et al. (3) that our spectrophotometric assay over-estimated the enzyme activity and that no differences exist between wild type and mutant strains. In this paper we also confirm the de-activation of the trehalose synthase complex in response to a "glucose signal", and present trehalose-6-phosphate synthase and trehalase activities in different strains measured during all phases of growth on glucose.

Colorimetry↗
PubMed1990

Effect of dimethylsulfoxide on signal transduction in mutants of Saccharomyces cerevisiae.

1. As the first part of a study of pesticide toxicity we report the effects of the solvent dimethylsulfoxide (DMSO) on signal transduction in mutants of Saccharomyces cerevisiae. 2. The enzymes of trehalose metabolism, which are activated and deactivated by a "glucose signal" and by heat shock treatment, were chosen as targets for this study. 3. DMSO was shown to be able to permeate glucose and cAMP. The effects of glucose and cAMP were enhanced by pre-incubating the cells in the presence of DMSO. 4. No effects were observed during the heat shock, suggesting that the solvent acts on the cell membrane. 5. The results suggest that DMSO may be used as a vehicle for small molecules which do not easily penetrate yeast cell membranes, thus providing a new tool for biochemical and toxicological studies.

Cyclic AMP↗
PubMed1989

Determination of trehalose in biological samples by a simple and stable trehalase preparation.

A three step purification procedure for trehalase from Saccharomyces cerevisiae with a recovery of 76% of the original activity is presented. The enzyme was activated by a heat shock treatment prior to homogenization of the cells. A mutant strain deleted in SUC genes was used to avoid contamination by invertase. The lyophylized enzyme was stable for, at least, 5 months and could be used to determine trehalose in the range 25 to 500 nmol. The preparation was free of inspecific phosphatases allowing for trehalose determinations in yeast cell free extracts and in insect hemolymph.

Chromatography↗
PubMed1989

Identification of an ADPG-dependent trehalose synthase in Saccharomyces.

Uridine diphosphoglucose is not the sole donor for trehalose synthesis in yeast cells: an ADPG-dependent trehalose synthase, has been identified in mutant strains with undetectable UDPG-dependent trehalose-6-P synthase activity. Genetic and chromatographic studies indicate that the two activities correspond to different proteins. The apparent Km for the nucleotide is similar for both enzymes, and Mg2+ is also required for both activities; however, a striking difference was observed with respect to ATP.Mg activation. This newly determined enzymatic activity in Saccharomyces clarifies previous contradictory results with mutant strains that are able to accumulate trehalose during growth yet whose UDPG-dependent trehalose synthase activity is undetectable in vitro.

Chromatography↗
PubMed1989

Comparative studies between the glucose-induced phosphorylation signal and the heat shock response in mutants of Saccharomyces cerevisiae.

Addition of glucose to derepressed yeast cells, as well as a heat shock treatment, trigger the phosphorylation of trehalase and of trehalose-6-phosphate synthase. In the present paper, evidence is provided for the requirement of the RAS protein in the transduction of the glucose signal. On the other hand, a heat shock at 52 degrees C for 2 min was able to produce a significant phosphorylating effect even in mutant strains deficient in the GTP binding protein. Moreover, it was shown that this treatment does not affect exclusively the cAMP-dependent protein kinase. The use of a series of mutant strains confirmed that low levels of cAMP favor thermotolerance; the role of trehalose in yeast viability is also discussed.

Cyclic AMP↗
PubMed1989

Parallel changes in catabolite repression of haem biosynthesis and cytochromes in repression-resistant mutants of Saccharomyces cerevisiae.

Effects of three mutant genes, CAT1-2d, cat2-1 and hex2-3, on catabolite repression of mitochondrial cytochromes and the first two enzymes of haem biosynthesis were compared. The CAT1-2d mutation gave no resistance to glucose, whereas cat2-1 endowed both cytochromes and 5-aminolaevulinate dehydratase with resistance, but did not alter the effect of glucose on 5-aminolaevulinate synthase. The hex2-3 mutation caused repression resistance of cytochromes and of the two haem biosynthetic enzymes. hex2-3 strains also accumulated intracellular 5-aminolaevulinate. Co-inheritance of the latter traits, sensitivity to maltose inhibition and ability to grow on raffinose in the presence of 2-deoxyglucose, demonstrated that the pleiotropic phenotype is a function of the single gene hex2-3. Revertants which grew on maltose regained sensitivity to deoxyglucose and exhibited normal sensitivity of cytochromes and haem biosynthesis enzymes to repression. Addition of the hex1-18 mutation, which renders cytochromes resistant to repression, to a cat2-1 strain did not produce the same effect on 5-aminolaevulinate synthase as hex2-3. It is concluded that repression patterns of haem and cytochrome biosynthesis are substantially affected by hex2-3 and cat2-1 but not by CAT1-2d.

5-Aminolevulinate Synthetase↗