Filament formation in Saccharomyces cerevisiae--a review.

Many yeasts can produce filamentous elongated cells identifiable as hyphae, pseudohyphae or invasive filaments. Filament formation has been understood as a foraging response that occurs in nutrient-poor conditions. However, fusel alcohols were observed to induce filament formation in rich nutrient conditions in every yeast species examined. Fusel alcohols, e.g., 3-methyl-1-butanol (3Me-BuOH; 'isoamyl alcohol'), 2-methyl-1-propanol (isobutyl alcohol), (-)-2-methyl-1-butanol ('active amyl alcohol'), 2-phenylethanol and 3-(2-hydroxyethyl)indole (tryptophol) (the end products of leucine, valine, isoleucine, phenylalanine and tryptophan catabolism, respectively) are the end products of amino acid catabolism that accumulate when nutrients become limiting. Thus, yeast responds to its own metabolic by-products. Considerable effort was made to define the cell biological and biochemical changes that take place during 3Me-BuOH-induced filamentation. In Saccharomyces cerevisiae filaments contain significantly greater mitochondrial mass and increased chitin content in comparison with yeast-form cells. The global transcriptional response of S. cerevisiae during the early stages of 3Me-BuOH-induced filament formation has been described. Four ORFs displayed very significant (more than 10-fold) increases in their RNA species, and 12 ORFs displayed increases in transcription of more than 5-fold. The transcription of five genes (all of which encode transporters) decreased by similar amounts. Where examined, the activity of the proteins encoded reflected the transcriptional pattern of their respective mRNAs. To understand this regulation, studies were performed to see whether deletion or overexpression of key genes affects the ability to filament and invade solid YEPD medium. This has led to identification of those proteins that are essential for filament formation, repressors and those which are simply not required. It also leads to the conclusion that 3Me-BuOH-induced filament formation is not a foraging response but a response to reduced growth rate.

Are yeasts free-living unicellular eukaryotes?

Yeasts are defined as unicellular fungi, yet many recent observations suggest their whole lifestyle is anything but unicellular. This review surveys the evidence that yeasts are really social organisms with cell-to-cell communication.

The Arabidopsis CDC25 induces a short cell length when overexpressed in fission yeast: evidence for cell cycle function.

The putative mitotic inducer gene, Arath;CDC25 cloned in Arabidopsis thaliana, was screened for cell cycle function by overexpressing it in Schizosaccharomyces pombe (fission yeast). The expression pattern of Arath;CDC25 was also examined in different tissues of A. thaliana. Fission yeast was transformed with plasmids pREP1 and pREP81 with the Arath;CDC25 gene under the control of the thiamine-repressible nmt promoter. Using reverse transcription-polymerase chain reaction (RT-PCR), the expression of Arath;CDC25 was examined in seedlings, flower buds, mature leaves and stems of A. thaliana; actin (ACT2) was used as a control. In three independent transformants of fission yeast, cultured in the absence of thiamine (T), pREP1::Arath;CDC25 induced a highly significant reduction in mitotic cell length compared with wild type, pREP::Arath;CDC25 +T, and empty vector (pREP1 +/- T). The extent of cell shortening was greater using the stronger pREP1 compared with the weaker pREP81. However, Arath;CDC25 was expressed at low levels in all tissues examined. The data indicate that Arath;CDC25 can function as a mitotic accelerator in fission yeast. However, unlike other plant cell cycle genes, expression of Arath;CDC25 was not enhanced in rapidly dividing compared with non-proliferative Arabidopsis tissues.

Yeasts: providing questions and answers for modern biology.

Yeasts are to be found in virtually every conceivable niche on this planet and are amazingly varied in their shapes ('morphologies'), life cycles, metabolic capabilities, potentials for use in industrial processes, abilities to spoil food and drink or to act as dangerous human pathogens. This review describes four very different species of yeast to illustrate some of the diversity which exists and, in the case of one of them, Saccharomyces cerevisiae (the familiar baker's or brewer's yeast), the extent of both our knowledge and ignorance.

An investigation of the metabolism of isoleucine to active Amyl alcohol in Saccharomyces cerevisiae.

The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate. The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha-methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta-methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate.

An investigation of the metabolism of valine to isobutyl alcohol in Saccharomyces cerevisiae.

The metabolism of valine to isobutyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy and combined gas chromatography-mass spectrometry. The product of valine transamination, alpha-ketoisovalerate, had four potential routes to isobutyl alcohol. The first, via branched-chain alpha-ketoacid dehydrogenase to isobutyryl-CoA is not required for the synthesis of isobutyl alcohol because abolition of branched-chain alpha-ketoacid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isobutyl alcohol. The second route, via pyruvate decarboxylase, is the one that is used because elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant virtually abolished isobutyl alcohol production. A third potential route involved alpha-ketoisovalerate reductase, but this had no role in the formation of isobutyl alcohol from alpha-hydroxyisovalerate because cell homogenates could not convert alpha-hydroxyisovalerate to isobutyl alcohol. The final possibility, use of the pyruvate decarboxylase-like enzyme encoded by YDL080c, seemed to be irrelevant, because a strain with a disruption in this gene produced wild-type levels of isobutyl alcohol. Thus there are major differences in the catabolism of leucine and valine to their respective "fusel" alcohols. Whereas in the catabolism of leucine to isoamyl alcohol the major route is via the decarboxylase encoded by YDL080c, any single isozyme of pyruvate decarboxylase is sufficient for the formation of isobutyl alcohol from valine. Finally, analysis of the 13C-labeled products revealed that the pathways of valine catabolism and leucine biosynthesis share a common pool of alpha-ketoisovalerate.

A 13C nuclear magnetic resonance investigation of the metabolism of leucine to isoamyl alcohol in Saccharomyces cerevisiae.

The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy. The product of leucine transamination, alpha-ketoisocaproate had four potential routes to isoamyl alcohol. The first, via branched-chain alpha-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product. This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain alpha-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol. A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced. A third route utilizes alpha-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from alpha-hydroxyisocaproate because cell homogenates could not convert alpha-hydroxyisocaproate to isoamyl alcohol. The final possibility was that a pyruvate decarboxylase-like enzyme encoded by YDL080c appears to be the major route of decarboxylation of alpha-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent.

Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase.

Dihydrolipoamide dehydrogenase is a common component of mammalian multienzyme complexes that decarboxylate alpha-ketoacids and catabolize glycine. The common function is to reoxidize a reduced lipoate component of each complex, thereby preparing that lipoate for another round of catalysis. Regions within dihydrolipoamide dehydrogenase involved in association with other proteins of the complexes are poorly defined, and despite high amino acid sequence conservation through evolution, it is unknown if dihydrolipoamide dehydrogenases are functionally equivalent across species. To address this issue, we asked whether the human enzyme could restore function to the alpha-ketoacid dehydrogenase complexes in a yeast strain deficient in endogenous dihydrolipoamide dehydrogenase. This dihydrolipoamide dehydrogenase null mutant will not grow on non-fermentable carbon sources. The human enzyme expressed from a CEN plasmid complemented the growth phenotype and restored full activity to the pyruvate and alpha-ketoglutarate dehydrogenase complexes. Human dihydrolipoamide dehydrogenases with selected amino acid substitutions were then tested in the null strain for their ability to restore function. Substitutions tested represented naturally occurring candidate mutations identified in an individual with inactive dihydrolipoamide dehydrogenase. A K37E change had full function while a P453L change resulted in reduced dihydrolipoamide dehydrogenase abundance in the mitochondria and no detectable catalytic activity.

CEN14 sequences cause slower proliferation, reduced cell size and asporogeny in Saccharomyces cerevisiae.

The introduction of CEN14-based plasmids into haploid strains of Saccharomyces cerevisiae resulted in reduced proliferation rates and significantly smaller (20%) cell size than in untransformed control cells. This could be useful to those yeast biotechnology processes that require high levels of gene expression but little or no yeast growth and proliferation. In diploids similar plasmids caused asporogeny, which was possibly a consequence of the reduced cell size.

In Saccharomyces cerevisiae deletion of phosphoglucose isomerase can be suppressed by increased activities of enzymes of the hexose monophosphate pathway.

Saccharomyces cerevisiae mutants defective in the structural gene PGI1 lack phosphoglucose isomerase and hence cannot grow on glucose. Spontaneous mutants were isolated by selecting for the regained ability to grow on YEPD (yeast extract/peptone/glucose). Three complementation groups called spg29-31 (suppressor of pgi1 delta) were identified. The metabolism of [2-13C]glucose was studied by 13C NMR spectroscopy. This led to the conclusion that in a spg29 mutant suppression of the glycolytic defect was achieved by increased carbon flux through the hexose monophosphate pathway. The specific activities of enzymes of the hexose monophosphate pathway (except glucose-6-phosphate dehydrogenase) and NAD- and NADP-dependent glutamate dehydrogenase were increased in the bypass mutant.

Irreversible formation of pseudohyphae by haploid Saccharomyces cerevisiae.

Culturing haploid strains of Saccharomyces cerevisiae in liquid minimal medium with 2% ethanol and 2% leucine resulted in the formation of long anucleate pseudohyphae. This occurred only with the combination of ethanol as carbon source and leucine as nitrogen source and was independent of mating type. The transition to a pseudohyphal form observed under these conditions appears to be irreversible. These findings further extend our view of the developmental alternatives in this important model eukaryote.

Exercise on-transient gas exchange kinetics are slowed as a function of age.

The purpose was to characterize gas exchange kinetics following the on-transient of exercise in men aged 30-80 yr. Forty-six men completed square wave exercise tests from loadless cycling to subventilatory threshold (V(E)T) work rates with gas exchange measured breath-by-breath. Signal averaged data were fit with a monoexponential equation to derive time constants (tau) for gas exchange and ventilation (tau VO2, tau VCO2, tau VE) and heart rate (tau HR). There was a significant slowing of ventilation and gas exchange kinetics across age with linear regression yielding an increase of 0.67 s.yr-1 for tau VO2 (39 s in young to 61 s in old), 0.57 s.yr-1 for tau VCO2, and 0.65 s.yr-1 for tau VE, whereas tau HR (44 to 41 s) was not changed significantly. The slowed VO2 kinetics with age may reflect limitations in muscle blood flow or in control of the rate of oxidative metabolism. The less marked slowing of tau VCO2 compared with tau VO2 across age may reflect reduced CO2 storage capacity with loss of muscle tissue. The tau VE change across age was similar to that for tau VCO2 (tau VE/tau VCO2 unchanged). The present study demonstrated marked age-related slowing of gas exchange dynamics at exercise onset.

Purification and characterization of the branched chain alpha-ketoacid dehydrogenase complex from Saccharomyces cerevisiae.

Branched chain alpha-ketoacid dehydrogenase complex was purified from Saccharomyces cerevisiae by polyethylene glycol fractionation and chromatography on Sephacryl S-200, DEAE-cellulose and Sepharose CL-2B. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated the enzyme contained subunits of M(r) = 57,000, 52,000, 47,000 and 38,000. The specific activity of the purified enzyme was 0.82 mumol NADH/min/mg protein at 30 degrees C with 16 mM alpha-ketoisovalerate as substrate. The apparent Km values for alpha-ketoisovalerate, alpha-ketoisocaproate and alpha-keto-beta-methylvalerate were 21, 22, and 20 mM, respectively. The preparation was also able to oxidize the intermediates of threonine and methionine metabolism, alpha-keto-gamma-methiolbutyrate and alpha-ketobutyrate, with Km values of 13 and 8 mM, respectively.

Sequence of the Escherichia coli K-12 edd and eda genes of the Entner-Doudoroff pathway.

A 3.5-kb DNA fragment from the Clarke and Carbon Escherichia coli genomic clone, pLC37-44, was sequenced on both strands. Part of the zwf gene, encoding glucose-6-phosphate dehydrogenase, and all of the edd and eda genes, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, respectively, of the Entner-Doudoroff pathway, were identified. These data are compared with those of Egan et al. [J. Bacteriol. 174 (1992) 4638-4646] and important differences were noted.

A study of branched-chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched-chain amino acids in Saccharomyces cerevisiae.

The specific activity of branched-chain amino acid aminotransferase was highest when S. cerevisiae was grown in minimal medium containing a branched-chain amino acid as nitrogen source. Growth in complex media with glycerol or ethanol gave moderately high levels, whereas with glucose and fructose the specific activity was very low. Mutagenesis defined three genes (BAA1 to BAA3) required for branched-chain amino acid catabolism. The baa1 mutation reduced the specific activity of the aminotransferase, the stationary phase density in YEPD and caused gross morphological disturbance. Branched-chain amino acid aminotransferase is essential for sporulation.

Gas exchange dynamics with sinusoidal work in young and elderly women.

The objective of this study was to model the dynamics of, and interrelationships among, gas exchange, ventilation, and heart rate responses to sinusoidal forcing in young and elderly women. Nineteen females, 22-28 years (n = 10) and 62-73 years (n = 9) volunteered for the study. All experiments were conducted at work rates below the ventilatory threshold (TVE). A sine wave test consisted of 4 min of cycling (60 rpm) at a work rate equal to the mean of the limits of sinusoid (60% TVE) followed by 16-20 min of a sinusoidally varying work rate (30-90% of TVE) and ending with 4 min of cycling at 60% TVE. The periods were 0.75, 1.0, 1.5, 3.0, 6.0, 10.0 min, assigned randomly. The averaged data were used to determine amplitude and phase lag of the sinusoidal response of VO2, VCO2, VE, fH, PETCO2, and PETO2. Bode plots demonstrated that the dynamics of the cardiorespiratory responses were all well-described by a first-order exponential equation with a delay for both young and elderly subjects. The time constants were much longer in the elderly. Nevertheless, there appeared to be a strong link between the relative slowing of the four components of the gas transport system (VO2, VCO2, VE, and fH). This may suggest one single factor is reflected in the slowing of all components in older subjects.

Effects of chlorhexidine diacetate and cetylpyridinium chloride on whole cells and protoplasts of Saccharomyces cerevisiae.

Chlorhexidine diacetate (CHA) and the quaternary ammonium compound, cetylpyridinium chloride (CPC), were fungicidal to Saccharomyces cerevisiae A364A and CHA to its mannoprotein mutant LB6-5D. Both CHA and CPC induced leakage of K+ and pentose material from both strains and both agents induced protoplast lysis as well as interacting with crude cell sap. Differences were observed between CHA and CPC in protoplast lysis and in cell sap interaction. Q25 values for A364A with CHA as test agent were 5.6 and 1.6 (fungicidal activity, depending on method of calculation), 1.46 (cell sap interaction) and 0.77 (leakage of pentoses). A sub-lytic concentration of CHA reduced considerably protoplast regeneration. Although the plasma membrane is an important target site for both agents, interaction with other cellular components also contributes to fungal death.

Strength training alters contractile properties of the triceps brachii in men aged 65-78 years.

Voluntary and electrically evoked contractile properties were studied in the triceps brachii following a 24-week dynamic strength training program in ten men aged 65-78 years. Eight men of a similar age were control subjects. A resistance overload program was undertaken three times per week with subjects performing four sets of six to eight repetitions at 80% of their one repetition on maximum (1RM). Maximum voluntary contraction (MVC) and contractile properties were measured at 0, 12, and 24 weeks in the exercise group and at 0 and 24 weeks in the controls. The 1RM was used to assess dynamic strength at 0 and 24 weeks in the exercise group. Contractile measures consisted of supramaximal isometric twitch and post-activation twitch parameters. Muscle size was estimated from anthropometric measurements. Compared with the control group, the exercise group MVC increased by about 20% and time to peak tension was slowed by about 11%. Also in the exercise group the peak rate of torque development of the potentiated twitch was reduced by about 10%. Twitch potentiation was substantial in both groups (about 140%) and unaffected by training. The 1 RM increased by about 30%, and there was a non-significant positive change of 8.6% in the muscle plus bone cross-sectional area in the exercise group. The results show that the force generating capacity of the triceps brachii in these men can be significantly improved for up to 24 weeks using concentric overload training. Furthermore, the finding of slowed twitch properties and no change in peak twitch amplitude substantiate and extend the limited data currently available on intrinsic contractile changes in the elderly.(ABSTRACT TRUNCATED AT 250 WORDS)