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Kevin Verstrepen's lab meetings can be pretty boozy affairs. Twice a week, several members of his group at Belgium's University of Leuven and the Flanders Institute for Biotechnology gather around [...]

This study was designed to characterize the suppressant effect of yeast protein and purified peptides on energy intake. For this purpose, 5 experiments were carried out using adult male Wistar rats. Rats that consumed ad libitum a standard yeast protein diet are significantly less and were leaner over 21 d than rats that consumed ad libitum a standard milk protein diet (Expt. 1). Moreover, rats fed a high yeast protein load reduced their next meal and daily energy intake more than rats fed any other well-balanced, amino acid, high protein load (soy, total milk protein, or wheat gluten} and more than those fed a wheat starch diet (Expt. 2). Purified peptides from the yeast protein extract produced similar effects on subsequent energy intake (Expt. 3). Study of the behavioral satiety sequence showed that rats consuming P14-y or P55-y diets ad libitum did not acquire a conditioned food aversion (Expt. 4). Finally, a preliminary study of gastric emptying in rats fed yeast protein loads showed that yeast protein was emptied more rapidly through the pylorus than total milk protein during a meal, which may induce satiety (Expt. 5). Taken together, these experiments show that yeast proteins enhance satiety in rats more than other proteins.

A possible plasmid transfer between Candida glabrata and the Saccharomyces species was studied for evaluating the possibility of gene transfer between pathogenic and food-borne yeasts. The transfer could enable the spread of virulence factors and resistance to medical drugs also between distantly related yeast species and might help in 'transformation' of harmless saprophytes into potential causative agents of human infections.

G-protein-coupled receptors (GPCRs) are the major targets of today's medicines. To elucidate the mechanism of activation of GPCRs and the interaction of these receptors with their G proteins, mutagenesis studies have proven to be a powerful tool and have provided insight into the structure and function of GPCRs. Random mutagenesis is useful in this respect particularly when combined with a robust screening assay that is based on the functional properties of the mutants. In this article, the use of random mutagenesis combined with a functional screening assay in yeast is described and compared with alternative approaches such as site-directed mutagenesis per se, alanine/cysteine scanning and another screening assay, receptor selection and amplification technology (R-SAT). Screening in yeast of randomly mutated GPCRs has proven successful in the identification of ligands for orphan receptors and in high-throughput approaches. Moreover, it has provided substantial insight into G-protein coupling and receptor activation.

In the past, studies using the yeast Saccharomyces cerevisiae enabled major breakthroughs in the understanding of basic cellular and molecular processes. Today, the use of yeast is undergoing a 'rebirth' in both fundamental and applied research. Indeed, advances in yeast technology have paved the way for a variety of new genome-wide screening approaches. Experimental strategies using yeast aim to unravel disease-related molecular events and to discover novel medicinal compounds. In this article, the impact of yeast as an experimental tool for disease-related studies is summarized and the use of yeast in high-throughput screenings for pharmacological purposes is evaluated. The recently applied and promising approach of so-called humanized yeast systems is also discussed.

By using the maximum likelihood method, we made a genome-wide comparison of the evolutionary rates in the lineages leading to the laboratory strain (S288c) and a wild strain (YJM789) of Saccharomyces cerevisiae and found that genes in the laboratory strain tend to evolve faster than in the wild strain. The pattern of elevated evolution suggests that relaxation of selection intensity is the dominant underlying reason, which is consistent with recurrent bottlenecks in the S. cerevisiae laboratory strain population. Supporting this conclusion are the following observations: (i) the increases in nonsynonymous evolutionary rate occur for genes in all functional categories; (ii) most of the synonymous evolutionary rate increases in $288c occur in genes with strong codon usage bias; (iii) genes under stronger negative selection have a larger increase in nonsynonymous evolutionary rate; and (iv) more genes with adaptive evolution were detected in the laboratory strain, but they do not account for the majority of the increased evolution. The present discoveries suggest that experimental and possible industrial manipulations of the laboratory strain of yeast could have had a strong effect on the genetic makeup of this model organism. Furthermore, they imply an evolution of laboratory model organisms away from their wild counterparts, questioning the relevancy of the models especially when extensive laboratory cultivation has occurred. In addition, these results shed light on the evolution of livestock and crop species that have been under human domestication for years. model organism | slightly deleterious mutation | yeast evolution

Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure. Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding. Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19. Results for the a12 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures. Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones. bakers' yeast | mitochondria | splicing factor

A method for the simultaneous determination of methionine (Met) and selenomethionine (SeMet) in yeast using species-specific isotope dilution (ID) gas chromatography/mass spectrometry (GC/MS) is described. Samples were digested by refluxing for 16 h with 4 M methanesulfonic acid. Analytes were derivatized with methyl chloroformate and extracted into chloroform for GC/MS analysis. In addition to use of commercially available 13Cenriched Met and SeMet spikes for species specific ID analysis, a [sup.74]Se-enriched SeMet spike was also available for comparison of results. In selective ion monitoring mode, the intensities of ions at m/z 221, 222, 269, 270, and 263 were used to calculate the 221/222, 269/270, and 269/263 ion ratios for quantification of Met and SeMet. Concentrations of 5959 [+ or -] 33 and 3404 [+ or -] 12 [micro]g [g.sup.-1] (one standard deviation, n = 6) with relative standard deviations of 0.55 and 0.36% for Met and SeMet, respectively, were obtained using [sup.13]C-enriched spikes. A concentration of 3417 [+ or -] 8 [micro]g [g.sup.-1] (one standard deviation, n = 6) was obtained using the [sup.74]Se-enriched SeMet spike. The concentration of SeMet measured in the yeast is equivalent to 66.43 [+ or -] 0.24% of total Se and 30.31 [+ or -] 0.11% of total Met is in the form of SeMet. Method detection limits (three times the standard deviation) of 3.4 and 1.0[micro]g [g.sup.-1] were estimated for Met and SeMet, respectively, based on a 0.25-g subsample of yeast with 1 mL of extract used for derivatization. A similar concentration of 5930 [+ or -] 29 [micro]g [g.sup.-1] (one standard deviation, n = 4) for Met and a lower concentration of 2787 [+ or -] 49 [micro]g [g.sup.-1] (one standard deviation, n = 4) for SeMet were obtained for this yeast sample using species-specific ID analysis based on GC/MS with [sup.13]C-enriched Met and SeMet spikes when a 2-h open microwave digestion approach using 8 M methanesulfonic acid was used.

DNA 3'-phosphatase mediates the removal of 3'-phosphate ends generated by the Escherichia coli fpg apurinic/apyrimidinic (AP) lyase following the incision of AP site. The protein does not cleave DNA bearing AP sites, indicating that it is not an AP endonuclease or AP lyase.

Here we describe the phenotypic characterization of the cta[4.sup.+] gene, encoding a novel member of the P4 family of P-type ATPases of fission yeast. The cta4[DELTA] mutant is temperature sensitive and cold sensitive lethal and displays several morphological defects in cell polarity and cytokinesis. Microtubules are generally destabilized in cells lacking Cta4p. The microtubule length is decreased, and the number of microtubules per cell is increased. This is concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell. These defects are likely due to a general imbalance in cation homeostasis. Immunofluorescence microscopy and membrane fractionation experiments revealed that green fluorescent protein-tagged Cta4 localizes to the ER. Fluorescence resonance energy transfer experiments in living cells using the yellow cameleon indicator for [Ca.sup.2+] indicated that Cta4p regulates the cellular [Ca.sup.2+] concentration. Thus, our results reveal a link between cation homeostasis and the control of cell shape, microtubule dynamics, and cytokinesis, and appoint [Ca.sup.2+] as a key ion in controlling these processes.

The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade. Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood. The pheromone-responsive G protein α subunit (Gα) of yeast down-regulates the mating signal, but its targets are unknown. We have found that Gα binds directly to the mating-specific MAPK in yeast cells responding to pheromone. This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Gα-MAPK pathway., The pheromone response system of S. cerevisiae, which enables haploid cells of opposite mating type to sense one another's presence and to differentiate into gametes, uses signaling molecules found in [...]

The use of auxotrophic strains of Saccharomyces cerevisiae in quantitative physiology, functional genome analysis, and industrial processes is reviewed. Topics include applications, genetic complementation vs. nutritional supplementation, design of synthetic culture media, stability of transformed strains and cross-feeding, and phenotype masking.

A working model for iron-sulfur cluster assembly protein is presented using the recombinat purified yeast protein. Data suggest that yeast protein is sparingly soluble at ambient temperatures, is sensitive to proton-mediated degradation, and forms a complex with the electron transport protein ferredoxin in both apo and holo states.

Research examines 36 polypeptides of yeast type 1 protein phosphatase capable of interacting with the Glc7p protein complexes from yeast. Data indicate that the Glc7p is a component of the cleavage and polyadenylation factor complex and mediates type 1 protein phosphatase dephosphorylation in the regulation mRNA 3'-end formation and processing.

Research reveals that delta-gluconolactone is anaerobically metabolized initially to glucose, which is mediated by the NADPH-linked glucose dehydrogenase. This is followed by glycolysis and the pentose phosphate pathway, the primary dissimilatory pathway for delta-gluconolactone metabolism in Saccharomyces bulderi.

The mouse mutants mocha and pearl are deficient in the AP-3 [delta] and [beta]3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the [beta]3 and [mu]3 subunits coassemble into a heterodimer, whereas the [sigma]3 subunit remains monomeric. In pearl cells, the [delta] and [sigma]3 subunits coassemble into a heterodimer, whereas [mu]3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of [beta]3 and [mu]3 can interact with each other. Pearl cell lines were generated that express [beta]3A, [beta]3B, a [beta]3A[beta]2 chimera, two [beta]3A deletion mutants, and a [beta]3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only [beta]3A, [beta]3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The [beta]3A[beta]2 chimera and the [beta]3A short deletion mutant gave partial functional rescue, whereas the [beta]3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of [beta]3 are important for function, but the clathrin binding site is not needed.

At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent 'switch' to turn on effectors or as an EF-Tu-like 'assembly factor' using the GTPase cycle to assemble a macromolecular structure.

Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules. Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole, including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected. Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile. The vps41[DELTA] and apl6[DELTA] mutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally. Furthermore, a vps10[DELTA] sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles. These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface. Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

Results show that centromeres are enriched in the histone deposition complex chromatin assembly factor I (CAF-I) and Hir proteins, suggesting that these proteins directly influence centromeric chromatin formation. Data indicate that the kinetochore-related function of CAF-1 and Hir proteins overlaps.

This review unfolds the story of the unfolded protein response (UPR) process encountered with surface and secreted proteins in yeast and mammalian systems. The UPR begins when protein maturation, which involves protein folding, is interrupted at the endoplasm reticulum. Data indicate that three gene products can initiate the UPR in yeast and mammals.

Results show that yeast cofilin differentiates between various phosphatidylinositides with PI(4,5)P(sub)2 binding site exhibiting residues in helix 3 and other parts of the molecules. Mutant analysis reveals biological role for the residues and their involvement in actin and PI(4,5)P(sub)2 binding.

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ~245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p: This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ~0.5 [micro] M. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

Results reveal that Saccharomyces cerevisiae 2'-phosphotransferase exhibits a high apparent affinity for ligand ligated tRNA and low turnover rate. The enzyme efficiently removes 2'-phosphate from substrates and bears resemblance to its Escherichia coli homolog.

Electron microscopic analysis reveals that yeast ribosome-nascent chain containing complexes and associated trimeric Sec61 complex bind to the protein-conducting channel of translating 80S ribosome. A model is proposed for the two functional states of the translating ribosome-channel complex.

Results indicate that the negative regulator of G(sub)1 cyclin Cln3, Whi3 exhibits an RNA-recognition motif, which binds the CLN3 mRNA localizing to discrete cytoplasmic foci. Furthermore, Whi3 restrains Cln3 function during meiosis, filamentaion, and mating in budding yeast.

Results show that the divalent metal magnesium, activating the phosphoryl transfer in yeast pyruvate kinase, has a role in modulating multiligand interactions that are involved in the allosteric behavior of the enzyme.

Acetohydroxyacid synthase mediates biosynthesis of isoleucine and valine, using thiamin pyrophosphate as a cofactor. Thiamin auxotrophic phenotype of yeast results from an altered dependence for the cofactor due to thi1 mutation, which leads to a substitution of aspartate 176 that is involved in thiamine pyrophosphate binding by the enzyme.

Research reveals that Cryptococcus neoformans populations in tissue undergo dynamic morphological changes during the course of murine pulmonary infection. Data indicate that cells show increased cell wall thickness, melanization, and black color. The heterogeneous cell populations assume giant forms.

Research demonstrates that kinetochores comprising of molecular motors and microtubule associated proteins collectively mediate binding of chromosomes to spindle microtubules, imposition of tension to the paired sister kinetochores, and correct chromosome movement during mitosis.

Research shows that the yeast prion [PSI(sup)+] is induced from gain-of-function properties of New1p and Rnq1p aggregates. The Gln/Asn-rich prion domains of these proteins control the susceptibility of [PSI(sup)+] induction and aggregation of human glutamine expansion disease protein.

Research demonstrates that Sit4p-mediated regulatory mechanism plays a crucial role in the modulation of the ABC transporter KIPdr5p of Kluyveromyces lactis. Data further show that Sit4p gene regulation is a posttranslational event because mutations in SIT4 do not affect either the transcription of KIPDR5 or the steady-state level of KIPdr5P.

Nuclear magnetic resonance spectoscopic data reveal no change in the geometry of the magnetic axes for cytochrome c consequent upon binding to cytochrome c peroxidase. Data further suggest that the changes in the ferric protein are due to diffeences in the binding mode.

A highly fluorinated peptide system was design, synthesized and characterized, in terms of the coiled coil area of the yeast transcription factor GCN4.

Research describes six morphogenetic genes in Yarrowia lipolytica involved in hyphal formation and its filamentous morphology. Results show that a Ras-cyclic AMP pathway forms the hyphae and processing abnormalities of the extracellular and the parietal proteins lead to morphogenetic defects.

Zygosaccharomyces bailii appears to be one of the few yeasts that can grow in the complete absence of oxygen. For this reason, oxygen limitation would not prevent the organism from spoiling food.

Researchers have engineered a strain of yeast that displays a-agglutinin and alpha-agglutinin on the cell surface. This yeast grows normally at high glucose concentrations, but agglutinates and sediments completely when glucose concentrations are very low. This can be used industrially to separate the cells from the supernatant.

High frequency time domain EPR and ENDOR spectroscopy were used to study the tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast.

This review discusses the recent developments in cell wall biogenesis of yeast in terms of the molecular architecture and composition and biosynthetic genes and their regulation. Also aspects of cell polarity and signal transduction between external and internal stimuli during growth and proliferation are presented.

The performance of baker's yest for beta-keto ester reductions was improved using recombinant DNA methods to alter enzyme levels.

Research examines the intramolecular interaction and molecular basis of the propagation mechanism of yeast prion Ure2p. Results demonstrate that Ure2p dimerizes via its C-terminal region and its N-terminal facilitates assembly of the protein and subsequent formation of the amyloid fibrils.

Results demonstrate that cysteine221 of the yeast pyruvate decarboxylase mediates the substrate binding and transmittal of the information to the active site thiamin diphosphate. Data indicate that cysteine221 has a role in the structure and mechanism of pyruvate decarboxylase catalysis as revealed by site-directed mutagenesis studies.