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In the budding yeast Saccharomyces cerevisiae, self-recognition and the thereby promoted aggregation of thousands of cells into protective flocs is mediated by a family of cell-surface adhesins, the flocculins (Flo). Based on this social behavior FLO genes fulfill the definition of 'greenbeard' genes, which direct cooperation toward other carriers of the same gene. The process of flocculation plays an eminent role in the food industry for the production of beer and wine. However, the precise mode of flocculin-mediated surface recognition and the exact structure of cognate ligands have remained elusive. Here, we present structures of the adhesion domain of a flocculin complexed to its cognate ligands derived from yeast high-mannose oligosaccharides at resolutions up to 0.95 [Angstrom]. Besides a PA14-like architecture, the Flo5A domain reveals a previously undescribed lectin fold that utilizes a unique DcisD calcium-binding motif for carbohydrate binding and that is widely spread among pro- and eukaryotes. Given the high abundance of high-mannose oligosaccharides in yeast cell walls, the Flo5A structure suggests a model for recognition, where social non-self-instead of unsocial self-interactions are favored. altruism | molecular recognition | fungal development | atomic resolution doi: 10.1073/pnas.1013210108.

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR). However, how myosin II is targeted to the division site and promotes AMR assembly, and how the AMR coordinates with membrane trafficking during cytokinesis, remains poorly understood. Here we show that Myo1 is a two-headed myosin II in Saccharomyces cerevisiae, and that Myo1 localizes to the division site via two distinct targeting signals in its tail that act sequentially during the cell cycle. Before cytokinesis, Myo1 localization depends on the septin-binding protein Bni5. During cytokinesis, Myo1 localization depends on the IQGAP Iqg1. We also show that the Myo1 tail is sufficient for promoting the assembly of a 'headless' AMR, which guides membrane deposition and extracellular matrix remodeling at the division site. Our study establishes a biphasic targeting mechanism for myosin II and highlights an underappreciated role of the AMR in cytokinesis beyond force generation. doi/ 10.1083/jcb.201005134

The parameters that control nuclear size and shape are poorly understood. In yeast, unregulated membrane proliferation, caused by deletion of the phospholipid biosynthesis inhibitor SPO7, leads to a single nuclear envelope 'flare' that protrudes into the cytoplasm. This flare is always associated with the asymmetrically localized nucleolus, which suggests that the site of membrane expansion is spatially confined by an unknown mechanism. Here we show that in spo7[DELTA] cells, mutations in vesicle-trafficking genes lead to multiple flares around the entire nucleus. These mutations also alter the distribution of small nucleolar RNA-associated nucleolar proteins independently of their effect on nuclear shape. Both single- and multi-flared nuclei have increased nuclear envelope surface area, yet they maintain the same nuclear/cell volume ratio as wild-type cells. These data suggest that, upon membrane expansion, the spatial confinement of the single nuclear flare is dependent on vesicle trafficking. Moreover, flares may facilitate maintenance of a constant nuclear/cell volume ratio in the face of altered membrane proliferation. doi/ 10.1083/jcb.201006083

Lte1 is known as a regulator of mitotic progression in budding yeast. Here we demonstrate phosphorylation-dependent inhibition of polarized bud growth during G2/M by Lte1. Cla4 activity first localizes Lte1 to the polarity cap and thus specifically to the bud. This localization is a prerequisite for subsequent Clb--Cdk-dependent phosphorylation of Lte1 and its relocalization to the entire bud cortex. There, Lte1 interferes with activation of the small GTPases, Ras and Bud1. The inhibition of Bud1 prevents untimely polarization until mitosis is completed and Cdc14 phosphatase is released. Inhibition of Bud1 and Ras depends on Lte1's GEF-like domain, which unexpectedly inhibits these small G proteins. Thus, Lte1 has dual functions for regulation of mitotic progression: it both induces mitotic exit and prevents polarized growth during mitotic arrest, thereby coupling cell cycle progression and morphological development. doi/ 10.1083/jcb.201005070

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the elF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm. doi/ 10.1083/jcb.201003001

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2'deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdUlabelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes. DOI 10.1099/mic.0.035519-0

Sulphite is widely used as a preservative in foods and beverages for its antimicrobial and antioxidant activities, particularly in winemaking where SO2 is frequently added. Thus, sulphite resistance mechanisms have been extensively studied in the fermenting yeast Saccharomyces cerevisiae. Sulphite detoxification, involving a plasma membrane protein encoded by the SSU1 gene, is the most efficient resistance mechanism in S. cerevisiae. In this study, we characterized the unusual expression pattern of SSU1 in the wine strain 71B. We provide, for the first time, evidence of SSU1 induction by sulphite. The study of SSU1 expression during fermentation and in different growth conditions showed that sulphite is the main regulator of SSU1 expression, explaining its specific pattern. Combining analyses of gene expression and growth behaviour in response to sulphite, we found that 71 B displayed unique behavioural patterns in response to sulphite pre-adaptation that may be explained by changes in SSU1 expression. Examination of the genomic organization of the SSU1 locus and sequencing of the region revealed three different alleles in 71 B, two of which corresponded to translocated VIII-XVI forms. The lack of differences between promoter regions suggests that this inducible SSU1 expression pattern is due to modification of regulatory/signalling pathways. DOI 10.1099/mic.0.036723-0

Boundaries between euchromatic and heterochromatic regions until now have been associated with chromatin-opening activities. Here, we identified an unexpected role for histone deacetylation in this process. Significantly, the histone deacetylase (HDAC) Rpd3 was necessary for boundary formation in Saccharomyces cerevisiae. rpd3[DELTA] led to silent information regulator (SIR) spreading and repression of subtelomeric genes. In the absence of a known boundary factor, the histone acetyltransferase complex SAS-I, rpd3[DELTA] caused inappropriate SIR spreading that was lethal to yeast cells. Notably, Rpd3 was capable of creating a boundary when targeted to heterochromatin. Our data suggest a mechanism for boundary formation whereby histone deacetylation by Rpd3 removes the substrate for the HDAC Sir2, so that Sir2 no longer can produce O-acetyl-ADP ribose (OAADPR) by consumption of [NAD.sup.+] in the deacetylation reaction. In essence, OAADPR therefore is unavailable for binding to Sir3, preventing SIR propagation. gene silencing | histone deacetylase | O-acetyl-ADP-ribose | Sas2 | Sir3 www.pnas.org/cgi/doi/10.1073/pnas.0909169107

During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 [right arrow] R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. [COX2.sup.W56R] can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3'-UTR that localizes mRNA translation to the mitochondria, such as yeast [ATP2.sup.3'-UTR]. Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity. cytochrome coxidase | mitochondria | Saccharomyces cerevisiae doi/ 10.1073/pnas.1000735107

Inositol is essential in eukaryotes, and must be imported or synthesized. Inositol biosynthesis in Saccharomyces cerevisiae is controlled by three non-essential genes that make up the inositol regulon: SclNO2 and SclNO4, which together encode a heterodimeric transcriptional activator, and ScOPI1, which encodes a transcriptional repressor. ScOpi1p inhibits the Sclno2-Sclno4p activator in response to extracellular inositol levels. An important gene controlled by the inositol regulon is SclNO1, which encodes inositol-3-phosphate synthase, a key enzyme in inositol biosynthesis. In the pathogenic yeast Candida albicans, homologues of the S. cerevisiae inositol regulon genes are 'transcriptionally rewired'. Instead of regulating the CAINO1 gene, CAINO2 and CAINO4 regulate ribosomal genes. Another Candida species that is a prevalent cause of infections is Candida glabrata; however, C. glabrata is phylogenetically more closely related to S. cerevisiae than C. albicans. Experiments were designed to determine if C. glabrata homologues of the inositol regulon genes function similarly to S. cerevisiae or are transcriptionally rewired. CglNO2, CglNO4 and CgOPI1 regulate CglNO1 in a manner similar to that observed in S. cerevisiae. However, unlike in S. cerevisiae, CgOPI1 is essential. Genetic data indicate that CgOPI1 is a repressor that affects viability by regulating activation of a target of the inositol regulon. DOI 10.1099/mic.0.030072-0

The DNA mismatch repair system (MMR) identifies replication errors and damaged bases in DNA and functions to preserve genomic integrity. MutS performs the task of locating mismatched base pairs, loops and lesions and initiating MMR, and the fundamental question of how this protein targets specific sites in DNA is unresolved. To address this question, we examined the interactions between Saccharomyces cerevisiae Msh2-Msh6, a eukaryotic MutS homolog, and DNA in real time. The reaction kinetics reveal that Msh2-Msh6 binds a variety of sites at similarly fast rates ([k.sub.ON] ~ N [10.sup.7] [M.sup.-1] [s.sup.-1]), and its selectivity manifests in differential dissociation rates; e.g., the protein releases a 2-Aminopurine:T base pair approximately 90-fold faster than a G:T mismatch. On releasing the 2-Ap:Tsite, Msh2-Msh6 is able to move laterally on DNA to locate a nearby G:T site. The long-lived Msh2-Msh6 x G:T complex triggers the next step in MMR--formation of an ATP-bound clamp--more effectively than the short-lived Msh2-Msh6 x 2-Ap:T complex. Mutation of Glu in the conserved Phe-X-Glu DNA binding motif stabilizes Msh2-[MSh6.sub.E339A] x 2-Ap:T complex, and the mutant can signal 2-Ap:T repair as effectively as wild-type Msh2-Msh6 signals G:T repair. These findings suggest a targeting mechanism whereby Msh2-Msh6 scans DNA, interrogating base pairs by transient contacts and pausing at potential target sites, and the longer the pause the greater the likelihood of MMR. ATPase activity | pre-steady-state kinetics www.pnas.orglcgi/doi/10.1073/pnas.0908302107

Carbonic anhydrases (CAs) are metalloenzymes that catalyse the interconversion of carbon dioxide and bicarbonate with high efficiency. This reaction is fundamental to biological processes such as respiration, photosynthesis, pH homeostasis, C[O.sub.2] transport and electrolyte secretion. CAs are distributed among all three domains of life, and are currently divided into five evolutionarily unrelated classes ([alpha], [beta], [gamma], [delta] and [zeta]). Fungal CAs have only recently been identified and characterized in detail. While Saccharomyces cerevisiae and Candida albicans each have only one [beta]-CA, multiple copies of [beta]-CA- and [alpha]-CA-encoding genes are found in other fungi. Recent work demonstrates that CAs play an important role in the C[O.sub.2]-sensing system of fungal pathogens and in the regulation of sexual development. This review focuses on CA functions in S. cerevisiae, the fungal pathogens C. albicans and Cryptococcus neoformans, and the filamentous ascomycete Sordaria macrospora. DOI 10.1099/mic.0.032581-0

The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Only sla2 strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis of C. albicans infections. DOI 10.1099/mic.0.033233-0

Glucose repression of the tricarboxylic acid (TCA) cycle in Saccharomyces cerevisiae was investigated under different environmental conditions using [sup.13]C-tracer experiments. Real-time quantification of the volatile metabolites ethanol and C[O.sub.2] allowed accurate carbon balancing. In all experiments with the wild-type, a strong correlation between the rates of growth and glucose uptake was observed, indicating a constant yield of biomass. In contrast, glycerol and acetate production rates were less dependent on the rate of glucose uptake, but were affected by environmental conditions. The glycerol production rate was highest during growth in high-osmolarity medium (2.9 mmol [g.sup.-1] [h.sup.-1]), while the highest acetate production rate of 2.1 mmol [g.sup.-1] [h.sup.-1] was observed in alkaline medium of pH 6.9. Under standard growth conditions (25 g glucose [l.sup.-1], pH 5.0, 30[degrees]C) S. cerevisiae had low fluxes through the pentose phosphate pathway and the TCA cycle. A significant increase in TCA cycle activity from 0.03 mmol [g.sup.-1] [h.sup.-1] to about 1.7 mmol [g.sup.-1] [h.sup.-1] was observed when S. cerevisiae grew more slowly as a result of environmental perturbations, including unfavourable pH values and sodium chloride stress. Compared to experiments with high glucose uptake rates, the ratio of C[O.sub.2] to ethanol increased more than 50%, indicating an increase in flux through the TCA cycle. Although glycolysis and the ethanol production pathway still exhibited the highest fluxes, the net flux through the TCA cycle increased significantly with decreasing glucose uptake rates. Results from experiments with single gene deletion mutants partially impaired in glucose repression (hxk2, grr1) indicated that the rate of glucose uptake correlates with this increase in TCA cycle flux. These findings are discussed in the context of regulation of glucose repression. DOI 10.1099/mic.0.030213-0

Cells constantly adjust the sizes and shapes of their Corganelles according to need. In this study, we examine endoplasmic reticulum (ER) membrane expansion during the unfolded protein response (UPR)in the yeast Saccharomyces cerevisiae. We find that membrane expansion occurs through the generation of ER sheets, requires UPR signaling, and is driven by lipid biosynthesis. Uncoupling ER size control and the UPR reveals that mem brane expansion alleviates ER stress independently of an increase in ER chaperone levels. Converting the sheets of the expanded ER into tubules by reticulon overexpression does not affect the ability of cells to cope with ER stress, showing that ER size rather than shape is the key factor. Thus, increasing ER size through membrane synthesis is an integral yet distinct part of the cellular program to overcome ER stress. doi 10.1083/jcb.200907074

In Saccharomyces cerevisiae, peroxisomal inheritance from mother cell to bud is conducted by the class V myosin motor, Myo2p. However, homologues of S. cerevisiae Myo2p peroxisomal receptor, Inp2p, are not readily identifiable outside the Saccharomycetaceae family. Here, we demonstrate an unexpected role for Pex3 proteins in peroxisome inheritance. Both Pex3p and Pex3Bp are peroxisomal integral membrane proteins that function as peroxisomal receptors for class V myosin through direct interaction with the myosin globular tail. In cells lacking Pex3Bp, peroxisomes are preferentially retained by the mother cell, whereas most peroxisomes gather and are transferred en masse to the bud in cells overexpressing Pex3Bp or Pex3p. Our results reveal an unprecedented role for members of the Pex3 protein family in peroxisome motilily and inheritance in addition to their well-established role in peroxisome biogenesis at the endoplasmic reticulum. Our results point to a temporal link between peroxisome formation and inheritance and delineate a general mechanism of peroxisome inheritance in eukaryotic cells. doi 10.1083/jcb.200902117

Trehalose accumulation is a common response to several stresses in the yeast Saccharomyces cerevisiae. This metabolite protects proteins and membrane lipids from structural damage and helps cells to maintain integrity. Based on genetic studies, degradation of trehalose has been proposed as a required mechanism for growth recovery after stress, and the neutral trehalase Nth1p as the unique degradative activity involved. Here we constructed a collection of mutants for several trehalose metabolism and transport genes and analysed their growth and trehalose mobilization profiles during experiments of saline stress recovery. The behaviour of the triple [DELTA]nth 1 [DELTA]nth2[DELTA]ath1 and quadruple [DELTA]nth1[DELTA]nth2[DELTA]ath1[DELTA]agtl mutant strains in these experiments demonstrates the participation of the three known yeast trehalases Nth1p, Nth2p and Ath1p in the mobilization of intracellular trehalose during growth recovery after saline stress, rules out the participation of the Agt1p [H.sup.+]-disaccharide symporter, and allows us to propose the existence of additional new mechanisms for trehalose mobilization after saline stress.

Cadmium is a highly toxic environmental contaminant that has been implicated in various disorders. A major mechanism for cadmium detoxification in the yeast Saccharomyces cerevisiae relies on extrusion via Pca1, a P-type ATPase. While an N-terminal degron targets Pca1 for degradation before its secretion to the plasma membrane, cadmium in the growth media rapidly upregulates Pca1 by preventing its turnover. Here we show that the endoplasmic reticulum-associated degradation (ERAD) system, known for its role in quality control of secretory proteins, is unexpectedly responsible for the regulation of Pca1 expression by cadmium. Direct cadmium sensing at the ER by a degron in Pca1 leads to an escape of Pca1 from ERAD. This regulated conversion of an ERAD substrate to a secretory competent state in response to a cellular need illustrates a mechanism for expressional control of a plasma membrane protein. Yeast has likely evolved this mode of regulation for a rapid response against cadmium toxicity at the expense of constant synthesis and degradation of Pca1. ERAD of a portion of secretory proteins might occur via signal-dependent regulatory mechanisms as demonstrated for Pca1. ERAD | degron | P-type ATPase | Pca1 yeast

The widespread prokaryotic toxin-antitoxin (TA) systems involve conditional interaction between two TA proteins. The interaction between the Epsilon and Zeta proteins, constituting the TA system of plasmid pSM19035 from Streptococcus pyogenes, was detected in vivo using a yeast two-hybrid system. As we showed using Saccharomyces cerevisiae, the Zeta toxin hybrid gene also exerts its toxic effects in a dose-dependent manner in eukaryotic cells. Analysis of mutant proteins in the two-hybrid system demonstrated that the N-terminal part of Zeta and the N-terminal region of Epsilon are involved in the interaction. The N-terminal region of the Zeta protein and its ATP/GTP binding motif were found to be responsible for the toxicity.

We studied the function of the cyclin-dependent kinase Cdc28 (Cdk1) in the DNA damage response and maintenance of genome stability using Saccharomyces cerevisiae. Reduced Cdc28 activity sensitizes cells to chronic DNA damage, but Cdc28 is not required for cell viability upon acute exposure to DNA-damaging agents. Cdc28 is also not required for activation of the DNA damage and replication checkpoints. Chemical--genetic analysis reveals that CDC28 functions in an extensive network of pathways involved in maintenance of genome stability, including homologous recombination, sister chromatid cohesion, the spindle checkpoint, postreplication repair, and telomere maintenance. In addition, Cdc28 and Mre11 appear to cooperate to prevent mitotic catastrophe after DNA replication arrest. We show that reduced Cdc28 activity results in suppression of gross chromosomal rearrangements (GCRs), indicating that Cdc28 is required for formation or recovery of GCRs. Thus, we conclude that Cdc28 functions in a genetic network that supports cell viability during DNA damage while promoting the formation of GCRs.

Several adhesins are induced by pheromones during mating in Saccharomyces cerevisiae, including Aga1p, Aga2p, Sag1p (Ag[alpha]1p), and Fig2p. These four proteins all participate in or influence a well-studied agglutinin interaction mediated by Aga1p-Aga2p complexes and Sag1p; however, they also play redundant and essential roles in mating via an unknown mechanism. Aga1p and Fig2p both contain repeated, conserved WCPL and CX4C domains. This study was directed toward understanding the mechanism underlying the collective requirement of agglutinins and Fig2p for mating. Apart from the well-known agglutinin interaction between Aga2p and Sag1p, three more pairs of interactions in cells of opposite mating type were revealed by this study, including bilateral heterotypic interactions between Aga1p and Fig2p and a homotypic interaction between Fig2p and Fig2p. These four pairs of adhesin interactions are collectively required for maximum mating efficiency and normal zygote morphogenesis. GPI-less, epitope-tagged forms of Aga1p and Fig2p can be co-immunoprecipitated from the culture medium of mating cells in a manner dependent on the WCPL and CX4C domains in the R1 repeat of Aga1p. Using site-directed mutagenesis, the conserved residues in Aga1p that interact with Fig2p were identified. Aga1p is involved in two distinct adhesive functions that are independent of each other, which raises the possibility for combinatorial interactions of this protein with its different adhesion receptors, Sag1 and Fig2p, a property of many higher eukaryotic adhesins. DOI: 10.1534/genetics.108.100073

Ribosomal RNA (rRNA) is transcribed from the ribosomal DNA (rDNA) genes by RNA polymerase I (Pal I). Despite being responsible for the majority of transcription in growing cells, Pal l regulation is poorly understood compared to Pal I1. To gain new insights into rDNA transcriptional regulation, we developed a genetic assay in Saccharomyces cerevisiae that detects alternations in transcription from the centromereproximal rDNA gene of the tandem array. Changes in Pal I transcription at this gene alter the expression of an adjacent, modified URA3 reporter cassette (mUtL43) such that reductions in Pal I transcription induce growth on synthetic media lacking uracil. Increases in Pal I transcription induce growth on media containing 5-FOA. A transposon mutagenesis screen was performed with the reporter strain to identify genes that play a role in modulating rDNA transcription. Mutations in 68 different genes were identified, several of which were already known to function in chromatin modification and the regulation of Pal II transcription. Among the other classes of genes were those encoding proteasome subunits and multiple kinases and phosphatases that function in nutrient and stress signaling pathways. Fourteen genes were previously uncharacterized and have been named as regulators of rDNA transcription (RRT). DOI: 10.1534/genetics.108.100313

The detection of protein-protein interactions through two-hybrid assays has revolutionized our understanding of biology. The remarkable impact of two-hybrid assay platforms derives from their speed, simplicity, and broad applicability. Yet for many organisms, the need to express test proteins in Saccharomyces cerevisiae or Escherichia coli presents a substantial barrier because variations in codon specificity or bias may result in aberrant protein expression. In particular, nonstandard genetic codes are characteristic of several eukaryotic pathogens, for which there are currently no genetically based systems for detection of protein-protein interactions. We have developed a protein-protein interaction assay that is carried out in native host cells by using GFP as the only foreign protein moiety, thus circumventing these problems. We show that interaction can be detected between two protein pairs in both the model yeast S. cerevisiae and the fungal pathogen Candida albicans. We use computational analysis of microscopic images to provide a quantitative and automated assessment of confidence. DOI 10.1534/genetics.109.101162

In this contribution we resolve the long-standing dispute whether or not the Monod constant (Ks), describing the overall affinity of an organism for its growth-limiting substrate, can be related to the affinity of the transporter for that substrate (KM). We show how this can be done via the control of the transporter on the specific growth rate; they are identical if the transport step has full control. The analysis leads to the counter-intuitive result that the affinity of an organism for its substrate is expected to be higher than the affinity of the enzyme that facilitates its transport. Experimentally, we show this indeed to be the case for the yeast Saccharomyces cerevisiae, for which we determined a KM value for glucose more than two times higher than the Ks value in glucose-limited chemostat cultures. Moreover, we calculated that at glucose concentrations of 0.03 and 0.29 mM, the transport step controls the specific growth rate at 78 and 49 %, respectively.

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1 p association with the plasmid partitioning locus STB are consistent with Kip1 p being an authentic component of the plasmid partitioning complex. Kip1-STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1 p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1 p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1 p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1 p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a 'partitioning center' represents yet another facet of the benign parasitism of the yeast plasmid.

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided [approximately equal to] 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches. fatty acid elongation | S. cerevisiae | shotgun lipidomics

[O.sup.6]-methylguanine ([O.sup.6]meG) and related modifications of guanine in double-stranded DNA are functionally severe lesions that can be produced by many alkylating agents, including N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent carcinogen. [O.sup.6]meG is repaired through its demethylation by the [O.sup.6]-alkylguanine-DNA alkyltransferase (AGT). This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. AGT proteins remove methyl and other alkyl groups from an alkylated [O.sup.6] in guanine by transferring the adduct to an active-site cysteine residue. The resulting S-alkyl-Cys of AGT is not restored back to Cys, so repair proteins of this kind can act only once. We report here that S. cerevisiae Mgt1 is cotargeted for degradation, through a degron near its N terminus, by 2 ubiquitin-mediated proteolytic systems, the Ubr1/Rad6-dependent N-end rule pathway and the Ufd4/Ubc4-dependent ubiquitin fusion degradation (UFD) pathway. The cotargeting of Mgt1 by these pathways is synergistic, in that it increases not only the yield of polyubiquitylated Mgt1, but also the processivity of polyubiquitylation. The N-end rule and UFD pathways comediate both the constitutive and MNNG-accelerated degradation of Mgt1. Yeast cells lacking the Ubr1 and Ufd4 ubiquitin ligases were hyperresistant to MNNG but hypersensitive to the toxicity of overexpressed Mgt1. We consider ramifications of this discovery for the control of DNA repair and mechanisms of substrate targeting by the ubiquitin system. N-end rule | proteolysis | Ubr1 | Ufd4 | yeast

The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartment-specific measurements of intracellular pH (pHi) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH ([pH.sub.cyt]) and mitochondrial pH ([pH.sub.mit]) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant [pH.sub.cyt] of 7.2 and a constant [pH.sub.mit] of 7.5 in cells exponentially growing on glucose. We show that [pH.sub.cyt] and [pH.sub.mit] are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, [pH.sub.i] decrease coincided with growth stasis. Additionally, pHi and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pHi and cellular energy generation, and therefore a relation between [pH.sub.i] and growth.

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like [beta]-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint ([T.sub.m]) of 45 [degrees]C, 62[degrees]C, and 72[degrees]C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability ([T.sub.m] of 50[degrees]C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by [approximately equal to]5[degrees]C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations ([T.sub.m] 50-55[degrees]C; 1 K/min), followed by unfolding of domains 1 and 3 ([approximately equal to]65-70[degrees]C; 1 K/min). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family. spectroscopy | calorimetry | cupredoxin | copper-binding protein

In Saccharomyces cerevisiae, fermentable carbon sources such as glucose and fructose are preferred and elicit glucose repression of genes needed to metabolize non-fermentable carbon sources such as glycerol, ethanol and acetate. Different sets of transcription factors are needed to adjust to specific carbon conditions. For example, Migl and Mig2 repress the transcription of gluconeogenic and respiratory genes in the presence of abundant glucose, while the transcriptional activation of these genes depends on transcription factors such as Adrl and Cat8 Here we show that Yp1230w, which we renamed to Nsfl (nutrient and stress factor 1), is expressed and localizes to the nucleus under non-fermentable carbon conditions to activate gene transcription. Specifically, the transcriptional activation of ACS1, CIT2 and IDH1 is shown to be partially dependent on intact NSF1. Similarly, the transcriptional activation of ENA 1 is impaired in the nsflA mutant in response to high concentrations of NaCl, implying that NSF1 is also needed for the yeast response to sodium stress. The carbon- and NaCl-mediated transcriptional activation of ENA 1 is dependent on Nsf1. This finding implies that the yeast response to nonfermentable carbon and salt stress is at least partially dependent on NSF1.

Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10-Cdc10- Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel 'railroad tracks.' Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures. electron microscopy | yeast | complexes | GTP

The ability to visualize cellular events by linking them to color or fluorescence changes has been an invaluable tool for biology. We describe a novel plasmid-borne color marker whose expression in yeast leads to purple-colored cells that are also brightly fluorescent. This dominant marker provides a useful tool for rapidly screening plasmid maintenance using a visual or fluorescence assay in both Saccharomyces cerevisiae and Candida albicans.

Origination of new genes is an important mechanism generating genetic novelties during the evolution of an organism. Processes of creating new genes using preexisting genes as the raw materials are well characterized, such as exon shuffling, gene duplication, retroposition, gene fusion, and fission. However, the process of how a new gene is de novo created from noncoding sequence is largely unknown. On the basis of genome comparison among yeast species, we have identified a new de novo protein-coding gene, BSC4 in Saccharomyces cerevisiae. The BSC4 gene has an open reading frame (ORF) encoding a 132-amino-acid-long peptide, while there is no homologous ORF in all the sequenced genomes of other fungal species, including its closely related species such as S. paradoxus and S. mikatae. The functional protein-coding feature of the BSC4 gene in S. cerevisiae is supported by population genetics, expression, proteomics, and synthetic lethal data. The evidence suggests that BSC4 may be involved in the DNA repair pathway during the stationary phase of S. cerevisiae and contribute to the robustness of S. cerevisiae, when shifted to a nutrient-poor environment. Because the corresponding noncoding sequences in S. paradoxus, S. mikatae, and S. bayanus also transcribe, we propose that a new de novo protein-coding gene may have evolved from a previously expressed noncoding sequence.

Histone acetylation levels are regulated through the opposing activities of histone acetyltransferases (HATs) and deacetylases (HDACs). While much is known about gene-specific control of histone acetylation, little is understood about how total or cellular levels of histone acetylation are regulated. To identify regulators of cellular levels of histone acetylation, we developed an immunofluorescence-based approach to screen the single-gene deletion library of Saccharomyces cerevisiae for strains with significant reductions in cellular histone acetylation levels. Of the 4848 mutants screened, we identified 63 strains with considerable cellular hypoacetylation of N-terminal lysines in histones H3 and H4. The cellular hypoacetylation was validated for subsets of the identified strains through secondary screens including mass spectrometric analysis of individual lysines and chromatin immunoprecipitation of specific genomic loci. Among the identified mutants were several members of the Ccr4-Not complex, V-type ATPases, and vacuolar protein-sorting complexes as well as genes with unknown functions. We show that Gcn5, a major HAT in yeast, has diminished histone acetyltransferase activity in particular mutants, providing a plausible explanation for reduction of cellular acetylation levels in vivo. Our findings have revealed unexpected and novel links between histone acetylation, Gcn5 HAT activity, and diverse processes such as transcription, cellular ion homeostasis, and protein transport.

A study is conducted to show that the Tmt1-dependent methylation of 3-isopropylmalate in Saccharomyces cerevisiae is not directly related to the leucine biosynthetic pathway. Results indicate that 3-isopropylmalate methyl ester signals yeast to switch to invasive growth during amino acid starvation.

The Saccharomyces cerevisiae non-Mendelian genetic element [PS[I.sup.+]] is the prion form of the translation termination factor Sup35p. The ability of [PS[I.sup.+]] to propagate efficiently has been shown previously to depend upon the action of protein chaperones. In this article we describe a genetic screen that identifies an array of mutants within the two major cytosolic Hsp70 chaperones of yeast, Ssa1p and Ssa2p, which impair the propagation of [PS[I.sup.+]]. All but one of the mutants was located within the ATPase domain of Hsp70, which highlights the important role of regulation of Hsp70-Ssa ATP hydrolysis in prion propagation. A subset of mutants is shown to alter Hsp70 function in a way that is distinct from that of previously characterized Hsp70 mutants that alter [PS[I.sup.+]] propagation and supports the importance of interdomain communication and Hsp70 interaction with nucleotide exchange factors in prion propagation. Analysis of the effects of Hsp70 mutants upon propagation of a second yeast prion [URE3] further classifies these mutants as having general or prion-specific inhibitory properties.

Gic1 and Gic2 are two Cdc42/Rac interactive binding (CRIB) domain-containing effectors of Cdc42-GTPase that promote polarized cell growth in S. cerevisiae. To identify novel genes that functionally interact with Gic1 and Gic2, we screened for high-copy suppressors of a gic1 gic2 temperature-sensitive strain. We identified two pairs of structurally related genes, SKG6-TOS2 and VHS2-MLF3. These genes have been implicated in polarized cell growth, but their functions have not previously been characterized. We found that overproduction of Skg6 and Tos2 in wild-type cells causes aberrant localization of Cdc3 septin and actin structures as well as defective recruitment of Hof1 and impaired formation of the septum at the mother-bud neck. These data suggest a negative regulatory function for Skg6 and Tos2 in cytokinesis. Consistent with this model, deletion of SKG6 suppresses the growth defects associated with loss of HOF1, a positive regulator of cytokinesis. Our analysis of the second pair of gic1 gic2 suppressors, VHS2 and MLF3, suggests that they regulate polarization of the actin cytoskeleton and cell growth and function in a pathway distinct from and parallel to GIC1 and GIC2.

RecQ DNA helicases and Topo III topoisomerases have conserved genetic, physical, and functional interactions that are consistent with a model in which RecQ creates a recombination-dependent substrate that is resolved by Topo III. The phenotype associated with Topo III loss suggests that accumulation of a RecQ-created substrate is detrimental. In yeast, mutation of the TOP3 gene encoding Topo III causes pleiotropic defects that are suppressed by deletion of the RecQ homolog Sgs1. We searched for gene dosage suppressors of top3 and identified Pill, a DNA helicase that acts with polarity opposite to that of Sgs1. Pif1 overexpression suppresses multiple top3 defects, but exacerbates sgs1 and sgs1 top3 defects. Furthermore, Pif1 helicase activity is essential in the absence of Top3 in an Sgs1-dependent manner. These data clearly demonstrate that Pif1 helicase activity is required to counteract Sgs1 helicase activity that has become uncoupled from Top3. Pif1 genetic interactions with the Sgs1-Top3 pathway are dependent upon homologous recombination. We also find that Pif1 is recruited to DNA repair foci and that the frequency of these foci is significantly increased in top3 mutants. Our results support a model in which Pif1 has a direct role in the prevention or repair of Sgs1-induced DNA damage that accumulates in top3 mutants.

A novel role for Rad53 in the initiation of DNA replication that is independent of checkpoint or deoxynucleotide regulation is proposed. Rad53 kinase is part of a signal transduction pathway involved in the DNA damage and replication checkpoints, while Cdc7-Dbf4 kinase (DDK) is important for the initiation of DNA replication. In addition to the known cdc7-rad53 synthetic lethality, rad53 mutations suppress mcm5-bob1, a mutation in the replicative MCM helicase that bypasses DDK's essential role. Rad53 kinase activity but neither checkpoint FHA domain is required. Conversely, Rad53 kinase can be activated without DDK. Rad53's role in replication is independent of both DNA and mitotic checkpoints because mutations in other checkpoint genes that act upstream or downstream of RAD53 or in the mitotic checkpoint do not exhibit these phenotypes. Because Rad53 binds an origin of replication mainly through its kinase domain and rad53 null mutants display a minichromosome loss phenotype, Rad53 is important in the initiation of DNA replication, as are DDK and Mcm2-7 proteins. This unique requirement for Rad53 can be suppressed by the deletion of the major histone H3/H4 gene pair, indicating that Rad53 may be regulating initiation by controlling histone protein levels and/or by affecting origin chromatin structure.

In Saccharomyces cerevisiae the cAMP-dependent protein kinase A pathway antagonizes the cellular response to stress. It is shown here that the cellular content of Cdc25p, the upstream activator of Ras and adenylyl cyclase, decays upon various stresses such as heat shock and oxidative and ethanol shocks, whereas its phosphorylation level and its localization are unaffected. In parallel with the reduction of Cdc25p, the maximal capacity of the cell to accumulate cAMP decreases when its feedback regulation is abolished. A deletion of CDC25 prevents this decrease. Paradoxically, in wild-type cells, with normal feedback regulation, the level of cAMP, which is much lower, is not reduced but is rather increased upon stress. These observations are consistent with a role of Cdc25p in sensing and transducing stress to downstream targets, either through a cAMP-independent pathway or by large fluctuations in the cAMP content of the cell.

Most fungal cell walls are constructed with significant amounts of chitin, a linear polysaccharide that contributes mechanical resistance to the structure. In the yeast Saccharomyces cerevisiae, chitin is synthesized by three different isozymes, each of which has a separate cellular function. In this yeast, the most important role of chitin is in cytokinesis, when a thin primary septum is synthesized by chitin synthase II to separate mother and daughter cells. If no primary septum can be formed, an irregular remedial septum is synthesized, a process that relies on chitin synthase III. It was found that, with osmotic stabilization, S. cerevisiae tolerates a loss of all chitin synthase activities. Chitin-deficient mutants display a cytokinesis defect which leads to the formation of cell chains with incompletely separated cytoplasms. In these mutants septa are formed rarely. The few septa found are bulky structures which contain inclusions of cytoplasm. Nuclear division proceeds under these conditions, demonstrating that there is no cell cycle arrest triggered by a failure to form a septum between mother and daughter cell. A genetic suppressor arises quickly in chitin-deficient mutants, giving rise to the synthesis of chitin-free remedial septa. The suppressed chitin-free mutants grow well without osmotic stabilization and display hyper-resistance against the chitin-synthase inhibitor polyoxin D.

Kre1p is a cell surface O-glycoprotein involved in a late stage of 1,6-[beta]-glucan formation in the yeast Saccharomyces cerevisiae. Disruption of KRE1 leads to a 40% reduction in the overall 1,6-[beta]-glucan content of the cell wall. This paper shows that in a yeast [DELTA]kre1 null mutant, neither an N-terminal-truncated Kre1p nor Kre1p variants lacking a C-terminal glycosylphcepatidylinositol (GPI) attachment site are capable of achieving normal function in glucan assembly, while full-length Kre1p completely complements a [DELTA]kre1 null mutation and restores cell wall 1,6- [beta] -glucan content up to wild-type level. In a yeast gpi1 mutant, a green-fluorescent-protein-tagged Kre1p derivative is secreted into the medium, indicating an at least transient GPI-anchoring stage of Kre1p during its processing within the yeast secretory pathway. In contrast to the severe defect in cell wall [beta]-D-glucan, the amount of cell wall mannoproteins is not significantly decreased in [DELTA]kre1 disruptant, as could be confirmed in competition assays by investigating toxin binding to isolated cell wall mannoproteins. Since the yeast [DELTA]kre1 mutant differed in its sensitivity to zygocin and K28, two killer viral protein toxins that use different cell wall mannoprotein populations as a primary toxin receptor, it can be concluded that in a yeast [DELTA]kre1 background, mannoproteins do not differ significantly in total amount from a [Kre1.sup.+] wild-type but rather in their expression and distribution at the cell surface. Taken together, these data favour and suggest a structural, rather than enzymic, function of Kre1p in yeast cell wall assembly.

The Saccharomyces cerevisiae spore wall is a multilaminar coat that surrounds individual spores and protects them from environmental insult. Scanning electron microscopy reveals that the four spores of an ascus are connected by interspore bridges. Transmission electron microscopy of spores indicates that these bridges are continuous with the outer layers of the spore wall. In chs3 mutants, which lack the chitosan and dityresine layers of the spore wall, bridges are absent. By contrast, in dit1 mutants, which lack only the dityrosine layer, bridges are present, suggesting that the bridges may be composed of chitosan. Interspore bridges are shown to be necessary to hold spores together after release from the ascus. A function for these bridges in the maintenance of heterozygous markers in a homothallic yeast population is proposed.

Aging is believed to be a nonadaptive process that escapes the force of natural selection. Here, we challenge this dogma by showing that yeast laboratory strains and strains isolated from grapes undergo an age- and pH-dependent death with features of mammalian programmed cell death (apoptosis). After 90-99% of the population dies, a small mutant subpopulation uses the nutrients released by dead cells to grow. This adaptive regrowth is inversely correlated with protection against superoxide toxicity and life span and is associated with elevated age-dependent release of nutrients and increased mutation frequency. Computational simulations confirm that premature aging together with a relatively high mutation frequency can result in a major advantage in adaptation to changing environments. These results suggest that under conditions that model natural environments, yeast organisms undergo an altruistic and premature aging and death program, mediated in part by superoxide. The role of similar pathways in the regulation of longevity in organisms ranging from yeast to mice raises the possibility that mammals may also undergo programmed aging.

The mechanisms involved in the establishment of the resistance of the medium-chain fatty acids (octanoic and decanoic acids) are characterized. The comparison of the transcriptional responses to the responses caused by other lipophilic compound has shown the role of several transcription factors.

X-ray crystallography had defined to the structure of a large fragment of the bovine peripheral stalk, which has included 75% of subunit d, of the ATP synthase from Saccharomyces cerevisiae. The studies have shown that the C-terminal region of subunit d has spanned the gap from [F.sub.1] to [F.sub.0], thus reinforcing the section of the peripheral stalk.

The structure of thiole synthase (Thi4) from Saccharomyces cerevisiae was determined to 1.8 angstrom resolution. Thi4 exists as an octamer with two monomers in the asymmetric unit and reveals the presence of a tightly bound adenosine diphospho-5-[(beta)-ethyl)]-4-methylthiazole-2-carboxylic acid at the active site and also the first protein structure with a GR2 domain that binds NAD instead of FAD.

A report is presented that Zip1 is a binding protein for small ubiquitin-related modifier (SUMO)-conjugated products and Zip3 is a SUMO E3 ligase. The results indicate that at early meiotic prophase, Zip1 interacts with Zip3-independent Smt3 conjugates to promote nonhomologous centromere coupling.

The identification of membrane proteins involved in endoplasmic reticulum (ER) network formation is discussed. The major components shaping the tubular ER are the reticulons and the interacting protein DP1/Yop 1p.

A study is conducted on the several stress conditions undergone by the Saccharomyces cerevisiae cells throughout alcoholic fermentation. It is observed the transcriptomatic and proteomic comparison between two commercial strains showed different fermentive behaviors that could be related to differences in the mRNA and protein profiles.