Search

A principal objective of cellular biology is to identify the function of proteins as they interact with their environment. Cellular localization provides context for the function and interactions of proteins. In this study, we seek to identify the localization of three proteins within Saccharomyces cerevisiae through C-terminal green fluorescent protein epitope tagging and subsequent fluorescent microscopy. As 75% of the yeast proteome has been classified, this project seeks to fill the remaining gap in unidentified cellular localization by investigating the proteins fsh3, gid10, and ade13. The analysis of high-resolution localization and comparative phylogenetic data reveals potential cellular environments and protein functions., Acknowledgements Sophia McNeill, Dr. Richard Adams, Dr. Indiren Pillay, and Dr. Ashok Hegde Recommended Citation Authors Lewis S. Barr (*), Georgia College and State University Follow Ellen France, Georgia College [...]

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.

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

Circadian timing is a fundamental biological process, underlying cellular physiology in animals, plants, fungi, and cyanobacteria. Circadian clocks organize gene expression, metabolism, and behavior such that they occur at specific times of day. The biological clocks that orchestrate these daily changes confer a survival advantage and dominate daily behavior, for example, waking us in the morning and helping us to sleep at night. The molecular mechanism of circadian clocks has been sketched out in genetic model systems from prokaryotes to humans, revealing a combination of transcriptional and posttranscriptional pathways, but the clock mechanism is far from solved. Although Saccharomyces cerevisiae is among the most powerful genetic experimental systems and, as such, could greatly contribute to our understanding of cellular timing, it still remains absent from the repertoire of circadian model organisms. Here, we use continuous cultures of yeast, establishing conditions that reveal characteristic clock properties similar to those described in other species. Our results show that metabolism in yeast shows systematic circadian entrainment, responding to cycle length and zeitgeber (stimulus) strength, and a (heavily damped) free running rhythm. Furthermore, the clock is obvious in a standard, haploid, auxotrophic strain, opening the door for rapid progress into cellular clock mechanisms. entrainment | oscillation | yeast | temperature cycle doi/10.1073/pnas.0907902107

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

Targeted mRNA trafficking and local translation may play a significant role in controlling protein localization. Here we examined for the first time the localization of all ([approximately equal to] 50) mRNAs encoding peroxisomal proteins (mPPs) involved in peroxisome biogenesis and function. By using the bacteriophage MS2-CP RNA-binding protein (RBP) fused to multiple copies of GFP, we demonstrated that >40 endogenously expressed mPPs tagged with the MS2 aptamer form fluorescent RNA granules in vivo. The use of different RFP-tagged organellar markers revealed 3 basic patterns of mPP granule localization: to peroxisomes, to the endoplasmic reticulum (ER), and nonperoxisomal. Twelve mPPs (i.e., PEX1, PEX5, PEX8, PEX11-15, DCI1, NPY1, PCS60, and POX1) had a high percentage (52%-80%) of mRNA colocalization with peroxisomes. Thirteen mPPs (i.e., AAT2, PEX6, MDH3, PEX28, etc.) showed a low percentage (30%-42%) of colocalization, and 1 mPP (PEX3) preferentially localized to the ER. The mPPs of the nonperoxisomal pattern (i.e., GPD1, PCD1, PEX7) showed < mRNA localization | peroxisomes doi/10.1073/pnas.0910754106

The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the a or [beta] subunits of [F.sub.1], or the Atp11p and Atp12p chaperones that promote [F.sub.1] assembly, have normal levels of the bicistronic ATPS/ATP6 mRNAs but fail to synthesize Atp6p and Atp8p. [F.sub.1] mutants are also unable to express [ARG8.sup.m] when this normally nuclear gene is substituted for ATP6 or ATP8 in mitochondrial DNA. Translational activation by [F.sub.1] is also supported by the ability of ATP22, an Atp6p-specific translation factor, to restore Atp6p and to a lesser degree Atp8p synthesis in the absence of [F.sub.1]. These results establish a mechanism by which expression of ATP6 and ATP8 is translationally regulated by [F.sub.1] to achieve a balanced output of two compartmentally separated sets of ATP synthase genes. F1-ATPase | mitochondria | Saccharomyces cerevisiae | translational regulation www.pnas.org/cgi/doi/10.1073/pnas.0910351106

RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Socchoromyces cerevisiae. Here, we show that RNAi is present in other budding yeast species, including Soccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, Which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' messenger RNA revers. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a previously unknown class of Dicer proteins, bring the toot of RNAi to the study of budding yeasts, and bring the toots of budding yeast to the study of RNAi. 10.1126/science.1176945

Ser-5 phosphorylation of the RNA polymerase II (Pol II) C-terminal domain by TFIIH kinase has been implicated in critical steps in mRNA synthesis, such as Pol II promoter escape and mRNA 5'-capping. However, the general requirement and precise role of TFIIH kinase in Pol II transcription still remain elusive. Here we use a chemical genetics approach to show that, for a majority of budding-yeast genes, specific inhibition of the yeast TFIIH kinase results in a dramatic reduction in both mRNA level and Ser-5 C-terminal domain phosphorylation. Surprisingly, inhibition of TFIIH kinase activity only partially affected both Pol II density and Ser-2 phosphorylation level. The discrepancy between mRNA level and Pol II density is attributed to the defective 5'-capping, which results in the destabilization of mRNAs. Therefore, contrary to the current belief, our study points strongly toward a minor role of TFIIH kinase in Pol II transcription, and a more significant role in mRNA capping in budding yeast. chemical genetics | Kin28 | mRNA capping

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

Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast [p150.sup.Glued] homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering. microtubule | motor protein | nuclear segregation | p150Glued | single molecule

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 Gpg1 protein is a G[gamma] subunit mimic implicated in the G-protein glucose-signaling pathway in Saccharomyces cerevisiae, and its function is largely unknown. Here we report that Gpg1 blocks the maintenance of [[PSI.sup.+]], an aggregated prion form of the translation termination factor Sup35. Although the GPG1 gene is normally not expressed, over-expression of GPG1 inhibits propagation of not only [[PSI.sup.+]] but also [[PIN.sup.+]], [URE3] prions, and the toxic polyglutamine aggregate in S. cerevisiae. Over-expression of Gpg1 does not affect expression and activity of Hsp104, a protein-remodeling factor required for prion propagation, showing that Gpg1 does not target Hsp104 directly. Nevertheless, prion elimination by Gpg1 is weakened by over-expression of Hsp104. Importantly, Gpg1 protein is prone to self-aggregate and transiently colocalized with Sup35NM-prion aggregates when expressed in [[PSI.sup.+]] cells. Genetic selection and characterization of loss-of-activity gpg1 mutations revealed that multiple mutations on the hydrophobic one-side surface of predicted [alpha]-helices of the Gpg1 protein hampered the activity. Prion elimination by Gpg1 is unaffected in the gpa2[DELTA] and gpb1[DELTA] strains lacking the supposed physiological G-protein partners of Gpg1. These findings suggest a general inhibitory interaction of the Gpg1 protein with other transmissible and nontransmissible amyloids, resulting in prion elimination. Assuming the ability of Gpg1 to form G-protein heterotrimeric complexes, Gpg1 is likely to play a versatile function of reversing the prion state and modulating the G-protein signaling pathway. [[PIN.sup.+]] | [[PSI.sup.+]] | [URE3] | Gpg1 | yeast prion

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

The Saccharomyces cerevisiae Mec1--Ddc2 checkpoint kinase complex (the ortholog to human ATR-ATRIP) is an essential regulator of genomic integrity. The S. cerevisiae BRCT repeat protein Dpb11 functions in the initiation of both DNA replication and cell cycle checkpoints. Here, we report a genetic and physical interaction between Dpb11 and Mec1--Ddc2. A C-terminal domain of Dpb11 is sufficient to associate with Mec1--Ddc2 and strongly stimulates the kinase activity of Mec1 in a Ddc2-dependent manner. Furthermore, Mec1 phosphorylates Dpb11 and thereby amplifies the stimulating effect of Dpb11 on Mec1--Ddc2 kinase activity. Thus, Dpb11 is a functional ortholog of human TopBP1, and the Mec1/ATR activation mechanism is conserved from yeast to humans. ATR | checkpoint | DNA damage | TopBP1

Acetylation of histone proteins by the yeast Spt-Ada-Gcn5-acetyl-tansferase (SAGA) complex has served as a paradigm for understanding how posttranslational modifications of chromatin regulate eukaryotic gene expression. Nonetheless, it has been unclear to what extent the structural complexity of the chromatin substrate modulates SAGA activity. By using chromatin model systems, we have found that SAGA-mediated histone acetylation is highly cooperative (cooperativity constant of 1.97 [+ or -] 0.15), employing the binding of multiple noncontiguous nucleosomes to facilitate maximal acetylation activity. Studies with various chromatin substrates, including those containing novel asymmetric histone octamers, indicate that this cooperativity occurs only when both H3 histone tails within a nucleosome are properly oriented and unacetylated. We propose that modulation of maximal SAGA activity through this dual-tail recognition could facilitate coregulation of spatially proximal genes by promoting cooperative nucleosome acetylation between genes. chromatin | enzymology | protein modification | transcription | S. cerevisiae

Prp8 stands out among hundreds of splicing factors as a key regulator of spliceosome activation and a potential cofactor of the splicing reaction. We present here the crystal structure of a 274-residue domain (residues 1,822-2,095) near the C terminus of Saccharomyces cerevisiae Prp8. The most striking feature of this domain is a [beta]-hairpin finger protruding out of the protein (hence, this domain will be referred to as the [beta]-finger domain), resembling many globular ribosomal proteins with protruding extensions. Mutations throughout the [beta]-finger change the conformational equilibrium between the first and the second catalytic step. Mutations at the base of the [beta]-finger affect U4/U6 unwinding-mediated spliceosome activation. Prp8 may insert its E-finger into the first-step complex (U2/U5/U6/pre-mRNA) or U4/U6.U5 tri-snRNP and stabilize these complexes. Mutations on the [beta]-finger likely alter these interactions, leading to the observed mutant phenotypes. Our results suggest a possible mechanism of how Prp8 regulates spliceosome activation. These results also demonstrate an analogy between a spliceosomal protein and ribosomal proteins that insert extensions into folded rRNAs and stabilize the ribosome.

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 yeast Saccharomyces cerevisiae has developed specialized mechanisms that enable growth on suboptimal nitrogen sources. Exposure of yeast cells to poor nitrogen sources or treatment with the Tor kinase inhibitor rapamycin elicits activation of Gln3 and transcription of nitrogen catabolite-repressed (NCR) genes whose products function in scavenging and metabolizing nitrogen. Here, we show that mutations in class C and D Vps components, which mediate Golgi-to-endosome vesicle transport, impair nuclear translocation of Gln3, NCR gene activation, and growth in poor nitrogen sources. In nutrient-replete conditions, a significant fraction of Gln3 is peripherally associated with light membranes and partially colocalizes with Vps10-containing foci. These results reveal a role for Golgi-to-endosome vesicular trafficking in TORC1-controlled nuclear translocation of Gln3 and support a model in which Tor-mediated signaling in response to nutrient cues occurs in these compartments. These findings have important implications for nutrient sensing and growth control via mTor pathways in metazoans. rapamycin action | Tor signaling

The Saccharomyces cerevisiae phosphatidylcholine/phosphatidylinositol transfer protein Sec14p is required for Golgi apparatusderived vesicular transport through coordinate regulation of phospholipid metabolism. Sec14p is normally essential. The essential requirement for SEC14 can be bypassed by inactivation of (i) the CDP-choline pathway for phosphatidylcholine synthesis or (ii) KES1, which encodes an oxysterol binding protein. A unique screen was used to determine genome-wide genetic interactions for the essential gene SEC14 and to assess whether the two modes of 'sec14 bypass' were similar or distinct. The results indicate that inactivation of the CDP-choline pathway allows cells with inactivated SEC14 to live through a mechanism distinct from that of inactivation of KES1. We go on to demonstrate an important biological function of Kes1p. Kes1p regulates Golgi apparatus-derived vesicular transport by inhibiting the function of Pik1p-generated Golgi apparatus phosphatidylinositol-4-phosphate (PI-4P). Kes1p affects both the availability and level of Golgi apparatus PI-4P. A set of potential PI-4P-responsive proteins that include the Rab GTPase Ypt31p and its GTP exchange factor are described. KES1 | Saccharomyces cerevisiae | SEC14 | vesicular transport

Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective high-throughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-[beta]-D-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels. We estimate that our approach should be able to provide milligram quantities for at least one-quarter of all membrane proteins from both yeast and higher eukaryotic organisms. GFP-based fusion technology | structural genomics

We study how the dynamic equilibrium of the reversible protein--protein-binding network in yeast Saccharomyces cerevisiae responds to large changes in abundances of individual proteins. The magnitude of shifts between free and bound concentrations of their immediate and more distant neighbors in the network is influenced by such factors as the network topology, the distribution of protein concentrations among its nodes, and the average binding strength. Our primary conclusion is that, on average, the effects of a perturbation are strongly localized and exponentially decay with the network distance away from the perturbed node, which explains why, despite globally connected topology, individual functional modules in such networks are able to operate fairly independently. We also found that under specific favorable conditions, realized in a significant number of paths in the yeast network, concentration perturbations can selectively propagate over considerable network distances (up to four steps). Such 'action-at-a-distance' requires high concentrations of heterodimers along the path as well as low free (unbound) concentration of intermediate proteins. dissociation constant | genetic interactions | law of mass action | small-world networks | binding equilibrium

Synergistically interacting gene mutations reveal buffering relationships that provide growth homeostasis through their compensation of one another. This analysis in Saccharomyces cerevisiae revealed genetic modules involved in tricarboxylic acid cycle regulation (RTG1, RTG2, RTG3), threonine biosynthesis (HOM3, HOM2, HOM6, THR1, THR4), amino acid permease trafficking (LST4, LST7), and threonine catabolism (GLY1). These modules contribute to a molecular circuit that regulates threonine metabolism and buffers deficiency in deoxyribonucleotide biosynthesis. Phenotypic, genetic, and biochemical evidence for this buffering circuit was obtained through analysis of deletion mutants, titratable alleles of ribonucleotide reductase genes, and measurements of intracellular deoxyribonucleotide pool concentrations. This circuit provides experimental evidence, in eukaryotes, for the presence of a high-flux backbone of metabolism, which was previously predicted from in silico modeling of global metabolism in bacteria. This part of the high-flux backbone appears to buffer deficiency in ribonucleotide reductase by enabling a compensatory increase in de novo purine biosynthesis that provides additional rate-limiting substrates for dNTP production and DNA synthesis. Hypotheses regarding unexpected connections between these metabolic pathways were facilitated by genome-wide but also highly quantitative phenotypic assessment of interactions. Validation of these hypotheses substantiates the added benefit of quantitative phenotyping for identifying subtleties in gene interaction networks that modulate cellular phenotypes. genetic buffering | high-flux backbone of metabolism | protein trafficking | ribonucleotide reductase | mitochondria-to-nucleus retrograde signaling pathway

Mitochondrial carriers are believed widely to be dimers both in structure and function. However, the structural fold is a barrel of six transmembrane [alpha]-helices without an obvious dimerisation interface. Here, we show by negative dominance studies that the yeast mitochondrial ADP/ATP carrier 2 from Saccharomyces cerevisiae (AAC2) is functional as a monomer in the mitochondrial membrane. Adenine nucleotide transport by wild-type AAC2 is inhibited by the sulfhydryl reagent 2-sulfonatoethyl-methanethiosulfonate (MTSES), whereas the activity of a mutant AAC2, devoid of cysteines, is unaffected. Wild-type and cysteine-less AAC2 were coexpressed in different molar ratios in yeast mitochondrial membranes. After addition of MTSES the residual transport activity correlated linearly with the fraction of cysteine-less carrier present in the membranes, and so the two versions functioned independently of each other. Also, the cysteine-less and wild-type carriers were purified separately, mixed in defined ratios and reconstituted into liposomes. Again, the residual transport activity in the presence of MTSES depended linearly on the amount of cysteine-less carrier. Thus, the entire transport cycle for ADP/ATP exchange is carried out by the monomer. mechanism | membrane protein | negative dominance studies | oligomeric state | transport

Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins. DNA microarray | posttranscriptional gene regulation | RNA-binding protein

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 ([alpha], oligomeric state unknown) and a small subunit R2 ([[beta].sub.2]). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [[(Fe).sub.2]-Y*] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer ([beta][beta]'), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, [beta][beta]' is redistributed from the nucleus to the cytoplasm in a checkpoint-dependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in [beta][beta]' nuclear localization: the importin [beta] homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of [beta][beta]' nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with [beta][beta]'. Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of [beta][beta]' by Kap122 or as an anchor to retain [beta][beta]' in the nucleus. DNA-damage checkpoint | subcellular redistribution

Interpreting data from large-scale protein interaction experiments has been a challenging task because of the widespread presence of random false positives. Here, we present a network-based statistical algorithm that overcomes this difficulty and allows us to derive functions of unannotated proteins from large-scale interaction data. Our algorithm uses the insight that if two proteins share significantly larger number of common interaction partners than random, they have close functional associations. Analysis of publicly available data from Saccharomyces cerevisiae reveals >2,800 reliable functional associations, 29% of which involve at least one unannotated protein. By further analyzing these associations we derive tentative functions for 81 unannotated proteins with high certainty. Our method is not overly sensitive to the false positives present in the data. Even after adding 50% randomly generated interactions to the measured data set, we are able to recover almost all (~89%) of the original associations.

Siderophores are low molecular weight compounds, synthesized and secreted by microorganisms, that specifically bind ferric iron with exceptionally high affinity. Microbes capture these compounds and take up the bound iron through specific, high-affinity systems. Saccharomyces cerevisiae can take up iron bound to siderophores through the transporters of the ARN family; however, the mechanism by which the siderophore-bound iron enters the cell via these transporters is not known. Here we describe how ferrichrome, a siderophore of the hydroxamate class, is taken up by Arn1p. Arn1p exhibits two surface binding sites for ferrichrome, one that is similar in affinity to the [K.sub.T] for uptake and one of a much higher affinity that is specific for the metallated form of ferrichrome. Ferrichrome may gain access to the higher-affinity site through endocytosis. Tracer studies using [sup.14]C-labeled ferrichrome bound to either iron(III) or aluminum(III), a nonreducible ligand for ferrichrome, indicate that ferrichrome enters the cell as the intact metallosiderophore and accumulates in the cytosol. Both ferrichrome chelates were relatively stable within the cell, and metal-free ferrichrome did not accumulate, indicating a role for ferrichrome in intracellular iron storage. Iron stored as ferrichrome was readily mobilized to meet the metabolic needs of the cell.

Recent research in the field of nanometer-scale electronics has focused on the operating principles of small-scale devices and schemes to realize useful circuits. In contrast to established 'top-down' fabrication techniques, molecular self-assembly is emerging as a 'bottom-up' approach for fabricating nanostructured materials. Biological macromolecules, especially proteins, provide many valuable properties, but poor physical stability and poor electrical characteristics have prevented their direct use in electrical circuits. Here we describe the use of self-assembling amyloid protein fibers to construct nanowire elements. Self-assembly of a prion determinant from Saccharomyces cerevisiae, the N-terminal and middle region (NM) of Sup35p, produced 10-nm-wide protein fibers that were stable under a wide variety of harsh physical conditions. Their lengths could be roughly controlled by assembly conditions in the range of 60 nm to several hundred micrometers. A genetically modified NM variant that presents reactive, surface-accessible cysteine residues was used to covalently link NM fibers to colloidal gold particles, These fibers were placed across gold electrodes, and additional metal was deposited by highly specific chemical enhancement of the colloidal gold by reductive deposition of metallic silver and gold from salts. The resulting silver and gold wires were [approximately equal to] 100 nm wide. These biotemplated metal wires demonstrated the conductive properties of a solid metal wire, such as low resistance and ohmic behavior. With such materials it should be possible to harness the extraordinary diversity and specificity of protein functions to nanoscale electrical circuitry.

We describe a comparative mathematical framework for two genome-scale expression data sets. This framework formulates expression as superposition of the effects of regulatory programs, biological processes, and experimental artifacts common to both data sets, as well as those that are exclusive to one data set or the other, by using generalized singular value decomposition. This framework enables comparative reconstruction and classification of the genes and arrays of both data sets. We illustrate this framework with a comparison of yeast and human cell-cycle expression data sets. DNA microarrays | cell cycle | yeast Saccharomyces cerevisiae | human HeLa cell line

We propose MOTIF REGRESSOR for discovering sequence motifs up-stream of genes that undergo expression changes in a given condition. The method combines the advantages of matrix-based motif finding and oligomer motif-expression regression analysis, resulting in high sensitivity and specificity. MOTIF REGRESSOR is particularly effective in discovering expression-mediating motifs of medium to long width with multiple degenerate positions. When applied to Saccharomyces cerevisiae, MOTIF REGRESSOR identified the ROX1 and YAP1 motifs from Rox1p and Yap1p overexpression experiments, respectively; predicted that Gcn4p may have increased activity in YAP1 deletion mutants; reported a group of motifs (including GCN4, PH04, MET4, STRE, USR1, RAP1, M3A, and M3B) that may mediate the transcriptional response to amino acid starvation; and found all of the known cell-cycle regulation motifs from 18 expression microarrays over two cell cycles. sequence motif discovery | microarray data | correlation | transcription regulation

To elucidate the essential functions of acetyl-CoA carboxylase (ACC1/FAS3) in Saccharomyces cerevisiae, a temperature-sensitive mutant ([acc1.sup.ts]) was constructed. When the [acc1.sup.ts] cells were synchronized in [G.sub.1] phase with [alpha]-factor at the permissive temperature of 24[degrees]C and then released from the blockade and incubated at the restrictive temperature of 37[degrees]C, 95% of the cell population became arrested at the [G.sub.2]/M phase of the cell cycle despite the presence of fatty acids ([C.sub.14]-[C.sub.26]) in the medium. These cells developed large undivided nuclei, and the spindles of the arrested mutant cells were short. Shifting the [G.sub.2] arrested cells back to the permissive temperature resulted in a reversal of the cell-cycle arrest, with cells initiating mitosis. However, after 3 h of incubation at 37[degrees]C, [G.sub.2] arrested mutant cells lost viability and displayed a uniquely altered nuclear envelope. Using [[1-.sup.14]C acetate as a precursor for fatty acids synthesis, we identified the phospholipids and sphingolipids derived from [acc1.sup.ts] cells and wild-type cells at 24[degrees]C and 37[degrees]C, respectively. The levels of inositol-ceramides [IPC, MIPC, and [M(IP).sub.2]C and very long-chain fatty acids [C.sub.24] and [C.sub.26] declined sharply in the [G.sub.2]/M arrested cells because of ACC inactivation. Shifting the [acc1.sup.ts] cells to 24[degrees]C after 2 h of incubation at 37[degrees]C resulted in reactivation of the ACC and elevation of the ceramides and very long-chain fatty acid syntheses with normal cell-cycle progression. In contrast, synthesis of wild-type inositol-ceramides, [C.sub.24] and [C.sub.26], fatty acids were elevated on incubation at 37[degrees]C and declined when the cells shifted to the permissive temperature of 24[degree]C. ACC1 | VLCF | very long-chain fatty acid

The mRNA and protein expression in Saccharomyces cerevisiae cultured in rich or minimal media was analyzed by oligonucleotide arrays and quantitative multidimensional protein identification technology. The overall correlation between mRNA and protein expression was weakly positive with a Spearman rank correlation coefficient of 0.45 for 678 loci. To place the data sets in a proper biological context, a clustering approach based on protein pathways and protein complexes was implemented. Protein expression levels were transcriptionally controlled for not only single loci but for entire protein pathways (e.g., Met, Arg, and Leu biosynthetic pathways). In contrast, the protein expression of loci in several protein complexes (e.g., SPT, COPI, and ribosome) was posttranscriptionally controlled. The coupling of the methods described provided insight into the biology of S. cerevisiae and a clustering strategy by which future studies should be based.

Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1[DELTA]/ ssd1[DELTA] yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.

DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with [T.sup.-][B.sup.-] severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells. Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination. Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue in Saccharomyces cerevisiae functions in NHEJ. These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.

Narnavirus 23S RNA is a persistent positive-stranded RNA virus found in yeast Saccharomyces cerevisiae. The viral genome (2.9 kb) only encodes its RNA-dependent RNA polymerase, p104. Here we report the generation of 23S RNA virus, with high frequency, from a vector containing the entire viral cDNA sequence. When the conserved GDD (Gly-Asp-Asp) motif of RNA-dependent RNA polymerase was modified, the vector failed to generate the virus, indicating that an active p104 is essential for replication. Successful launching required transcripts having the proper viral 3' terminus generated in vivo. This was accomplished through in vivo processing of the primary transcripts by the hepatitis delta virus antigenomic ribozyme directly fused to the 3' terminus of the 23S RNA genome. Although the primary transcripts also contained extra nucleotides at their 5' ends derived from the vector, the launched virus possessed the authentic 5' terminus of the viral genome without these extra nucleotides. Modifications of the genome sequence at the 5' and 3' termini abolished viral generation, indicating that the viral genome has cis-acting signals for replication at both termini. The great ease to generate the virus will facilitate the identification of these cis-acting signals. Furthermore, the virus, once generated, can be transmitted to daughter cells indefinitely without the vector or any selection, which makes the 23S RNA virus-launching system particularly useful for investigating the basis for RNA virus persistence.

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of 'natively unfolded' proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.

Previous studies in Saccharomyces cerevisiae have defined an essential role for the Dbf4-Cdc7 kinase complex in the initiation of DNA replication presumably by phosphorylation of target proteins, such as the minichromosome maintenance (Mcm) complex. We have examined the phosphorylation of the Mcm complex by the Dfp1-Hsk1 kinase, the Schizosaccharomyces pombe homologue of Dbf4-Cdc7. In vitro, the purified Dfp1-Hsk1 kinase efficiently phosphorylated Mcm2p. In contrast, Mcm2p, present in the six-subunit Mcm complex, was a poor substrate of this kinase and required Cdc23p (homologue of Mcm10p) for efficient phosphorylation. In the presence of Cdc23p, Dfp1-Hsk1 phosphorylated the Mcm2p and Mcm4p subunits of the Mcm complex. Cdc23p interacted with both the Mcm complex and Dfp1-Hsk1 by selectively binding to the Mcm4/6/7 subunits and Dfp1p, respectively. The N terminus of Cdc23p was found to interact directly with Dfp1-Hsk1 and was essential for phosphorylation of the Mcm complex. Truncated derivatives of Cdc23p that complemented the temperature-sensitive phenotype of cdc23 mutant cells also stimulated the phosphorylation of Mcm complex, implying that this activity might be a critical role of Cdc23p in vivo. These results suggest that Cdc23p participates in the activation of prereplicative complex by recruiting the Dfp1-Hsk1 kinase and stimulating the phosphorylation of the Mcm complex. DNA replication | scaffold protein