S. cerevisiae

Proteins, 2004 Nov 15, 57(3), 481 - 92
Predictive reconstruction of the mitochondrial iron-sulfur cluster assembly metabolism . II . Role of glutaredoxin Grx5; Alves R et al.; Grx5 is a Saccharomyces cerevisiae glutaredoxin involved in iron-sulfur cluster (FeSC) biogenesis . Previous work suggests that Grx5 is involved in regulating protein cysteine glutathionylation, prompting several questions about the systemic role of Grx5 . First, is the regulation of mixed protein-glutathione disulfide bridges in FeSC biosynthetic proteins by Grx5 sufficient to account for the observed phenotypes of the Deltagrx5 mutants? If so, does Grx5 regulate the oxidation state of mixed protein-glutathione disulfide bridges in FeSC biogenesis in general? Alternatively, can the Deltagrx5 mutant phenotypes be explained if Grx5 acts on just one or a few of the FeSC biogenesis proteins?In the first part of this article, we address these questions by building and analyzing a mathematical model of FeSC biosynthesis . We show that, independent of the tested parameter values, the dynamic behavior observed in cells depleted of Grx5 can only be qualitatively reproduced if Grx5 acts by regulating the initial assembly of FeSC in scaffold proteins . This can be achieved by acting on the cysteine desulfurase (Nfs1) activity and/or on scaffold functionality.In the second part of this article, we use structural bioinformatics methods to evaluate the possibility of interaction between Grx5 and proteins involved in FeSC biogenesis . Based on such methods, our results indicate that the proteins with which Grx5 is more likely to interact are consistent with the kinetic modeling results.Thus, our theoretical studies, combined with known Grx5 biochemistry, suggest that Grx5 acts on FeSC biosynthesis by regulating the redox state of important cysteine residues in Nfs1 and/or in the scaffold proteins where FeSC initially assemble . (c) 2004 Wiley-Liss, Inc.

J Biol Chem, 2004 Dec 3, 279(49), 50754 - 63 Epub 2004 Dec 3.
The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells; Revollo JR et al.; Recent studies have revealed new roles for NAD and its derivatives in transcriptional regulation . The evolutionarily conserved Sir2 protein family requires NAD for its deacetylase activity and regulates a variety of biological processes, such as stress response, differentiation, metabolism, and aging . Despite its absolute requirement for NAD, the regulation of Sir2 function by NAD biosynthesis pathways is poorly understood in mammals . In this study, we determined the kinetics of the NAD biosynthesis mediated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat), and we examined its effects on the transcriptional regulatory function of the mouse Sir2 ortholog, Sir2alpha, in mouse fibroblasts . We found that Nampt was the rate-limiting component in this mammalian NAD biosynthesis pathway . Increased dosage of Nampt, but not Nmnat, increased the total cellular NAD level and enhanced the transcriptional regulatory activity of the catalytic domain of Sir2alpha recruited onto a reporter gene in mouse fibroblasts . Gene expression profiling with oligonucleotide microarrays also demonstrated a significant correlation between the expression profiles of Nampt- and Sir2alpha-overexpressing cells . These findings suggest that NAD biosynthesis mediated by Nampt regulates the function of Sir2alpha and thereby plays an important role in controlling various biological events in mammals.

Int J Biochem Cell Biol, 2005 Jan, 37(1), 48 - 53
Prk1p; Zeng G et al.; The protein kinase Prk1p (standing for p53 regulating kinase 1) of the yeast Saccharomyces cerevisiae is the prototype of a kinase family identified recently as important regulators of the actin cytoskeleton and endocytosis . These kinases all have a highly homologous serine/threonine kinase domain in their N-terminal region but share no significant homology in other regions . Prk1p also contains a proline-rich motif near its C-terminus that is required for the proper subcellular localization of the protein . The kinase activity of Prk1p has been confirmed by both in vitro and in vivo studies and shown to be essential for the protein's function . To date, several proteins that play essential roles in actin cytoskeleton organization and endocytosis have been identified as the regulatory targets of Prk1p . Phosphorylation on the {L/I/V/N}xx{Q/N/T/S}xTG motifs by Prk1p results in a down-regulation of the functions of these target proteins . The observation that many yeast proteins involved in the actin cytoskeleton organization and endocytosis contain the Prk1p phosphorylation motifs has led to the hypothesis that the Prk1p family of kinases are possibly the general regulators of the actin cytoskeleton and endocytosis in yeast.

Proteomics, 2004 Dec, 4(12), 3791 - 9
Statistical exploration of variation in quantitative two-dimensional gel electrophoresis data; Gustafsson JS et al.; Two-dimensional gel electrophoresis is a major technique in global analysis at the protein level . This paper presents an examination of spot volume data from three gel sets with radioactively labeled yeast Saccharomyces cerevisiae proteins . A strong variance versus mean dependence in data was found to be stabilized by applying a shifted logarithmic transformation . However, transformed data showed a remaining substantial variance heterogeneity for different proteins . Furthermore, examination of studentized residuals revealed that transformed data were approximately normally distributed and that there were spatial correlations among the measurement errors in the gel.

Oncogene, 2004 Sep 20, 23(43), 7283 - 9
Does the reservoir for self-renewal stem from the ends?
Harrington L.
Stem cell research is a burgeoning field with an alluring potential for therapeutic intervention, and thus begs a critical understanding of the long-term consequences of stem cell replacement . Operationally, a stem cell may be defined as a rarely dividing cell with the capacity for self-renewal throughout the lifetime of the organism, and an ability to reconstitute its appropriate lineages via proliferation and differentiation . In many differentiated normal and cancer cell types, the maintenance of telomeres plays a pivotal role in their continued division potential . Taken together with the presence of the enzymatic activity responsible for telomere addition, telomerase, in several progenitor cell lineages, it is presumed that telomere maintenance will be critical for the replenishment of stem cells or their successors . The purpose of this review is to discuss the role of telomere length maintenance in self-renewal, and the consequent challenges and potential pitfalls to the manipulation of normal and cancer-derived stem cells.

Oncogene, 2004 Sep 20, 23(43), 7256 - 66
Cell cycle inhibitors in normal and tumor stem cells; Cheng T; Emerging data suggest that stem cells may be one of the key elements in normal tissue regeneration and cancer development, although they are not necessarily the same entity in both scenarios . As extensively demonstrated in the hematopoietic system, stem cell repopulation is hierarchically organized and is intrinsically limited by the intracellular cell cycle inhibitors . Their inhibitory effects appear to be highly associated with the differentiation stage in stem/progenitor pools . While this negative regulation is important for maintaining homeostasis, especially at the stem cell level under physiological cues or pathological insults, it constrains the therapeutic use of adult stem cells in vitro and restricts endogenous tissue repair after injury . On the other hand, disruption of cell cycle inhibition may contribute to the formation of the so-called 'tumor stem cells' (TSCs) that are currently hypothesized to be partially responsible for tumorigenesis and recurrence of cancer after conventional therapies . Therefore, understanding how cell cycle inhibitors control stem cells may offer new strategies not only for therapeutic manipulations of normal stem cells but also for novel therapies selectively targeting TSCs.

Oncogene, 2004 Sep 20, 23(43), 7247 - 55
Are genetic determinants of asymmetric stem cell division active in hematopoietic stem cells?
Faubert A, Lessard J, Sauvageau G.
Stem cells have acquired a golden glow in the past few years as they represent possible tools for reversing the damage wreak on organs . These cells are found not only in major regenerative tissues, such as the epithelia, blood and testes, but also in 'static tissues', such as the nervous system and liver, where they play a central role in tissue growth and maintenance . The mechanism by which stem cells maintain populations of highly differentiated, short-lived cells seems to involve a critical balance between alternate fates: daughter cells either maintain stem cell identity or initiate differentiation . Recent studies in lower organisms have unveiled the regulatory mechanisms of asymmetric stem cell divisions . In these models, the surrounding environment likely provides key instructive signals for the cells to choose one fate over another . Our understanding now extends to the intrinsic mechanisms of cell polarity that influence asymmetrical stem cell divisions . This article focuses on the genetic determinants of asymmetric stem cell divisions in lower organisms as a model for studying the process of self-renewal of mammalian hematopoietic stem cells.

Nat Genet, 2004 Oct, 36(10), 1059 - 60 Epub 2004 Sep 19.
Regulatory evolution across the protein interaction network; Lemos B et al.; Protein-protein interactions may impose constraints on both structural and regulatory evolution . Here we show that protein-protein interactions are negatively associated with evolutionary variation in gene expression . Moreover, interacting proteins have similar levels of variation in expression, and their expression levels are positively correlated across strains . Our results suggest that interacting proteins undergo similar evolutionary dynamics, and that their expression levels are evolutionarily coupled . These patterns hold for organisms as diverse as budding yeast and fruit flies.

Plant Physiol, 2004 Oct, 136(2), 3104 - 13 Epub 2004 Sep 17.
Molecular and functional characterization of a family of amino acid transporters from Arabidopsis; Su YH et al.; More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants . Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family . Currently, mainly transporters of the amino acid transporter family have been characterized . Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily . CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane . Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8 . The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem . CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.

J Biol Chem, 2004 Nov 19, 279(47), 49439 - 46 Epub 2004 Sep 16.
Loss of proliferative capacity and induction of senescence in oxidatively stressed human fibroblasts; Chen JH et al.; Cellular senescence can result from short, dysfunctional telomeres, oxidative stress, or oncogene expression, and may contribute to aging . To investigate the role of cellular senescence in aging it is necessary to define the time-dependent molecular events by which it is characterized . Here we investigated changes in levels of key proteins involved in cell cycle regulation, DNA replication, and stress resistance in senescing human fibroblasts following oxidative stress . An immediate response in stressed cells was dephosphorylation of retinoblastoma (Rb) and cessation of DNA synthesis . This was followed by sequential induction of p53, p21, and p16 . Increase in hypophosphorylated Rb and induction of p53 and p21 by a single stress treatment was transient, whereas sustained induction or dephosphorylation were achieved by a second stress . Down-regulation of the critical DNA replication initiation factor Cdc6 occurred early after stress concurring with p53 induction, and was followed by a decrease in Mcm2 levels . A late event in the stress-induced molecular sequence was the induction of SOD1, catalase, and HSP27 coinciding with development of the fully senescent phenotype . Our data suggest that loss of proliferative capacity in oxidatively stressed cells is a multistep process regulated by time-dependent molecular events that may play differential roles in induction and maintenance of cellular senescence.

Bioinformatics, 2005 Jan 15, 21(2), 239 - 47 Epub 2004 Sep 17.
Predicting protein functions with message passing algorithms; Leone M et al.; MOTIVATION: In the last few years, a growing interest in biology has been shifting toward the problem of optimal information extraction from the huge amount of data generated via large-scale and high-throughput techniques . One of the most relevant issues has recently emerged that of correctly and reliably predicting the functions of a given protein with that of functions exploiting information coming from the whole network of proteins physically interacting with the functionally undetermined one . In the present work, we will refer to an 'observed' protein as the one present in the protein-protein interaction networks published in the literature . METHODS: The method proposed in this paper is based on a message passing algorithm known as Belief Propagation, which accepts the network of protein's physical interactions and a catalog of known protein's functions as input, and returns the probabilities for each unclassified protein of having one chosen function . The implementation of the algorithm allows for fast online analysis, and can easily be generalized into more complex graph topologies taking into account hypergraphs, i.e . complexes of more than two interacting proteins . RESULTS: Benchmarks of our method are the two Saccharomyces cerevisiae protein-protein interaction networks and the Database of Interacting Proteins . The validity of our approach is successfully tested against other available techniques . CONTACT: leone@isiosf.isi.it SUPPLEMENTARY INFORMATION: http://isiosf.isi.it/~pagnani.

Oncol Rep, 2004 Oct, 12(4), 939 - 43
Identification and characterization of CDC50A, CDC50B and CDC50C genes in silico; Katoh Y et al.; Bni1p, implicated in cell polarity control and microtubule regulation during yeast budding, is the Saccharomyces cerevisiae homolog of human Formin-homology proteins, such as FMN1, FMN2, FHOD1, FHOD3, FHDC1, GRID2IP, FMNL1, FMNL2, FMNL3, DIAPH1, DIAPH2, DIAPH3, DAAM1 and DAAM2 . Cdc50p is necessary for subcellular localization of Bni1p and asymmetrical cell division . Lem3p and Ynr048wp are yeast homologs of Cdc50p; however, mammalian homologs of Cdc50p remained to be identified . Here, we identified and characterized CDC50A (TMEM30A), CDC50B (TMEM30B) and CDC50C (TMEM30C) genes by using bioinformatics . C6orf67 and FLJ33850 were representative human CDC50A and CDC50B cDNAs, respectively . Complete coding sequence of CDC50C cDNA was determined by assembling seven exons within AC129803.3 genome sequence . CDC50A, CDC50B and CDC50C genes were mapped to human chromosome 6q14.1, 14q23.1 and 3q12, respectively . Human CDC50A mRNA was expressed in embryonic stem (ES) cells, placenta, brain and chondrosarcoma, while CDC50B mRNA was expressed in pancreatic islet, kidney, prostate as well as in lung carcinoid, parathyroid tumor, bladder tumor, meningioma and pancreatic cancer . Mouse Cdc50a (2010200I23), Cdc50b (9130011B11) and Cdc50c (4933401B01) cDNAs were also identified . Mammalian CDC50 homologs, including human CDC50A (361 aa), CDC50B (351 aa), CDC50C (341 aa), mouse Cdc50a (364 aa), Cdc50b (353 aa) and Cdc50c (342 aa), were two-transmembrane-spanning proteins with one extracellular loop . Membrane topology and extracellular loop containing three Cys residues and one Asn-linked glycosylation site were evolutionarily conserved among mammalian CDC50 homologs and yeast Cdc50p homologs . Mammalian CDC50 homologs were predicted components of phospholipid-translocators just like yeast Cdc50p and Lem3p.

Eur J Biochem, 2004 Oct, 271(19), 3821 - 31
The highly conserved extracellular peptide, DSYG(893-896), is a critical structure for sodium pump function; Becker S et al.; The peptide sequence DSYG(893-896) of the sheep sodium pump alpha 1 subunit is highly conserved among all K(+)-transporting P-type ATPases . To obtain information about its function, single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity, ion recognition, and alpha/beta subunit interactions . Mutants of Ser894 or Tyr895 were all active . Conservative phenylalanine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild-type enzyme . Replacement of the same amino acid by cysteine, however, produced heat-sensitive enzymes, indicating that the aromatic group contributes to the stability of the enzyme . Mutants of the neighbouring Ser894 recognized K(+) with altered apparent affinities . Thus, the Ser894-->Asp mutant displayed a threefold higher apparent affinity for K(+) (EC(50) = 1.4 +/- 0.06 mm) than the wild-type enzyme (EC(50) = 3.8 +/- 0.33 mm) . In contrast, the mutant Ser894-->Ile had an almost sixfold lower apparent affinity for K(+) (EC(50) = 21.95 +/- 1.41 mm) . Mutation of Asp893 or Gly896 produced inactive proteins . When an anti-beta 1 subunit immunoglobulin was used to co-immunoprecipitate the alpha 1 subunit, neither the Gly896-->Arg nor the Gly896-->Ile mutant could be visualized by subsequent probing with an anti-alpha 1 subunit immunoglobulin . On the other hand, co-immunoprecipitation was obtained with the inactive Asp893-->Arg and Asp893-->Glu mutants . Thus, it might be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and/or ion translocation . The results obtained here demonstrate the importance of the highly conserved peptide DSYG(893-896) for the function of alpha/beta heterodimeric P-type ATPases.

EMBO J, 2004 Oct 13, 23(20), 3939 - 49 Epub 2004 Sep 16.
Multiple SNARE interactions of an SM protein: Sed5p/Sly1p binding is dispensable for transport; Peng R et al.; Sec1/Munc18 (SM) proteins are central to intracellular transport and neurotransmitter release but their exact role is still elusive . Several SM proteins, like the neuronal N-Sec1 and the yeast Sly1 protein, bind their cognate t-SNAREs with high affinity . This has been thought to be critical for their function . Here, we show that various mutant forms of Sly1p and the Golgi-localized syntaxin Sed5p, which abolish their high-affinity interaction, are fully functional in vivo, indicating that the tight interaction of the two molecules per se is not relevant for proper function . Mutant Sly1p unable to bind Sed5p is excluded from core SNARE complexes, also demonstrating that Sly1p function is not directly coupled to assembled SNARE complexes thought to execute membrane fusion . We also find that wild-type Sly1p and mutant Sly1p unable to bind Sed5p directly interact with selected ER-to-Golgi and intra-Golgi nonsyntaxin SNAREs . The newly identified, direct interactions of the SM protein with nonsytaxin SNAREs might provide a molecular mechanism by which SNAREs can be activated to engage in pairing and assemble into fusogenic SNARE complexes.

Nature, 2004 Sep 16, 431(7006), 308 - 12
Genomic analysis of regulatory network dynamics reveals large topological changes; Luscombe NM et al.; Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids . It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically . Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae . We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics . We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations . In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network . A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions . By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors) . Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy . We anticipate that many of the concepts presented here--particularly the large-scale topological changes and hub transience--will apply to other biological networks, including complex sub-systems in higher eukaryotes.

Genes Dev, 2004 Sep 15, 18(18), 2243 - 8
Slik Sterile-20 kinase regulates Moesin activity to promote epithelial integrity during tissue growth; Hipfner DR et al.; The Drosophila Sterile-20 kinase Slik promotes tissue growth during development by stimulating cell proliferation and by preventing apoptosis . Proliferation within an epithelial sheet requires dynamic control of cellular architecture . Epithelial integrity fails in slik mutant imaginal discs . Cells leave the epithelium and undergo apoptosis . The abnormal behavior of slik mutant cells is due to failure to phosphorylate and activate Moesin, which leads to excess Rho1 activity . This is distinct from Slik's effects on cell proliferation, which are mediated by Raf . Thus Slik acts via distinct pathways to coordinate cell proliferation with epithelial cell behavior during tissue growth.

Acta Oncol, 2004, 43(6), 585 - 9
Decreased Hrad6B expression in lung cancer; Sasaki H et al.; Human homologues of yeast Rad 6 (Hrad6B) encode ubiquitin-conjugating enzymes and complement the DNA repair and UV mutagenesis defects of Saccharomyces cerevisiae rad6 mutant . There is a larger subgroup with reduced DNA repair capacity that is likely to be at increased cancer risk . The authors investigated Hrad6B expression in lung cancer . An attempt was made to determine the influence of Hrad6B expression on clinicopathological features for patients with lung cancer who had undergone surgery . Expression of Hrad6B messenger RNA was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 110 lung carcinomas and adjacent histological non-malignant lung samples from patients for whom follow-up data were available using LightCycler . The Hrad6B/glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA expression was significantly decreased in the lung cancer tissue (4.078+/-5.674) as compared with the non-malignant lung tissue (10.495+/-12.976, p<0.001) . There was no relationship between Hrad6B/GAPDH expression and age, clinical stages, T-status, N-status, and pathological subtypes in lung cancer tissues . Hrad6B/GAPDH mRNA levels in males (3.521+/-4.280) and in females (6.420+/-8.167) were significantly different (p=0.0443) in lung cancer tissues, but not in non-malignant lung tissues . Heavy smokers had a slight non-significant tendency (p=0.0857) towards lower Hrad6B/GAPDH mRNA levels (3.453+/-4.743) in their lung cancer tissues as compared with light or non-smokers . Thus decreased Hrad6B mRNA expression might be a biomarker for decreased DNA repair capacity and the dysfunction of Hrad6B might play a role in the tobacco-related oncogenesis of lung cancer.

Cell, 2004 Sep 17, 118(6), 699 - 713
Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins; Lisby M et al.; DNA repair is an essential process for preserving genome integrity in all organisms . In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci) . Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae . The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs . Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins . Later and only in S and G2 phase, the homologous recombination machinery assembles at the site . Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse . The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

Cell, 2004 Sep 17, 118(6), 666 - 8
Watching the DNA repair ensemble dance; Lukas J et al.; Repair of damaged DNA is a dynamic process that requires careful orchestration of a multitude of enzymes, adaptor proteins, and chromatin constituents . In this issue of Cell, Lisby et al . (2004) provide a visual glimpse into how the diverse signaling and repair machines are organized in space and time around the deadliest genetic lesions--the DNA double-strand breaks.

Mol Cell Biol, 2004 Oct, 24(19), 8487 - 503
Characterization of karyopherin cargoes reveals unique mechanisms of Kap121p-mediated nuclear import; Leslie DM et al.; In yeast there are at least 14 members of the beta-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm . Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions . Here, we have focused on the identification of nuclear import cargoes for the essential yeast beta-karyopherin, Kap121p . Using an overlay blot assay and coimmunopurification studies, we have identified 30 putative Kap121p cargoes . Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis . Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs . Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function . Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p . Together, our data elucidate additional levels of complexity in these nuclear transport pathways.

Mol Cell Biol, 2004 Oct, 24(19), 8332 - 41
TOR controls transcriptional and translational programs via Sap-Sit4 protein phosphatase signaling effectors; Rohde JR et al.; The Tor kinases are the targets of the immunosuppressive drug rapamycin and couple nutrient availability to cell growth . In the budding yeast Saccharomyces cerevisiae, the PP2A-related phosphatase Sit4 together with its regulatory subunit Tap42 mediates several Tor signaling events . Sit4 interacts with other potential regulatory proteins known as the Saps . Deletion of the SAP or SIT4 genes confers increased sensitivity to rapamycin and defects in expression of subsets of Tor-regulated genes . Sap155, Sap185, or Sap190 can restore these responses . Strains lacking Sap185 and Sap190 are hypersensitive to rapamycin, and this sensitivity is Gcn2 dependent and correlated with a defect in translation, constitutive eukaryotic initiation factor 2alpha hyperphosphorylation, induction of GCN4 translation, and hypersensitivity to amino acid starvation . We conclude that Tor signals via Sap-Sit4 complexes to control both transcriptional and translational programs that couple cell growth to amino acid availability.

J Virol, 2004 Oct, 78(19), 10348 - 59
Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus up-regulates transcription of human telomerase reverse transcriptase promoter through interaction with transcription factor Sp1; Verma SC et al.; Telomerase is required for the maintenance of telomere length and is an important determinant for cell immortalization . In human cells, telomerase activity is due to the expression of its enzymatic subunit, human telomerase reverse transcriptase (hTERT) . The expression of hTERT is not typically detectable in healthy somatic human cells but is present in cancerous tissues and immortalized cells . We have previously shown that hTERT promoter activity is up-regulated by the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) . LANA is expressed in all forms of human malignancies associated with KSHV . The hTERT promoter sequence located at positions -130 to +5 contains several Sp1 binding motifs and was shown be important for up-regulation by LANA . In this report, we demonstrate that hTERT promoter activity is due to the direct interaction of LANA with Sp1 . The interaction of LANA with Sp1 was demonstrated through in vitro binding experiments and coimmunoprecipitation and is supported by the colocalization of these two molecules in the nuclei of KSHV-infected cells . Moreover, LANA modulates Sp1-mediated transcription in transient GAL4 fusion reporter assays . Mapping of the regions involved in binding and transcriptional activation showed that the amino terminus of LANA is the major site for interaction and up-regulation but that it can cooperate with the carboxy terminus to enhance these functions . An analysis of Sp1 binding to its cognate sequence corroborated the binding data . Together, our results suggest that the interaction of LANA with Sp1 up-regulates the telomerase promoter, potentially contributing to the immortalization of KSHV-infected cells.

J Agric Food Chem, 2004 Sep 22, 52(19), 5902 - 6
Characterization of European wine glycerol: stable carbon isotope approach; Calderone G et al.; Glycerol of about 170 European wines was analyzed using gas chromatography-combustion-isotope ratio mass spectrometry technique . (13)C/(12)C isotopic ratio measurements were performed to characterize glycerol's delta(13)C values of genuine wine samples from European Union wine-producing countries . Glycerol was also successfully dosed using an internal reference, 1,5-pentanediol.

J Biol Chem, 2004 Nov 19, 279(47), 48767 - 73 Epub 2004 Sep 13.
Cell-free reconstitution of transport from the trans-golgi network to the late endosome/prevacuolar compartment; Blanchette JM et al.; Vesicle-mediated transport between the trans-Golgi network (TGN) and the late endosome/prevacuolar compartment (PVC) is an essential step in lysosomal/vacuolar biogenesis . In addition, localization of integral membrane proteins to the TGN requires continual cycles of vesicular transport between the TGN and endosomal compartments . Genetic and biochemical analyses in yeast have identified a variety of proteins required for TGN-to-PVC transport . However, the precise mechanisms of vesicle formation, transport, and fusion have not been fully elucidated . To study the steps of TGN-to-PVC transport in mechanistic detail, we have developed a cell-free assay to monitor delivery of the processing protease Kex2p from the TGN to PVC compartments containing a Kex2p substrate . Transport is time-, temperature-, and ATP-dependent and requires the t-SNARE Pep12p . Moreover, cell-free delivery of Kex2p to the PVC results in the co-integration of Kex2p into PVC membranes containing the Kex2p substrate as determined by co-immunoisolation of Kex2p and the substrate using antibody against the Kex2p cytosolic tail . This work represents the first cell-free reconstitution and biochemical analysis of the essential vacuolar/lysosomal sorting step TGN to late endosome transport.

J Mol Biol, 2004 Oct 1, 342(5), 1403 - 13
The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle; Richter K et al.; The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction . During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases . It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation . The specific role of the partner proteins in this process is unknown . Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1 . Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range . Two Sba1 molecules were found to bind per Hsp90 dimer . Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP . Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1 . Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.

Trends Genet, 2004 Oct, 20(10), 513 - 9
Coupling the mitochondrial transcription machinery to human disease; Shadel GS; Mitochondria are the central processing units for cellular energy metabolism and, in addition to carrying out oxidative phosphorylation, regulate important processes such as apoptosis and calcium homeostasis . Because mitochondria possess a genome that is central to their multiple functions, an understanding of the mechanism of mitochondrial gene expression is required to decipher the many ways mitochondrial dysfunction contributes to human disease . Towards this end, two human transcription factors that are related to rRNA methyltransferases have recently been characterized, providing new insight into the mechanism of mitochondrial transcription and a novel link to maternally inherited deafness . Furthermore, studies in the Saccharomyces cerevisiae model system have revealed a functional coupling of transcription and translation at the inner mitochondrial membrane, where assembly of the oxidative phosphorylation system commences . Defects in an analogous coupling mechanism in humans might underlie the cytochrome oxidase deficiency that causes a form of Leigh Syndrome.

Biochim Biophys Acta, 2004 Sep 17, 1693(3), 177 - 83
Calmodulin binds to and inhibits the activity of phosphoglycerate kinase; Myre MA et al.; Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis . Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK . Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind . The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency . Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition . Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells.

Plant J, 2004 Oct, 40(1), 22 - 34
Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins; Zimmermann IM et al.; In-depth analysis of protein-protein interaction specificities of the MYB protein family of Arabidopsis thaliana revealed a conserved amino acid signature ({DE}Lx(2){RK}x(3)Lx(6)Lx(3)R) as the structural basis for interaction between MYB and R/B-like BHLH proteins . The motif has successfully been used to predict new MYB/BHLH interactions for A . thaliana proteins, it allows to discriminate between even closely related MYB proteins and it is conserved amongst higher plants . In A . thaliana, the motif is shared by fourteen R2R3 MYB proteins and six 1R MYB proteins . It is located on helices 1 and 2 of the R3 repeat and forms a characteristic surface-exposed pattern of hydrophobic and charged residues . Single-site mutation of any amino acid of the signature impairs the interaction . Two particular amino acids have been determined to account for most of the interaction stability . Functional specificity of MYB/BHLH complexes was investigated in vivo by a transient DFR promoter activation assay . Residues stabilizing the MYB/BHLH interaction were shown to be critical for promoter activation . By virtue of proved and predicted interaction specificities, this study provides a comprehensive survey of the MYB proteins that interact with R/B-like BHLH proteins potentially involved in the TTG1-dependent regulatory interaction network . The results are discussed with respect to multi-functionality, specificity and redundancy of MYB and BHLH protein function.

EMBO J, 2004 Sep 29, 23(19), 3735 - 46 Epub 2004 Sep 09.
Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins; Chacinska A et al.; Mitochondria import nuclear-encoded precursor proteins to four different subcompartments . Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively . However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far . We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w) . Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10 . The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40 . The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes . We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.

EMBO J, 2004 Oct 27, 23(21), 4232 - 42 Epub 2004 Sep 09.
Members of the SAGA and Mediator complexes are partners of the transcription elongation factor TFIIS; Wery M et al.; TFIIS, an elongation factor encoded by DST1 in Saccharomyces cerevisiae, stimulates transcript cleavage in arrested RNA polymerase II . Two components of the RNA polymerase II machinery, Med13 (Srb9) and Spt8, were isolated as two-hybrid partners of the conserved TFIIS N-terminal domain . They belong to the Cdk8 module of the Mediator and to a subform of the SAGA co-activator, respectively . Co-immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell-free extracts . spt8Delta and dst1Delta mutants were sensitive to nucleotide-depleting drugs and epistatic to null mutants of the RNA polymerase II subunit Rpb9, suggesting that their elongation defects are mediated by Rpb9 . rpb9Delta, spt8Delta and dst1Delta were lethal in cells lacking the Rpb4 subunit . The TFIIS N-terminal domain is also strictly required for viability in rpb4Delta, although it is not needed for binding to RNA polymerase II or for transcript cleavage . It is proposed that TFIIS and the Spt8-containing form of SAGA co-operate to rescue RNA polymerase II from unproductive elongation complexes, and that the Cdk8 module temporarily blocks transcription during transcript cleavage.

J Biol Chem, 2004 Nov 19, 279(47), 49138 - 50 Epub 2004 Sep 08.
Degradation of the gluconeogenic enzymes fructose-1,6-bisphosphatase and malate dehydrogenase is mediated by distinct proteolytic pathways and signaling events; Hung GC et al.; The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is subjected to catabolite inactivation and degradation when glucose-starved cells are replenished with fresh glucose . In various studies, the proteasome and the vacuole have each been reported to be the major site of FBPase degradation . Because different growth conditions were used in these studies, we examined whether variations in growth conditions could alter the site of FBPase degradation . Here, we demonstrated that FBPase was degraded outside the vacuole (most likely in the proteasome), when glucose was added to cells that were grown in low glucose media for a short period of time . By contrast, cells that were grown in the same low glucose media for longer periods of time degraded FBPase in the vacuole in response to glucose . Another gluconeogenic enzyme malate dehydrogenase (MDH2) showed the same degradation characteristics as FBPase in that the short term starvation of cells led to a non-vacuolar degradation, whereas long term starvation resulted in the vacuolar degradation of this protein . The N-terminal proline is required for the degradation of FBPase and MDH2 for both the vacuolar and non-vacuolar proteolytic pathways . The cAMP signaling pathway and the phosphorylation of glucose were needed for the vacuolar-dependent degradation of FBPase and MDH2 . By contrast, the cAMP-dependent signaling pathway was not involved in the non-vacuolar degradation of these proteins, although the phosphorylation of glucose was required.

Biochem Biophys Res Commun, 2004 Aug 13, 321(1), 51 - 7
Homeostatic regulation of the proteasome via an Rpn4-dependent feedback circuit; Ju D et al.; The 26S proteasome is a complex protease consisting of at least 32 different subunits . Early studies showed that Rpn4 (also named Son1 and Ufd5) is a transcriptional activator of the Saccharomyces cerevisiae proteasome genes, and that Rpn4 is rapidly degraded by the 26S proteasome . These observations suggested that in vivo proteasome abundance may be regulated by an Rpn4-dependent feedback circuit . Here, we present direct evidence to support the Rpn4-proteasome feedback model . We show that proteasome expression is increased when proteasome activity is impaired, and that this increase is Rpn4-dependent . Moreover, we demonstrate that expression of a stable form of Rpn4 leads to elevation of proteasome expression . Our data also reveal that the Rpn4-proteasome feedback circuit is critical for cell growth when proteasome activity is compromised, and plays an important role in response to DNA damage . This study provides important insights into the mechanism underlying proteasome homeostasis.

Lipids, 2004 Apr, 39(4), 311 - 8
Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean; He X et al.; The oil from castor seed (Ricinus communis) contains 90% ricinoleate, a hydroxy FA that is used in producing numerous industrial products . Castor diacylglycerol acyltransferase (RcDGAT) is a critical enzyme, as it catalyzes the terminal step in castor oil biosynthesis in which the products contain two or three ricinoleoyl moieties . We have isolated a cDNA encoding RcDGAT from developing castor seeds . Analysis of the sequence reveals that this cDNA encodes a protein of 521 amino acids with a molecular mass of 59.9 kDa . Although there are regions of high similarity to other plant DGAT coding sequences, there are sequences that distinguish it as well . Southern blot analysis suggests that the castor genome contains a single copy of RcDGAT . Analysis by reverse transcription-PCR reveals that the accumulation of the mRNA reaches its highest level at 19 d after pollination and declines thereafter . Expression of the full-length cDNA for RcDGAT in the yeast Saccharomyces cerevisiae, strain INVSc1 results in sevenfold higher DGAT activity compared with controls . When different molecular species of DAG were provided as substrates to the microsomal mixture, the RcDGAT showed a greater preference to catalyze the transfer of oleate from {14C}oleoyl-CoA to diricinolein than to diolein and dipalmitolein . With the addition of 0.25 mM substrates, diricinolein gave 318 pmol/mg/min diricinoleoyloleoylglycerol (RRO), while diolein and dipalmitolein gave only about 195 pmol/mg/min of triolein (OOO) and 120 pmol/mg/min dipalmitoyleoylglycerol (PoPoO), respectively . This work will facilitate investigation of the role of RcDGAT in castor oil biosynthesis.

J Biol Chem, 2004 Nov 19, 279(47), 49542 - 50 Epub 2004 Sep 07.
The spindle pole body assembly component mps3p/nep98p functions in sister chromatid cohesion; Antoniacci LM et al.; For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister chromatids . Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate . Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p) . Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister chromatids are paired together, identifying the products of replication as sister chromatids . Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion . Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays . An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p . Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance . Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant . In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality . These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.

Brief Funct Genomic Proteomic, 2004 Aug, 3(2), 103 - 11
Translational profiling: the genome-wide measure of the nascent proteome; Beilharz TH et al.; Translation in eukaryotic cells is both physically and temporally separated from transcription . This provides cells with extended options to alter their proteome: (1) directly, by synchronizing translation with an altering transcriptional profile; (2) by imposing a changed translational control over transcripts already present in the transcriptome; or (3) by a combination of (1) and (2) . In this paper, recent findings in the controlled translation of the transcriptome using microarray analyses are reviewed . A guide to the current technologies and data analysis is also provided, and future directions in the study of translational control as the interface between the transcriptome and the proteome are outlined . This survey is focused on the yeast Saccharomyces cerevisiae, but the topics covered have universal relevance to the control of translation in eukaryotic cells.

Eur J Biochem, 2004 Sep, 271(18), 3741 - 51
RNA helicase A interacts with nuclear factor kappaB p65 and functions as a transcriptional coactivator; Tetsuka T et al.; RNA helicase A (RHA), a member of DNA and RNA helicase family containing ATPase activity, is involved in many steps of gene expression such as transcription and mRNA export . RHA has been reported to bind directly to the transcriptional coactivator, CREB-binding protein, and the tumor suppressor protein, BRCA1, and links them to RNA Polymerase II holoenzyme complex . Using yeast two-hybrid screening, we have identified RHA as an interacting molecule of the p65 subunit of nuclear factor kappaB (NF-kappaB) . The interaction between p65 and RHA was confirmed by glutathione-S transferase pull-down assay in vitro, and by co-immunoprecipitation assay in vivo . In transient transfection assays, RHA enhanced NF-kappaB dependent reporter gene expression induced by p65, tumor necrosis factor-alpha, or NF-kappaB inducing kinase . The mutant form of RHA lacking ATP-binding activity inhibited NF-kappaB dependent reporter gene expression induced by these activators . Moreover, depletion of RHA using short interfering RNA reduced the NF-kappaB dependent transactivation . These data suggest that RHA is an essential component of the transactivation complex by mediating the transcriptional activity of NF-kappaB .

Proc Natl Acad Sci U S A, 2004 Oct 5, 101(40), 14315 - 22 Epub 2004 Sep 07.
Sfp1 is a stress- and nutrient-sensitive regulator of ribosomal protein gene expression; Marion RM et al.; Yeast cells modulate their protein synthesis capacity in response to physiological needs through the transcriptional control of ribosomal protein (RP) genes . Here we demonstrate that the transcription factor Sfp1, previously shown to play a role in the control of cell size, regulates RP gene expression in response to nutrients and stress . Under optimal growth conditions, Sfp1 is localized to the nucleus, bound to the promoters of RP genes, and helps promote RP gene expression . In response to inhibition of target of rapamycin (TOR) signaling, stress, or changes in nutrient availability, Sfp1 is released from RP gene promoters and leaves the nucleus, and RP gene transcription is down-regulated . Additionally, cells lacking Sfp1 fail to appropriately modulate RP gene expression in response to environmental cues . We conclude that Sfp1 integrates information from nutrient- and stress-responsive signaling pathways to help control RP gene expression.

J Cell Biol, 2004 Sep 13, 166(6), 889 - 900 Epub 2004 Sep 07.
Robust cell polarity is a dynamic state established by coupling transport and GTPase signaling; Wedlich-Soldner R et al.; Yeast cells can initiate bud formation at the G1/S transition in a cue-independent manner . Here, we investigate the dynamic nature of the polar cap and the regulation of the GTPase Cdc42 in the establishment of cell polarity . Using analysis of fluorescence recovery after photobleaching, we found that Cdc42 exchanged rapidly between the polar caps and cytosol and that this rapid exchange required its GTPase cycle . A previously proposed positive feedback loop involving actomyosin-based transport of the Cdc42 GTPase is required for the generation of robust cell polarity during bud formation in yeast . Inhibition of actin-based transport resulted in unstable Cdc42 polar caps . Unstable polarity was also observed in mutants lacking Bem1, a protein previously implicated in a feedback loop for Cdc42 activation through a signaling pathway . When Bem1 and actin were both inhibited, polarization completely failed . These results suggest that cell polarity is established through coupling of transport and signaling pathways and maintained actively by balance of flux.

Hum Gene Ther, 2004 Sep, 15(9), 887 - 95
Functional expression of the single subunit NADH dehydrogenase in mitochondria in vivo: a potential therapy for complex I deficiencies; Seo BB et al.; It has been reported that defects of mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) are involved in many human diseases (such as encephalomyopathies and sporadic Parkinson's disease) . However, no effective remedies have been established for complex I deficiencies . We have adopted a gene therapy approach utilizing the NDI1 gene that codes for the single subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1) . Our earlier experiments show that the Ndi1 protein can replace or supplement the functionality of complex I in various cultured cells . For this approach to be useful, it is important to demonstrate in vivo that the mature protein is correctly placed in mitochondria . In this study, we have attempted in vivo expression of the NDI1 gene in skeletal muscles and brains (substantia nigra and striatum) of rodents . In all tissues tested, the Ndi1 protein was identified in the injected area by immunohistochemical staining at 1-2 weeks after the injection . Sustained expression was observed for at least 7 months . Double-staining of the sections using antibodies against Ndi1 and F(1)-ATPase revealed that the expressed Ndi1 protein was predominantly localized to mitochondria . In addition, the tissue cells expressing the Ndi1 protein stimulated the NADH dehydrogenase activity, suggesting that the expressed Ndi1 is functionally active . It was also confirmed that the Ndi1 expression induced no inflammatory response in the tissues examined . The data indicate that the NDI1 gene will be a promising therapeutic tool in the treatment of encephalomyopathies and neurodegenerative diseases caused by complex I impairments.

Biochem J, 2005 Jan 1, 385(Pt 1), 265 - 77
Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer; Raicu V et al.; We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(T)) forming complexes, and the average number (n) of those protein molecules in each complex . The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer . The approach and sensitivity limits are well suited for in vivo microscopic investigations . This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 alpha-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae . A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E(app)) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency) . We determined E by using a new method that relies on E(app) measurements for two donor/acceptor pairs, Ste2p-CFP/Ste2p-YFP and Ste2p-GFP/Ste2p-YFP . From E(app) and E we determined alpha(T) approximately 1 and n approximately 2 for Ste2 proteins . Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization.

Bioessays, 2004 Sep, 26(9), 955 - 62
Adding to the ends: what makes telomerase processive and how important is it?
Lue NF.
Telomerase is a cellular reverse transcriptase responsible for telomere maintenance in most organisms . It does so by adding telomere repeats onto pre-existing ends using an integral RNA component as template . Compared to "prototypical" reverse transcriptases, telomerase is unique in being able to repetitively copy a short templating RNA segment, thus adding multiple copies of the repeat to the DNA substrate following a single binding event . This uniquely processive property hints at the intricate conformational alterations that the enzyme must choreograph during its reaction cycles . Recent studies have identified distinct structural elements within both the RNA and protein components of telomerase that modulate enzyme processivity . Pharmacological and genetic analysis suggest that telomerase processivity is a significant determinant of telomere length . Because telomere maintenance and the lack thereof have been linked to tumor progression and aging, further investigation of telomerase processivity may lead to novel medical intervention strategies.

Biochem Biophys Res Commun, 2004 Oct 8, 323(1), 339 - 44
Oligomeric Aip2p/Dld2p modifies the protein conformation of both properly folded and misfolded substrates in vitro; Hachiya NS et al.; Oligomeric actin-interacting protein 2 (Aip2p) {Nat . Struct . Biol . 2 (1995) 28}/D-lactate dehydrogenase protein 2 (Dld2p) {Yeast 15 (1999) 1377, Biochem . Biophys . Res . Commun . 295 (2002) 910} exhibits the unique grapple-like structure with an ATP-dependent opening {Biochem . Biophys . Res . Commun . 320 (2004) 1271}, which is required for the F-actin conformation modifying activity in vitro and in vivo {Biochem . Biophys . Res . Commun . 319 (2004) 78} . To further investigate the molecular nature of oligomeric Aip2p/Dld2p, the substrate specificity of its binding and protein conformation modifying activity was examined . In the presence of 1mM ATP or AMP-PNP, oligomeric Aip2p/Dld2p bound to all substrates so far examined, and modified the conformation of actin, DNase I, the mature form of invertase, prepro-alpha-factor, pro-alpha-factor, and mitochondrial superoxide dismutase, as determined by the trypsin susceptibility assay . Of note, the activity could modify even the conformation of pathogenic highly aggregated polypeptides, such as recombinant prion protein in beta-sheet form, alpha-synuclein, and amyloid beta (1-42) in the presence of ATP . The in vivo protein conformation modifying activity, however, depends on the growth stage; the most significant substrate modification activity was observed in yeast cells at the log phase, suggesting the presence of a cofactor/s in yeast cells, where F-actin is supposed to be a major target in vivo . These data further support our previous notion that the oligomeric Aip2p/Dld2p may belong to an unusual class of molecular chaperones {Biochem . Biophys . Res . Commun . 320 (2004) 1271}, which can target both properly folded and misfolded proteins in an ATP-dependent manner in vitro .

Biochem Biophys Res Commun, 2004 Oct 8, 323(1), 91 - 7
Interaction of the BMPR-IA tumor suppressor with a developmentally relevant splicing factor; Nishanian TG et al.; Inactivation of bone morphogenetic protein signaling via mutation of the BMPR-IA TGF-beta superfamily type I receptor causes familial juvenile polyposis, an inherited gastrointestinal cancer predisposition syndrome . In an effort to provide new insight into the mechanism(s) of BMP-mediated tumor suppression, we employed a yeast two-hybrid screen to identify novel proteins that interact with the intracellular domain of BMPR-IA . 30/31 interacting clones encoded SAP49, a splicing factor that has been shown to be required for normal development in Caenorhabditis elegans . The remaining interacting clone was FKBP12.6, a known TGF-beta type I receptor interactor . The interaction between BMPR-IA and SAP49 was confirmed via coimmunoprecipitation in human cells . Mutational analysis demonstrated that the GS domain of the receptor and the conserved proline-rich domain of SAP49 were required for the interaction . Co-localization studies suggested that the interaction may occur at the inner leaflet of the nuclear membrane . These data suggest that BMPR-IA may interact with and modulate the activity of a developmentally relevant splicing factor .

J Mol Biol, 2004 Sep 24, 342(4), 1115 - 29
Role of the putative structural protein Sed1p in mitochondrial genome maintenance; Phadnis N et al.; The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae . A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified . Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p . Sed1p is a cell surface protein highly expressed in the stationary phase . We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro . Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration . In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability . Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria . Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K . Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p . Our results point towards a role for Sed1p in mitochondrial genome maintenance.

Vet Immunol Immunopathol, 2004 Oct, 101(3-4), 203 - 10
Total serum immunoglobulin M levels are affected by immunomodulators in seabream (Sparus aurata L.); Cuesta A et al.; Immunoglobulin M (IgM) is a major component of the teleost humoral immune system . Despite the significance of IgM levels as an immune parameter, there are relatively few studies on changes induced in its total levels in serum . This study examines the effects of several immunomodulators (vitamin A, chitin, yeast cells or levamisole, which act as immunostimulants, and crowding, hypoxia or anaesthetics, which act as stressors) upon the total serum IgM levels of non-immunized gilthead seabream (Sparus aurata L.) . Total serum IgM levels of fish fed with the assayed immunostimulant-supplemented diets were statistically higher than those in fish fed a non-supplemented diet, especially in the case of levamisole . On the other hand, serum IgM levels of fish subjected to different stressors were not affected by crowding, hypoxia or certain anaesthetics . However, benzocaine and a narcotic dose of 2-phenoxyethanol provoked a great reduction, while quinaldine sulphate increased IgM levels to a significant degree . These results show how the seric IgM levels can be differently affected by some immunomodulators and the important role they may play in the regulation of total circulating IgM levels in seabream . The possibility of using total serum IgM for assessing immunostimulation, disease diagnosis and stress symptoms during fish farming is discussed.

Electrophoresis, 2004 Sep, 25(17), 2926 - 31
Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels; Hou WC et al.; Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column . For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min . The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min . The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min . The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels . This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources . Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast . This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.

Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13442 - 7 Epub 2004 Sep 03.
Distinct roles of transcription factors TFIIIB and TFIIIC in RNA polymerase III transcription reinitiation; Ferrari R et al.; Eukaryotic RNA polymerase (Pol) III is recruited to target promoters by a stable preinitiation complex containing transcription factors TFIIIC and TFIIIB . After the first transcription cycle, reinitiation proceeds through facilitated recycling, a process by which the terminating Pol III rapidly reloads onto the same transcription unit . Here, we show that Pol III is repeatedly recaptured in vitro by the first transcribed gene, even in the presence of a juxtaposed competitor promoter complex, thus suggesting that facilitated recycling is not merely due to a stochastic reassociation process favored by the small size of class III genes . The transcription factor requirements for facilitated reinitiation were investigated by taking advantage of Pol III templates that support both TFIIIC-dependent and TFIIIC-independent transcription . A TFIIIC-less transcription system, in which TFIIIB was reconstituted from recombinant TATA box-binding protein and Brf1 proteins and a crude fraction containing the Bdp1 component, was sufficient to direct efficient Pol III recycling on short ( approximately 100 bp) class III genes . Unexpectedly, however, on longer (>300 bp) transcription units, reinitiation in the presence of TFIIIB alone was compromised, and TFIIIC was further required to reestablish a high reinitiation rate . Transcription reinitiation was also severely impaired when recombinant Bdp1 protein replaced the corresponding crude fraction in reconstituted TFIIIB . The data reveal an unexpected complexity in the Pol III reinitiation mechanism and suggest the existence of a handing-back network between Pol III, TFIIIC, and TFIIIB on actively transcribed class III genes.

Proc Natl Acad Sci U S A, 2004 Sep 14, 101(37), 13478 - 82 Epub 2004 Sep 03.
Fast folding of a helical protein initiated by the collision of unstructured chains; Meisner WK et al.; To examine whether helix formation necessarily precedes chain collision, we have measured the folding of a fully helical coiled coil that has been specially engineered to have negligible intrinsic helical propensity but high overall stability . The folding rate approaches the diffusion-limited value and is much faster than possible if folding is contingent on precollision helix formation . Therefore, the collision of two unstructured chains is the initial step of the dominant kinetic pathway, whereas helicity exerts its influence only at a later step . Folding from an unstructured encounter complex may be efficient and robust, which has implications for any biological process that couples folding to binding.

J Biol Chem, 2004 Nov 19, 279(47), 49497 - 507 Epub 2004 Sep 03.
Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica; Saito-Nakano Y et al.; In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation . Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals . EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state . Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A ("prephagosomal vacuoles (PPV)") in the amoeba within an incubation period of 5-10 min . PPV emerged de novo physically and distinct from phagosomes . PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore . After EhRab5 dissociated from PPV, 5-10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes . Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation . In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis . Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes . Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis . Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes.

Eur J Cell Biol, 2004 Jul, 83(5), 175 - 83
The exosome, a molecular machine for controlled RNA degradation in both nucleus and cytoplasm; Raijmakers R et al.; One of the most important protein complexes involved in maintaining correct RNA levels in eukaryotic cells is the exosome, a complex consisting almost exclusively of exoribonucleolytic proteins . Since the identification of the exosome complex, seven years ago, much progress has been made in the characterization of its composition, structure and function in a variety of organisms . Although the exosome seems to accumulate in the nucleolus, it has been clearly established that it is also localized in cytoplasm and nucleoplasm . In accordance with its widespread intracellular distribution, the exosome has been implicated in a variety of RNA processing and degradation processes . Nevertheless, many questions still remain unanswered . What are the factors that regulate the activity of the exosome? How and where is the complex assembled? What are the differences in the composition of the nuclear and cytoplasmic exosome? What is the detailed structure of exosome subunits? What are the mechanisms by which the exosome is recruited to substrate RNAs? Here, we summarize the current knowledge on the composition and architecture of this complex, explain its role in both the production and degradation of various types of RNA molecules and discuss the implications of recent research developments that shed some light on the questions above and the mechanisms that are controlling the exosome.

J Biol Chem, 2004 Nov 19, 279(47), 49488 - 96 Epub 2004 Sep 01.
The Traf2- and Nck-interacting kinase as a putative effector of Rap2 to regulate actin cytoskeleton; Taira K et al.; Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific roles in cell signaling remain unknown . In the present study, we have affinity-purified from rat brain a Rap2-interacting protein of approximately 155 kDa, p155 . By liquid chromatography tandem mass spectrometry, we have identified p155 as Traf2- and Nck-interacting kinase (TNIK) . TNIK possesses an N-terminal kinase domain homologous to STE20, the Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase kinase, and a C-terminal regulatory domain termed the citron homology (CNH) domain . TNIK induces disruption of F-actin structure, thereby inhibiting cell spreading . In addition, TNIK specifically activates the c-Jun N-terminal kinase (JNK) pathway . Among our observations, TNIK interacted with Rap2 through its CNH domain but did not interact with Rap1 or Ras . TNIK interaction with Rap2 was dependent on the intact effector region and GTP-bound configuration of Rap2 . When co-expressed in cultured cells, TNIK colocalized with Rap2, while a mutant TNIK lacking the CNH domain did not . Rap2 potently enhanced the inhibitory function of TNIK against cell spreading, but this was not observed for the mutant TNIK lacking the CNH domain . Rap2 did not significantly enhance TNIK-induced JNK activation, but promoted autophosphorylation and translocation of TNIK to the detergent-insoluble cytoskeletal fraction . These results suggest that TNIK is a specific effector of Rap2 to regulate actin cytoskeleton.

J Biol Chem, 2004 Nov 12, 279(46), 47580 - 8 Epub 2004 Sep 01.
Processing and joining of DNA ends coordinated by interactions among Dnl4/Lif1, Pol4, and FEN-1; Tseng HM et al.; The repair of DNA double-strand breaks is critical for maintaining genetic stability . In the non-homologous end-joining pathway, DNA ends are brought together by end-bridging factors . However, most in vivo DNA double-strand breaks have terminal structures that cannot be directly ligated . Thus, the DNA ends are aligned using short regions of sequence microhomology followed by processing of the aligned DNA ends by DNA polymerases and nucleases to generate ligatable termini . Genetic studies in Saccharomyces cerevisiae have implicated the DNA polymerase Pol4 and the DNA structure-specific endonuclease FEN-1(Rad27) in the processing of DNA ends to be joined by Dnl4/Lif1 . In this study, we demonstrated that FEN-1(Rad27) physically and functionally interacted with both Pol4 and Dnl4/Lif1 and that together these proteins coordinately processed and joined DNA molecules with incompatible 5' ends . Because Pol4 also interacts with Dnl4/Lif1, our results have revealed a series of pair-wise interactions among the factors that complete the repair of DNA double-strand breaks by non-homologous end-joining and provide a conceptual framework for delineating the end-processing reactions in higher eukaryotes.

J Biol Chem, 2004 Oct 22, 279(43), 44239 - 42 Epub 2004 Sep 01.
Intrinsic promoter recognition by a "core" RNA polymerase; Matsunaga M et al.; Two classes of RNA polymerases transcribe RNA from promoters on DNA templates: promoter recognition-competent single polypeptides and multisubunit enzymes that require separable promoter recognition factors . Eukaryotic mitochondria utilize an unusual hybrid of these classes composed of a "core" RNA polymerase related to the single polypeptide enzymes plus a "specificity factor" necessary for promoter utilization . Using supercoiled or premelted templates, we have discovered that the yeast core mitochondrial RNA polymerase (Rpo41) has the intrinsic ability to initiate from promoters without its specificity factor (Mtf1) . Rpo41 requires the mitochondrial promoter sequence (ATATAAGTA) for this activity . On premelted templates addition of Mtf1 actually inhibits the promoter selective activity of Rpo41 . Mtf1 increases abortive relative to productive transcription by Rpo41, possibly by stabilizing the promoter complex and reducing escape into elongation . The requirement for Mtf1 on closed but not open templates indicates that Mtf1 facilitates melting but not recognition of promoters.

J Mol Biol, 2004 Sep 17, 342(3), 757 - 73
The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity; Charron C et al.; The ribosomal L7Ae protein of archaea has the peculiarity to be a component of the C/D and H/ACA snRNPs, that guide rRNA post-transcriptional modifications . Its yeast (Snu13p) and human (15.5kDa protein) homologs are only found in C/D snoRNPs and the (U4/U6, U5) spliceosomal tri-snRNP . By using a large variety of RNAs, we compared the RNA-binding specificities of the recombinant Pyrococcus abyssi L7Ae and Saccharomyces cerevisiae Snu13 proteins . Unlike Snu13p, protein L7Ae binds terminal loops closed by two A:G and G:A pairs and canonical K-turn structures with similar efficiencies, provided that the terminal loop contains at least 5nt . In contrast to Snu13p, binding of protein L7Ae to canonical K-turn structures is not dependent on the identity of the residue at position 2 in the bulge . The peculiar KT-15 motif of P . abyssi 23S rRNA, that is recognized by L7Ae, does not associate with Snu13p . To get more information on the P . abyssi L7Ae protein, we solved its X-ray structure at 1.9A resolution . In spite of their sequence divergence, the free P . abyssi and bound H . marismortui proteins were found to have highly similar structures . Only a limited number of side-chain conformational changes occur at the protein-RNA interface upon RNA binding . In particular, one ion pair that is formed by residues Glu43 and Lys46 in the free protein is disrupted in the ribosomal 50S subunit, so that, residue Glu43 can interact with the RNA residue G264 . The Glu43-Lys46 ion pair of protein L7Ae belongs to a complex network of ion pairs that may participate to protein thermostability.

Structure (Camb), 2004 Sep, 12(9), 1585 - 94
Crystal structure of A . fulgidus Rio2 defines a new family of serine protein kinases; LaRonde-LeBlanc N et al.; The RIO family of atypical serine/threonine kinases contains two subfamilies, Rio1 and Rio2, highly conserved from archaea to man . Both RIO proteins from Saccharomyces cerevisiae catalyze serine phosphorylation in vitro, and the presence of conserved catalytic residues is required for cell viability . The activity of Rio2 is necessary for rRNA cleavage in 40S ribosomal subunit maturation . We solved the X-ray crystal structure of Archaeoglobus fulgidus Rio2, with and without bound nucleotides, at 2.0 A resolution . The C-terminal RIO domain is indeed structurally homologous to protein kinases, although it differs from known serine kinases in ATP binding and lacks the regions important for substrate binding . Unexpectedly, the N-terminal Rio2-specific domain contains a winged helix fold, seen primarily in DNA-binding proteins . These discoveries have implications in determining the target and function of RIO proteins and define a distinct new family of protein kinases.

Mol Microbiol, 2004 Sep, 53(6), 1785 - 96
Fungicide activity through activation of a fungal signalling pathway; Kojima K et al.; Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis . Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway . The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose . The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil . The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress . Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth . In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants . Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1 . Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea . These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.

Mol Microbiol, 2004 Sep, 53(6), 1743 - 56
Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance; Bilsland E et al.; We demonstrate a role in oxidative and metal stress resistance for the MAPK-activated protein kinases Rck1 and Rck2 in Saccharomyces cerevisiae . We show that Hog1 is robustly phosphorylated in a Pbs2-dependent way during oxidative stress, and that Rck2 also is phosphorylated under these circumstances . Hog1 concentrates in the nucleus in oxidative stress . Hog1 localization is partially dependent on Rck2, as rck2 cells have more nuclear Hog1 than wild-type cells . We find several proteins with a role in oxidative stress resistance using Rck1 or Rck2 as baits in a two-hybrid screen . We identify the transcription factor Yap2 as a putative target for Rck1, and the Zn2+ transporter Zrc1 as a target for Rck2 . Yap2 is normally cytoplasmic, but rapidly migrates to the nucleus upon exposure to oxidative stress agents . In a fraction of untreated pbs2 cells, Yap2 is nuclear . Zrc1 co-immunoprecipitates with Rck2, and ZRC1 is genetically downstream of RCK2 . These data connect activation of the Hog1 MAPK cascade with effectors having a role in oxidative stress resistance.

Mol Microbiol, 2004 Sep, 53(6), 1625 - 40
Characterization of IDI-4, a bZIP transcription factor inducing autophagy and cell death in the fungus Podospora anserina; Dementhon K et al.; In filamentous fungi a cell death reaction occurs when hyphae of unlike genotype fuse . This phenomenon is referred to as heterokaryon incompatibility . In Podospora anserina, this cell death reaction was found to be associated with the transcriptional induction of a set of genes termed idi genes (for induced during incompatibility) and activation of autophagy . Herein, we describe the characterization of idi-4, a novel idi gene encoding a bZIP transcription factor . Expression of idi-4 is induced during cell death by incompatibility and in various stress conditions . Inactivation of idi-4 by gene replacement does not suppress incompatibility but we show that overexpression of idi-4 triggers cell death . Strains which undergo idi-4-induced cell death display cytological hallmarks of cell death by incompatibility notably induction of autophagy . We also report that increased expression of idi-4 leads to transcriptional induction of other idi genes such as idi-7, the orthologue of the yeast ATG8 autophagy gene . Together these results establish IDI-4 as one of the transcription factor regulating autophagy and cell fate in Podospora.

Mol Microbiol, 2004 Sep, 53(6), 1611 - 23
The gprA and gprB genes encode putative G protein-coupled receptors required for self-fertilization in Aspergillus nidulans; Seo JA et al.; The filamentous fungus Aspergillus nidulans possesses both asexual and sexual reproductive cycles . Sexual fruiting bodies (cleistothecia) can be formed in both homothallic (self) and heterothallic (outcross) conditions . In this study, we characterized two genes, gprA and gprB, that are predicted to encode putative G protein-coupled receptors (GPCRs) similar to fungal pheromone receptors . Deletion (Delta) of gprA or gprB resulted in the production of a few small cleistothecia carrying a reduced number of ascospores, whereas DeltagprADeltagprB eliminated fruiting body formation in homothallic conditions . However, nullifying gprA and/or gprB did not affect vegetative growth, asexual sporulation, Hulle cell formation or even cleistothecia formation in outcross, indicating that GprA and GprB are specifically required for self-fertilization . The gprA and gprB genes encode two transcripts and, for both genes, larger transcripts are detectable during vegetative growth and asexual development whereas smaller transcripts accumulate during sexual development . Upregulation of nsdD encoding a key sexual developmental activator resulted in the production of barren cleistothecia in the DeltagprADeltagprB mutant, suggesting that NsdD can partially rescue the developmental defects caused by deletion of GPCRs and that GprA/B-mediated signalling may activate other genes necessary for maturation of cleistothecia and ascosporogenesis . Deletion of gprA and/or gprB suppressed growth defects caused by DeltagprD, implying that GprA/B function downstream of GprD-mediated negative control of sexual development.

Protein Sci, 2004 Oct, 13(10), 2673 - 84 Epub 2004 Aug 31.
Crystal structure of Ski8p, a WD-repeat protein with dual roles in mRNA metabolism and meiotic recombination; Cheng Z et al.; Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3'-to-5' mRNA decay . In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein . We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 A resolution . The structure reveals that Ski8p folds into a seven-bladed beta propeller . Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the beta propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p . Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the beta propeller as being required for Ski8p binding to Ski3p and Spo11p . The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed.

Mol Cell Biol, 2004 Sep, 24(18), 8312 - 21
Role for Nhp6, Gcn5, and the Swi/Snf complex in stimulating formation of the TATA-binding protein-TFIIA-DNA complex; Biswas D et al.; The TATA-binding protein (TBP), TFIIA, and TFIIB interact with promoter DNA to form a complex required for transcriptional initiation, and many transcriptional regulators function by either stimulating or inhibiting formation of this complex . We have recently identified TBP mutants that are viable in wild-type cells but lethal in the absence of the Nhp6 architectural transcription factor . Here we show that many of these TBP mutants were also lethal in strains with disruptions of either GCN5, encoding the histone acetyltransferase in the SAGA complex, or SWI2, encoding the catalytic subunit of the Swi/Snf chromatin remodeling complex . These synthetic lethalities could be suppressed by overexpression of TOA1 and TOA2, the genes encoding TFIIA . We also used TFIIA mutants that eliminated in vitro interactions with TBP . These viable TFIIA mutants were lethal in strains lacking Gcn5, Swi2, or Nhp6 . These lethalities could be suppressed by overexpression of TBP or Nhp6, suggesting that these coactivators stimulate formation of the TBP-TFIIA-DNA complex . In vitro studies have previously shown that TBP binds very poorly to a TATA sequence within a nucleosome but that Swi/Snf stimulates binding of TBP and TFIIA . In vitro binding experiments presented here show that histone acetylation facilitates TBP binding to a nucleosomal binding site and that Nhp6 stimulates formation of a TBP-TFIIA-DNA complex . Consistent with the idea that Nhp6, Gcn5, and Swi/Snf have overlapping functions in vivo, nhp6a nhp6b gcn5 mutants had a severe growth defect, and mutations in both nhp6a nhp6b swi2 and gcn5 swi2 strains were lethal.

Mol Cell Biol, 2004 Sep, 24(18), 8255 - 63
Pak1 protein kinase regulates activation and nuclear localization of Snf1-Gal83 protein kinase; Hedbacker K et al.; Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae . This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase . We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the beta-subunit isoforms Gal83, Sip1, and Sip2 . Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2 . These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes . We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress . The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Delta mutant and depends on Pak1 only when Snf1 is present . Snf1-Gal83 is the only form of the kinase that localizes to the nucleus . These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

Mol Cell Biol, 2004 Sep, 24(18), 8104 - 12
Global role of TATA box-binding protein recruitment to promoters in mediating gene expression profiles; Kim J et al.; The recruitment of TATA box-binding protein (TBP) to promoters is one of the rate-limiting steps during transcription initiation . However, the global importance of TBP recruitment in determining the absolute and changing levels of transcription across the genome is not known . We used a genomic approach to explore the relationship between TBP recruitment to promoters and global gene expression profiles in Saccharomyces cerevisiae . Our data indicate that first, RNA polymerase III promoters are the most prominent binding targets of TBP in vivo . Second, the steady-state transcript levels of genes throughout the genome are proportional to the occupancy of their promoters by TBP, and changes in the expression levels of these genes are closely correlated with changes in TBP recruitment to their promoters . Third, a consensus TATA element does not appear to be a major determinant of either TBP binding or gene expression throughout the genome . Our results indicate that the recruitment of TBP to promoters in vivo is of universal importance in determining gene expression levels in yeast, regardless of the nature of the core promoter or the type of activator or repressor that may mediate changes in transcription . The primary data reported here are available at http://www.iyerlab.org/tbp.

Mol Cell Biol, 2004 Sep, 24(18), 8069 - 79
Sgt1 associates with Hsp90: an initial step of assembly of the core kinetochore complex; Bansal PK et al.; The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division . In budding yeast, Sgt1, together with Skp1, is required for assembly of the core kinetochore complex (CBF3) via Ctf13 activation . Formation of the active Ctf13-Skp1 complex also requires Hsp90, a molecular chaperone . We have found that Sgt1 interacts with Hsp90 in yeast . We also have determined that Skp1 and Hsc82 (a yeast Hsp90 protein) bind to the N-terminal region of Sgt1 that contains tetratricopeptide repeat motifs . Results of sequence and phenotypic analyses of sgt1 mutants strongly suggest that the N-terminal region containing the Hsc82-binding and Skp1-binding domains of Sgt1 is important for the kinetochore function of Sgt1 . We found that Hsp90's binding to Sgt1 stimulates the binding of Sgt1 to Skp1 and that Sgt1 and Hsp90 stimulate the binding of Skp1 to Ctf13, the F-box core kinetochore protein . Our results strongly suggest that Sgt1 and Hsp90 function in assembling CBF3 by activating Skp1 and Ctf13.

Mol Cell Biol, 2004 Sep, 24(18), 7976 - 86
The small nucle(ol)ar RNA cap trimethyltransferase is required for ribosome synthesis and intact nucleolar morphology; Colau G et al.; Nucleolar morphogenesis is a poorly defined process . Here we report that the Saccharomyces cerevisiae nucleolar trimethyl guanosine synthase I (Tgs1p), which specifically selects the m(7)G cap structure of snRNAs and snoRNAs for m(2,2,7)G conversion, is required not only for efficient pre-mRNA splicing but also for pre-rRNA processing and small ribosomal subunit synthesis . Mutational analysis indicates that the requirement for Tgs1p in pre-mRNA splicing, but not its involvement in ribosome synthesis, is dependent upon its function in cap trimethylation . In addition, we report that cells lacking Tgs1p showed a striking and unexpected loss of nucleolar structural organization . Tgs1p is not a core component of the snoRNP proteins; however, in vitro, the protein interacts with the KKD/E domain present at the carboxyl-terminal ends of several snoRNP proteins . Strains expressing versions of the snoRNPs lacking the KKD/E domain were also defective for nucleolar morphology and showed a loss of nucleolar compaction . We propose that the transient and functional interactions of Tgs1p with the abundant snoRNPs, through presumed interactions with the KKD/E domain of the snoRNP proteins, contribute substantially to the coalescence of nucleolar components . This conclusion is compatible with a model of self-organization for nucleolar assembly.

Cell, 2004 Sep 3, 118(5), 529 - 30
Centromere glue provides spindle cue; Deehan R et al.; During cell division, accurate distribution of the genome by the mitotic spindle requires that sister chromatids remain tethered until the right moment . Studies of an uncharacterized vertebrate protein, Sgo (Salic et al., 2004 {this issue of Cell}), reveal dual roles as a chromosome cohesion factor and a regulator of spindle microtubule dynamics.

J Am Chem Soc, 2004 Sep 8, 126(35), 11088 - 92
Magnetic field effects on cytochrome c-mediated bioelectrocatalytic transformations; Katz E et al.; Constant magnetic fields affect many biological transformations, but we lack mechanistic understanding of the processes . The magnetohydrodynamic effect may account for the enhancement of bioelectrocatalytic transformations at interfaces . This is exemplified by the bioelectrocatalyzed cytochrome c-mediated reduction of oxygen and oxidation of lactate in the presence of cytochrome oxidase and lactate dehydrogenase, respectively . We observe significant magnetic field effects on the rates of bioelectrochemical transformations (ca . 3-fold increase) at the functionalized interfaces at field strengths, B, up to 1 T . We show that the limiting current is proportional to the B(1/3)C*(4/3), where C is the concentration of electroactive species . The results may have important implications on the understanding of the magnetic field effects on natural biocatalytic processes at membranes and on the enhancement of biotransformations in biotechnology.

J Biol Chem, 2004 Oct 29, 279(44), 45693 - 700 Epub 2004 Aug 26.
The GTP-binding release factor eRF3 as a key mediator coupling translation termination to mRNA decay; Kobayashi T et al.; GTP is essential for eukaryotic translation termination, where the release factor 3 (eRF3) complexed with eRF1 is involved as the guanine nucleotide-binding protein . In addition, eRF3 regulates the termination-coupled events, eRF3 interacts with poly(A)-binding protein (Pab1) and the surveillance factor Upf1 to mediate normal and nonsense-mediated mRNA decay . However, the roles of GTP binding to eRF3 in these processes remain largely unknown . Here, we showed in yeast that GTP is essentially required for the association of eRF3 with eRF1, but not with Pab1 and Upf1 . A mutation in the GTP-binding motifs of eRF3 impairs the eRF1-binding ability without altering the Pab1- or Upf1-binding activity . Interestingly, the mutation causes not only a defect in translation termination but also delay of normal and nonsense-mediated mRNA decay, suggesting that GTP/eRF3-dependent termination exerts its influence on the subsequent mRNA degradation . The termination reaction itself is not sufficient, but eRF3 is essential for triggering mRNA decay . Thus, eRF3 is a key mediator that transduces termination signal to mRNA decay.

J Biol Chem, 2004 Oct 29, 279(44), 45701 - 7 Epub 2004 Aug 27.
Mitochondrial protein import . Requirement of presequence elements and tom components for precursor binding to the TOM complex; Esaki M et al.; Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex . The TOM complex possesses two presequence binding sites on the cytosolic side (the cis site) and on the intermembrane space side (the trans site) . Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to the cis and trans sites of the TOM complex . The N-terminal 14 residues of the presequence of subunit 9 of F(0)-ATPase are required for binding to the trans site . The interaction between the presequence and the cis site is not sufficient to anchor the precursor protein to the TOM complex . Tom7 constitutes or is close to the trans site and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import.

Oncogene, 2004 Oct 14, 23(47), 7761 - 71
DAL-1/4.1B tumor suppressor interacts with protein arginine N-methyltransferase 3 (PRMT3) and inhibits its ability to methylate substrates in vitro and in vivo; Singh V et al.; DAL-1 (differentially expressed in adenocarcinoma of the lung)/4.1B is a tumor suppressor gene on human chromosome 18p11.3 whose expression is lost in >50% of primary non-small-cell lung carcinomas . Based on sequence similarity, DAL-1/4.1B has been assigned to the Protein 4.1 superfamily whose members interact with plasma membrane proteins through their N-terminal FERM (4.1/Ezrin/Radixin/Moesin) domain, and cytoskeletal components via their C-terminal SAB (spectrin-actin binding) region . Using the DAL-1/4.1B FERM domain as bait for yeast two-hybrid interaction cloning, we identified protein arginine N-methyltransferase 3 (PRMT3) as a specific DAL-1/4.1B-interacting protein . PRMT3 catalyses the post-translational transfer of methyl groups from S-adenosyl-L-methionine to arginine residues of proteins . Coimmunoprecipitation experiments using lung and breast cancer cell lines confirmed this interaction in mammalian cells in vivo . In vitro binding assays demonstrated that this was an interaction occurring via the C-terminal catalytic core domain of PRMT3 . DAL-1/4.1B was determined not to be a substrate for PRMT3-mediated methylation but its presence inhibits the in vitro methylation of a glycine-rich and arginine-rich methyl-accepting protein, GST (glutathione-S-transferase-GAR (glycine- and arginine-rich), which contains 14 'RGG' consensus methylation sites . In addition, induced expression of DAL-1/4.1B in MCF-7 breast cancer cells showed that the DAL-1/4.1B protein significantly inhibits PRMT3 methylation of cellular substrates . These findings suggest that modulation of post-translational methylation may be an important mechanism through which DAL-1/4.1B affects tumor cell growth.

Acta Crystallogr D Biol Crystallogr, 2004 Sep, 60(Pt 9), 1622 - 4 Epub 2004 Aug 26.
Crystallization and preliminary X-ray analysis of carboxypeptidase Y inhibitor IC complexed with the cognate proteinase; Mima J et al.; Carboxypeptidase Y (CPY) inhibitor I(C) is a naturally occurring serine carboxypeptidase inhibitor from Saccharomyces cerevisiae, the sequence of which is not homologous with any other known proteinase inhibitor and is classified as the phosphatidylethanolamine-binding protein (PEBP) . I(C) has been crystallized in complex with the deglycosylated form of CPY by the hanging-drop vapour-diffusion technique with ammonium sulfate as a precipitant . The crystals of the complex belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 81.13, b = 186.6, c = 65.14 A . Diffraction data were collected to 2.7 A resolution . Structure determination of the complex is in progress by the molecular-replacement method using the structure of CPY as a search model.

Plant Physiol, 2004 Sep, 136(1), 2665 - 75 Epub 2004 Aug 27.
Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid; Mietkiewska E et al.; The fatty acid elongase {often designated FAE or beta-(or 3-) ketoacyl-CoA synthase} is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus) . Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases . This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids . A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns . Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo . Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family . To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco {Nicotiana tabacum}) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively . Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids . These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates . Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.

Nucleic Acids Res, 2004 Aug 27, 32(15), 4665 - 75 Print 2004.
Enzymatic switching for efficient and accurate translesion DNA replication; McCulloch SD et al.; When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) delta or epsilon, a switch occurs to allow translesion synthesis by DNA polymerase eta, followed by another switch that allows normal replication to resume . In the present study, we investigate these switches using Saccharomyces cerevisiae Pol delta, Pol epsilon and Pol eta and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis-syn thymine-thymine (TT) dimer . We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol eta and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.

J Biol Chem, 2004 Nov 12, 279(46), 47840 - 8 Epub 2004 Aug 28.
Insights into the role of an active site aspartate in Ty1 reverse transcriptase polymerization; Pandey M et al.; Long terminal repeat-containing retrotransposons encode reverse transcriptases (RTs) that replicate their RNA into integratable, double-stranded DNA . A mutant version of the RT from Saccharomyces cerevisiae retrotransposon Ty1, in which one of the three active site aspartates has been changed to asparagine (D211N), is still capable of in vitro polymerization, although it is blocked for in vivo transposition . We generated recombinant WT and D211N Ty1 RTs to study RT function and determine specific roles for the Asp(211) residue . Presteady-state kinetic analysis of the two enzymes shows that the D211N mutation has minimal effect on nucleotide binding but reduces the k(pol) by approximately 230-fold . The mutation reduces binding affinity for both Mn(2+) and Mg(2+), indicating that the Asp(211) side chain helps create a tight metal binding pocket . Although both enzymes are highly processive and tend to remain bound to their initial substrate, each shows distinctive patterns of pausing, attributable to interactions between metal ions and the active site residue . These results provide insights to specific roles for the Asp(211) residue during polymerization and indicate unusual enzymatic properties that bear on the Ty1 replication pathway.

J Virol, 2004 Sep, 78(18), 10045 - 53
Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin; Boshuizen JA et al.; Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children . Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection . To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used . NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7 . An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4 . Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization . After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids {aa} 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884) . Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145 . Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4 . Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation . NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction . In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4 . The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established .

J Virol, 2004 Sep, 78(18), 9947 - 53
The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo; Hedengren-Olcott M et al.; The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown . To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor . The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET . This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present . Likewise, the titer of infectious virus progeny decreased by 98.9% +/- 0.97% when the vvtetOG1L virus was propagated in the absence of TET . Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR . Modeling of G1L revealed a predicted structural similarity to the alpha-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (alpha-MPP) . The HXXEH motif of G1L perfectly overlaps the HXXDR motif of alpha-MPP in this model . These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to alpha-MPP . However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis .

J Virol, 2004 Sep, 78(18), 9731 - 9
Two African swine fever virus proteins derived from a common precursor exhibit different nucleocytoplasmic transport activities; Eulalio A et al.; African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs . The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor . Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells . The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm . The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor . In addition, p37 was shown to exhibit nuclear import activity in mammalian cells . The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome . Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities . These findings are significant for understanding the role these proteins play in the replication cycle of ASFV .

Nature, 2004 Sep 9, 431(7005), 221 - 5 Epub 2004 Aug 25.
Structure of the ESCRT-II endosomal trafficking complex; Hierro A et al.; The multivesicular-body (MVB) pathway delivers transmembrane proteins and lipids to the lumen of the endosome . The multivesicular-body sorting pathway has crucial roles in growth-factor-receptor downregulation, developmental signalling, regulation of the immune response and the budding of certain enveloped viruses such as human immunodeficiency virus . Ubiquitination is a signal for sorting into the MVB pathway, which also requires the functions of three protein complexes, termed ESCRT-I, -II and -III (endosomal sorting complex required for transport) . Here we report the crystal structure of the core of the yeast ESCRT-II complex, which contains one molecule of the Vps protein Vps22, the carboxy-terminal domain of Vps36 and two molecules of Vps25, and has the shape of a capital letter 'Y' . The amino-terminal coiled coil of Vps22 and the flexible linker leading to the ubiquitin-binding NZF domain of Vps36 both protrude from the tip of one branch of the 'Y' . Vps22 and Vps36 form nearly equivalent interactions with the two Vps25 molecules at the centre of the 'Y' . The structure suggests how ubiquitinated cargo could be passed between ESCRT components of the MVB pathway through the sequential transfer of ubiquitinated cargo from one complex to the next.

J Biol Chem, 2004 Oct 29, 279(44), 46286 - 94 Epub 2004 Aug 23.
ASH1 mRNA anchoring requires reorganization of the Myo4p-She3p-She2p transport complex; Gonsalvez GB et al.; One mechanism by which cells post-transcriptionally regulate gene expression is via intercellular and intracellular sorting of mRNA . In Saccharomyces cerevisiae, the localization of ASH1 mRNA to the distal tip of budding cells results in the asymmetric sorting of Ash1p to daughter cell nuclei . Efficient localization of ASH1 mRNA depends upon the activity of four cis-acting localization elements and also upon the activity of trans-factors She2p, She3p, and Myo4p . She2p, She3p, and Myo4p have been proposed to form an ASH1 mRNA localization particle . She2p directly and specifically binds each of the four ASH1 cis-acting localization elements, whereas She3p has been hypothesized to function as an adaptor by recruiting the She2p-mRNA complex to Myo4p, a type V myosin . The Myo4p-She3p-She2p heterotrimeric protein complex has been proposed to localize mRNA to daughter cells using polarized actin cables . Here we demonstrate that whereas the predicted Myo4p-She3p-She2p heterotrimeric complex forms in vivo, it represents a relatively minor species compared with the Myo4p-She3p complex . Furthermore, contrary to a prediction of the heterotrimeric complex model for ASH1 mRNA localization, ASH1 mRNA artificially tethered to She2p is not localized . Upon closer examination, we found that mRNA tightly associated with She2p is transported to daughter cells but is not properly anchored at the bud tip . These results are consistent with a model whereby anchoring of ASH1 mRNA requires molecular remodeling of the Myo4p-She3p-She2p heterotrimeric complex, a process that is apparently altered when mRNA is artificially tethered to She2p.

J Biol Chem, 2004 Nov 5, 279(45), 47139 - 47 Epub 2004 Aug 24.
Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly; Merkley N et al.; E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation . While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation . Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae . The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins . This domain is physically separated from its C-terminal domain by a 22-residue flexible tether . The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA) . NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin . This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.

FEBS Lett, 2004 Aug 27, 573(1-3), 45 - 50
Identification and characterization of a novel delta12-fatty acid desaturase gene from Rhizopus arrhizus; Wei D et al.; Based on the sequence information of Delta12-fatty acid desaturase genes (from Mucor circinelloides, Mortierella alpina, Mucor rouxii and Aspergillus nidulans), which were involved in the conversion from C18:1 to C18:2, a cDNA sequence putatively encoding a Delta12-fatty acid desaturase was isolated from Rhizopus arrhizus using the combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods . Sequence analysis indicated that it had an open reading frame (ORF) of 1170 bp, coding for 389 amino acid residues of 45 kDa, pI of the deduced protein was 7.01 . The deduced amino acid sequence of this cloned cDNA showed high identity to those filamentous fungal Delta12-desaturases mentioned above, including three conserved histidine-rich motifs and two hydrophobic domains . Functional identification was done heterologously in Saccharomyces cerevisiae strain INVScl . The result demonstrated that the deduced amino acid sequence exhibited Delta12-fatty acid desaturase activity, suggesting that this gene encoded for a membrane-bound desaturase, Delta12-fatty acid desaturase.

Cancer Lett, 2004 Sep 30, 213(2), 223 - 30
PEP8-TAT2, a membrane-permeable peptide, inhibits cyclin-Cdk2 activity in HeLa cells; Chun KH et al.; Here we show that the PEP8-TAT2 peptide is effectively transduced into HeLa cells and that it inhibits cellular cyclin-Cdk2 activity . Like the PEP8 peptide, the PEP8-TAT2 peptide inhibits Cdk2 activity in vitro with an IC50 value of 5 nM, as determined by an immuno-complex kinase assay . It also inhibits DNA synthesis in and proliferation of cultured HeLa cells by arresting cell cycle at the G1/S transition . Further, the PEP8-TAT2 peptide inhibits cell death-associated Cdk2 activity and thereby prevents apoptotic progression in paclitaxel-treated cells . We propose that this inhibitor peptide is an effective agent to suppress the proliferation of human cancer cells, as well as apoptotic progression, by blocking cellular cyclin-dependent Cdk kinase activity.

J Cell Biol, 2004 Aug 30, 166(5), 717 - 29 Epub 2004 Aug 23.
Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes; Luhtala N et al.; Ubiquitination directs the sorting of cell surface receptors and other integral membrane proteins into the multivesicular body (MVB) pathway . Cargo proteins are subsequently deubiquitinated before their enclosure within MVB vesicles . In Saccharomyces cerevisiae, Bro1 functions at a late step of MVB sorting and is required for cargo protein deubiquitination . We show that the loss of Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the removal of ubiquitin from MVB cargoes . Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes . We further demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes requires Bro1 . Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination by Doa4 in the MVB pathway.

J Cell Biol, 2004 Aug 30, 166(5), 621 - 7 Epub 2004 Aug 23.
Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly; Ishikawa D et al.; Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes . Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex . Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane . Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.

J Biol Chem, 2004 Nov 5, 279(45), 46527 - 35 Epub 2004 Aug 23.
Tor signaling and nutrient-based signals converge on Mks1p phosphorylation to regulate expression of Rtg1.Rtg3p-dependent target genes; Dilova I et al.; The heterodimeric bZip/HLH transcription factors Rtg1p and Rtg3p regulate the expression of a concise set of metabolic genes (termed RTG target genes) required for de novo biosynthesis of glutamate and glutamine . Several components have now been identified that control both the intracellular localization as well as activity of the Rtg1p.Rtg3p complex, yet the precise upstream regulatory signals involved remain unclear . For example, it has been proposed that Rtg1p.Rtg3p activity is repressed by glutamate, acting through the mitochondrial retrograde response pathway or, alternatively, by glutamine, acting through the Tor kinase pathway . Here we demonstrate that RTG target gene regulation is remarkably complex, with glutamate and glutamine as well as ammonia collaborating as potentially distinct signals to regulate RTG target gene expression . We show that both Tor and these nutrient-based signals converge on Mks1p, the immediate upstream inhibitor of Rtg1p.Rtg3p, and that a direct correlation exists between the degree of Mks1p phosphorylation and the extent of RTG target gene repression . Finally, we find that Tor- and glutamine-mediated RTG-target gene repression can be experimentally uncoupled, indicating that glutamine and Tor act, at least in part, independently to inhibit this pathway.

Cancer Cell, 2004 Aug, 6(2), 139 - 50
Dominant effects of an Msh6 missense mutation on DNA repair and cancer susceptibility; Yang G et al.; Mutations in DNA mismatch repair (MMR) genes cause hereditary nonpolyposis colorectal cancer (HNPCC), and MMR defects are associated with a significant proportion of sporadic cancers . MMR maintains genome stability and suppresses tumor formation by preventing the accumulation of mutations and by mediating an apoptotic response to DNA damage . We describe the analysis of a dominant MSH6 missense mutation in yeast and mice that causes loss of DNA repair function while having no effect on the apoptotic response to DNA damaging agents . Our results demonstrate that MSH6 missense mutations can effectively separate the two functions, and that increased mutation rates associated with the loss of DNA repair are sufficient to drive tumorigenesis in MMR-defective tumors.

BMC Bioinformatics . 2004 Aug 23;5(1):114.
Influence of microarrays experiments missing values on the stability of gene groups by hierarchical clustering; de Brevern AG et al.; BACKGROUND: Microarray technologies produced large amount of data . The hierarchical clustering is commonly used to identify clusters of co-expressed genes . However, microarray datasets often contain missing values (MVs) representing a major drawback for the use of the clustering methods . Usually the MVs are not treated, or replaced by zero or estimated by the k-Nearest Neighbor (kNN) approach . The topic of the paper is to study the stability of gene clusters, defined by various hierarchical clustering algorithms, of microarrays experiments including or not MVs . RESULTS: In this study, we show that the MVs have important effects on the stability of the gene clusters . Moreover, the magnitude of the gene misallocations is depending on the aggregation algorithm . The most appropriate aggregation methods (e.g . complete-linkage and Ward) are highly sensitive to MVs, and surprisingly, for a very tiny proportion of MVs (e.g . 1%) . In most of the case, the MVs must be replaced by expected values . The MVs replacement by the kNN approach clearly improves the identification of co-expressed gene clusters . Nevertheless, we observe that kNN approach is less suitable for the extreme values of gene expression . CONCLUSION: The presence of MVs (even at a low rate) is a major factor of gene cluster instability . In addition, the impact depends on the hierarchical clustering algorithm used . Some methods should be used carefully . Nevertheless, the kNN approach constitutes one efficient method for restoring the missing expression gene values, with a low error level . Our study highlights the need of statistical treatments in microarray data to avoid misinterpretation.

J Biol Chem, 2004 Nov 5, 279(45), 47391 - 401 Epub 2004 Aug 20.
Localized feedback phosphorylation of Ste5p scaffold by associated MAPK cascade; Flotho A et al.; Scaffold proteins play pivotal roles during signal transduction . In Saccharomyces cerevisiae, the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane . To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex . Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation . During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p . During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade . Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most . Pheromone induction or overexpression of the Ste4p Gbeta subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present . Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex . Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation . These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth . These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation.

J Biol Chem, 2004 Nov 5, 279(45), 47222 - 32 Epub 2004 Aug 20.
A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster; Seeds AM et al.; Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6) and PP-IP5 . Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster . Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators . We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4 . To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies . Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6 . Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either alpha or beta isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools . Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis . IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2 . Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers.

Plant Cell, 2004 Sep, 16(9), 2529 - 40 Epub 2004 Aug 19.
Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes; Favery B et al.; Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells . We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation . Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments . We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed . Suppression of the budding defect of the Saccharomyces cerevisiae bni1Delta bnr1Delta mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables . Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton . The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis . Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.

J Biol Chem, 2004 Oct 22, 279(43), 44656 - 66 Epub 2004 Aug 19.
Cdc123 and checkpoint forkhead associated with RING proteins control the cell cycle by controlling eIF2gamma abundance; Bieganowski P et al.; Eukaryotic initiation factor 2 (eIF2) is a central regulator of translational initiation in times of growth and times of stress . Here we discovered three new conserved regulators of eIF2 in Saccharomyces cerevisiae . cdc123, homolog of mammalian D123, is a new cell division cycle mutant with a G2 delay at permissive temperature and a terminal, mating-proficient G1 arrest point . Cdc123 protein is regulated by nutrient availability . CHF1 and CHF2, homologs of mammalian checkpoint forkhead associated with RING genes, are required for G2 delay and G1 arrest of cdc123-4 and promote G1 delay when over-expressed . Cell cycle delaying activity and the natural instability of Chf1 and Chf2 depend on the integrity of both domains and association with Cdc123 . Genetic analysis maps the Chf1 forkhead associated domain-binding site to the conserved Thr-274 of Cdc123, suggesting that mammalian D123 is a key target of Chfr . Gcd11, the gamma subunit of eIF2, is an additional Cdc123-interacting protein that is an essential target of the Cdc123 cell cycle promoting and Chf cell cycle arresting activity whose abundance is regulated by Cdc123, Chf1, and Chf2 . Loss of cdc123 activity promotes Chf1 and Chf2 accumulation and Gcd11 depletion, accounting for the essentiality of Cdc123 . The data establish the Cdc123-Chf-Gcd11 axis as an essential pathway for nutritional control of START that runs parallel to the Tor-Gcn2-Sui2 system of translational control.

Nature, 2004 Aug 19, 430(7002), 917 - 21
Structural basis for redox regulation of Yap1 transcription factor localization; Wood MJ et al.; The ability of organisms to alter their gene expression patterns in response to environmental changes is essential for viability . A central regulator of the response to oxidative stress in Saccharomyces cerevisiae is the Yap1 transcription factor . Upon activation by increased levels of reactive oxygen species, Yap1 rapidly redistributes to the nucleus where it regulates the expression of up to 70 genes . Here we identify a redox-regulated domain of Yap1 and determine its high-resolution solution structure . In the active oxidized form, a nuclear export signal (NES) in the carboxy-terminal cysteine-rich domain is masked by disulphide-bond-mediated interactions with a conserved amino-terminal alpha-helix . Point mutations that weaken the hydrophobic interactions between the N-terminal alpha-helix and the C-terminal NES-containing domain abolished redox-regulated changes in subcellular localization of Yap1 . Upon reduction of the disulphide bonds, Yap1 undergoes a change to an unstructured conformation that exposes the NES and allows redistribution to the cytoplasm . These results reveal the structural basis of redox-dependent Yap1 localization and provide a previously unknown mechanism of transcription factor regulation by reversible intramolecular disulphide bond formation.

J Biol Chem, 2004 Oct 22, 279(43), 44311 - 9 Epub 2004 Aug 17.
Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas; Koybasi S et al.; In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/mass spectroscopy . Interestingly, only one specific ceramide, C(18:0)-ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues . On the other hand, in nonsquamous tumor tissues, this selectivity for C18-ceramide was not detected . These data suggested the hypotheses that decreased levels of C18-ceramide might impart a growth advantage to HNSCC cells and that increased generation of C18-ceramide may be involved in the inhibition of growth . These roles were examined by reconstitution of C18-ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C18-ceramide . Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/mUOG1 resulted in increased levels of only C(18:0)-ceramide by approximately 2-fold, i.e . concentrations similar to those of normal head and neck tissues . Importantly, increased generation of C18-ceramide by mLAG1/mUOG1 inhibited cell growth (approximately 70-80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction . In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C18-ceramide in the regulation of growth of HNSCC.

Plant Mol Biol, 2004 Mar, 54(4), 583 - 96
Identification and characterization of several new members of the ZIP family of metal ion transporters in Medicago truncatula; Lopez-Millan AF et al.; To broaden our understanding of micronutrient metal transport in plants, we have identified cDNAs for six new metal transporters in the model legume Medicago truncatula . All of the predicted proteins have high similarity to the ZIP protein family, and have been designated MtZIP1, MtZIP3, MtZIP4, MtZIP5, MtZIP6, and MtZIP7 . The six predicted proteins ranged from 350 to 372 amino acids in length; sequence analysis revealed that all proteins contained eight transmembrane domains and the highly conserved ZIP signature motif . Most of the proteins also exhibited a histidine-rich region in the variable sequence between transmembrane domains III and IV . When MtZIPs were transformed into appropriate metal-uptake defective yeast mutants and grown on metal-limited media, MtZIP1, MtZIP5, and MtZIP6 proteins restored yeast growth on Zn-limited media, MtZIP4 and MtZIP7 proteins restored yeast growth on Mn-limited media, and MtZIP3, MtZIP5, and MtZIP6 proteins restored yeast growth on Fe-limited media . Therefore, we conclude that these proteins function as metal transporters in Medicago truncatula . The expression pattern for each gene was studied by semi-quantitative RT-PCR in roots and leaves from plants grown under various metal supplies . MtZIP1 transcripts were only detected in Zn-deficient roots and leaves . MtZIP3 and MtZIP4 expression was down regulated in leaves from Mn- and Fe-deficient plants and appeared to be upregulated under Zn-deficient conditions in both roots and leaves . MtZIP5 was upregulated in leaves under Zn and Mn deficiency . The expression of MtZIP6 and MtZIP7 was unaffected by the metal supply, at least in root and leaf tissues . Characterizing these proteins in a single organism will allow us to understand the interplay between various ZIP genes, and the role they play in the regulation/execution of plant metal homeostasis.

Plant Cell, 2004 Sep, 16(9), 2380 - 93 Epub 2004 Aug 17.
DNA replication licensing affects cell proliferation or endoreplication in a cell type-specific manner; Castellano Mdel M et al.; In eukaryotic cells, the function of DNA replication licensing components (Cdc6 and Cdt1, among others) is crucial for cell proliferation and genome stability . However, little is known about their role in whole organisms and whether licensing control interfaces with differentiation and developmental programs . Here, we study Arabidopsis thaliana CDT1, its regulation, and the consequences of overriding licensing control . The availability of AtCDT1 is strictly regulated at two levels: (1) at the transcription level, by E2F and growth-arresting signals, and (2) posttranscriptionally, by CDK phosphorylation, a step that is required for its proteasome-mediated degradation . We also show that CDC6 and CDT1 are key targets for the coordination of cell proliferation, differentiation, and development . Indeed, altered CDT1 or CDC6 levels have cell type-specific effects in developing Arabidopsis plants: in leaf cells competent to divide, cell proliferation is stimulated, whereas in cells programmed to undergo differentiation-associated endoreplication rounds, extra endocycles are triggered . Thus, we propose that DNA replication licensing control is critical for the proper maintenance of proliferative potential, developmental programs, and morphogenetic patterns.

J Biol Chem, 2004 Nov 5, 279(45), 46448 - 54 Epub 2004 Aug 16.
A biophysical analysis of the tetratricopeptide repeat-rich mitochondrial import receptor, Tom70, reveals an elongated monomer that is inherently flexible, unstable, and unfolds via a multistate pathway; Beddoe T et al.; Proteins destined for all submitochondrial compartments are translocated across the outer mitochondrial membrane by the TOM (translocase of the outer membrane) complex, which consists of a number of specialized receptor subunits that bind mitochondrial precursor proteins for delivery into the translocation channel . One receptor, Tom70, binds large, hydrophobic mitochondrial precursors . The current model of Tom70-mediated import involves multiple dimers of the receptor recognizing a single molecule of substrate . Here we show via a battery of biophysical and spectroscopic techniques that the cytosolic domain of Tom70 is an elongated monomer . Thermal and urea-induced denaturation revealed that the receptor, which unfolds via a multistate pathway, is a relatively unstable molecule undergoing major conformational change at physiological temperatures . The data suggest that the malleability of the monomeric Tom70 receptor is an important factor in mitochondrial import.

J Biol Chem, 2004 Oct 22, 279(43), 45226 - 34 Epub 2004 Aug 16.
ATP-binding cassette (ABC) transporters mediate nonvesicular, raft-modulated sterol movement from the plasma membrane to the endoplasmic reticulum; Li Y et al.; Little is known about the mechanisms of intracellular sterol transport or how cells maintain the high sterol concentration of the plasma membrane (PM) . Here we demonstrate that two inducible ATP-binding cassette (ABC) transporters (Aus1p and Pdr11p) mediate nonvesicular movement of PM sterol to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae . This transport facilitates exogenous sterol uptake, which we find requires steryl ester synthesis in the ER . Surprisingly, while expression of Aus1p and Pdr11p significantly increases sterol movement from PM to ER, it does not alter intracellular sterol distribution . Thus, ER sterol is likely rapidly returned to the PM when it is not esterified in the ER . We show that the propensity of PM sterols to be moved to the ER is largely determined by their affinity for sterol sphingolipid-enriched microdomains (rafts) . Our findings suggest that raft association is a primary determinant of sterol accumulation in the PM and that Aus1p and Pdr11p facilitate sterol uptake by increasing the cycling of sterol between the PM and ER.

Gene, 2004 Sep 1, 338(2), 177 - 86
Identification of a domain within human TAF(I)48, a subunit of Selectivity Factor 1, that interacts with helix 2 of TBP; Xu S et al.; RNA polymerase I transcription in human cells requires Selectivity Factor 1, a multisubunit complex composed of the TATA-box-binding protein (TBP) and three TBP-associated factors (TAFs) called TAF(I)48, TAF(I)63 and TAF(I)110 . Each of the Selectivity Factor 1 subunits binds directly to the other three components, but these interactions have not been characterized . This study is the initial identification and analysis of a TBP-binding domain within a Selectivity Factor 1 TAF . The interaction between human TBP and human TAF(I)48 was initially examined using the yeast two-hybrid assay, and a TBP-binding domain was identified in the carboxyl-terminus of human (h)TAF(I)48 . Consistent with this result, the hTAF(I)48 carboxyl-terminus was able to bind directly to TBP in protein-protein interaction assays . When mutations were introduced into the hTAF(I)48 carboxyl-terminus, we identified changes in uncharged and positive residues that affect its interaction with TBP . By examining TBP mutants, residues within and adjacent to helix 2 of TBP, previously demonstrated to interact with subunits of other TBP-containing complexes {Transcription Factor IID (TFIID) and TFIIIB} were also found to diminish its affinity for the carboxyl-terminus of hTAF(I)48 . The regions of hTAF(I)48 and TBP that interact are compared to those identified within other complexes containing TBP.

Cell, 2004 Aug 20, 118(4), 404 - 6
Modifying histones and initiating meiotic recombination; new answers to an old question; Maleki S et al.; It is well documented that the formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination is influenced by chromatin and larger scale chromosome organization, but the molecular nature of this influence has remained elusive . Several recent studies, including (this issue of Cell), shed light on this issue by revealing roles for posttranslational histone modifications in promoting DSB formation.

PLoS Biol . 2004 Aug;2(8):E235 . Epub 2004 Aug 17.
IRE1-independent gain control of the unfolded protein response; Leber JH et al.; Nonconventional splicing of the gene encoding the Hac1p transcription activator regulates the unfolded protein response (UPR) in Saccharomyces cerevisiae . This simple on/off switch contrasts with a more complex circuitry in higher eukaryotes . Here we show that a heretofore unrecognized pathway operates in yeast to regulate the transcription of HAC1 . The resulting increase in Hac1p production, combined with the production or activation of a putative UPR modulatory factor, is necessary to qualitatively modify the cellular response in order to survive the inducing conditions . This parallel endoplasmic reticulum-to-nucleus signaling pathway thereby serves to modify the UPR-driven transcriptional program . The results suggest a surprising conservation among all eukaryotes of the ways by which the elements of the UPR signaling circuit are connected . We show that by adding an additional signaling element to the basic UPR circuit, a simple switch is transformed into a complex response.

Mol Cell Biol, 2004 Sep, 24(17), 7769 - 78
GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination; Salas-Marco J et al.; Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3 . eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain . eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism . In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae . We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others . As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals . To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter . We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants . We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals . Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release .

Mol Cell Biol, 2004 Sep, 24(17), 7695 - 706
Glucose repression of STA1 expression is mediated by the Nrg1 and Sfl1 repressors and the Srb8-11 complex; Kim TS et al.; In the yeast Saccharomyces diastaticus, expression of the STA1 gene, which encodes an extracellular glucoamylase, is negatively regulated by glucose . Here we demonstrate that glucose-dependent repression of STA1 expression is imposed by both Sfl1 and Nrg1, which serve as direct transcriptional repressors . We show that Nrg1 acts only on UAS1, and Sfl1 acts only on UAS2, in the STA1 promoter . When bound to its specific site, Sfl1 (but not Nrg1) prevents the binding to UAS2 of two transcriptional activators, Ste12 and Tec1, required for STA1 expression . We also found that Sfl1 contributes to STA1 repression by binding to the promoter and inhibiting the expression of FLO8, a gene that encodes a third transcriptional activator involved in STA1 expression . In addition, we show that the levels of Nrg1 and Sfl1 increase in glucose-grown cells, suggesting that the effects of glucose are mediated, at least in part, through an increase in the abundance of these repressors . NRG1 and SFL1 expression requires the Srb8-11 complex, and correspondingly, the Srb8-11 complex is also necessary for STA1 repression . However, our evidence indicates that the Srb8-11 complex does not associate with either the SFL1 or the NRG1 promoter and thus plays an indirect role in activating NRG1 and SFL1 expression .

Mol Cell Biol, 2004 Sep, 24(17), 7503 - 13
The N-terminal regulatory domain of Stp1p is modular and, fused to an artificial transcription factor, confers full Ssy1p-Ptr3p-Ssy5p sensor control; Andreasson C et al.; Stp1p and Stp2p are homologous and redundant transcription factors that are synthesized as latent cytoplasmic proteins with N-terminal regulatory domains . In response to extracellular amino acids, the plasma membrane-localized Ssy1p-Ptr3p-Ssy5p (SPS) sensor induces an endoproteolytic processing event that cleaves away the N-terminal regulatory domains . The shorter forms of Stp1p and Stp2p are targeted to the nucleus, where they bind and activate the transcription of amino acid permease genes . A novel genetic screen, specifically designed to search for rare mutations that affect the SPS-sensing pathway, identified the F-box protein Grr1p as an obligatory factor required for Stp1p/Stp2p processing . Additionally, we have found that a null mutation in the ASI1 (amino acid sensor-independent) gene enables full-length unprocessed Stp1p/Stp2p to enter the nucleus and derepress SPS sensor-dependent genes . The N-terminal domains of Stp1p/Stp2p contain two conserved motifs that are required for proper nuclear exclusion and proteolytic processing . These motifs function in parallel; mutations that abolish processing inhibit signaling, whereas mutations that interfere with cytoplasmic retention result in constitutive derepression of SPS sensor-regulated genes independently of processing . The N-terminal domain of Stp1p is functionally autonomous and transferable to other transcription factors, where its presence confers ASI1-dependent nuclear exclusion and SPS sensor-induced proteolytic processing .

Mol Cell Biol, 2004 Sep, 24(17), 7419 - 34
The B-subunit of DNA polymerase alpha-primase associates with the origin recognition complex for initiation of DNA replication; Uchiyama M et al.; The B-subunit (p70/Pol12p) of the DNA polymerase alpha-primase (Polalpha-primase) complex is thought to have a regulatory role in an early stage of S phase . We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches . Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58 . A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polalpha-primase . Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70 . Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event . Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polalpha-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit . Our results thus suggest a role for the recruited Polalpha-primase in the initiation of both leading and lagging strands at the replication origins .

Genes Dev, 2004 Aug 15, 18(16), 2024 - 35
Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors; Yu MC et al.; Hmt1 is the major type I arginine methyltransferase in the yeast Saccharomyces cerevisiae and facilitates the nucleocytoplasmic transport of mRNA-binding proteins through their methylation . Here we demonstrate that Hmt1 is recruited during the beginning of the transcriptional elongation process . Hmt1 methylates Yra1 and Hrp1, two mRNA-binding proteins important for mRNA processing and export . Moreover, loss of Hmt1 affects interactions between mRNA-binding proteins and Tho2, a component of the TREX (transcription/export) complex that is important for transcriptional elongation and recruitment of mRNA export factors . Furthermore, RNA in situ hybridization analysis demonstrates that loss of Hmt1 results in slowed release of HSP104 mRNA from the sites of transcription . Genome-wide location analysis shows that Hmt1 is bound to specific functional gene classes, many of which are also bound by Tho2 and other mRNA-processing factors . These data suggest a model whereby Hmt1 affects transcriptional elongation and, as a result, influences recruitment of RNA-processing factors.

Biochemistry, 2004 Aug 24, 43(33), 10628 - 41
Crystal structure of the actin binding domain of the cyclase-associated protein; Dodatko T et al.; Cyclase-associated protein (CAP or Srv2p) is a modular actin monomer binding protein that directly regulates filament dynamics and has been implicated in a number of complex developmental and morphological processes, including mRNA localization and the establishment of cell polarity . The crystal structure of the C-terminal dimerization and actin monomer binding domain (C-CAP) reveals a highly unusual dimer, composed of monomers possessing six coils of right-handed beta-helix flanked by antiparallel beta-strands . Domain swapping, involving the last two strands of each monomer, results in the formation of an extended dimer with an extensive interface . This structural and biochemical characterization provides new insights into the organization and potential mechanistic properties of the multiprotein assemblies that integrate dynamic actin processes into the overall physiology of the cell . An unanticipated finding is that the unique tertiary structure of the C-CAP monomer provides a structural model for a wide range of molecules, including RP2 and cofactor C, proteins involved in X-linked retinitis pigmentosa and tubulin maturation, respectively, as well as several uncharacterized proteins that exhibit very diverse domain organizations . Thus, the unusual right-handed beta-helical fold present in C-CAP appears to support a wide range of biological functions.

Nature, 2004 Sep 9, 431(7005), 184 - 7 Epub 2004 Aug 15.
Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits; True HL et al.; Phenotypic plasticity and the exposure of hidden genetic variation both affect the survival and evolution of new traits, but their contributing molecular mechanisms are largely unknown . A single factor, the yeast prion {PSI(+)}, may exert a profound effect on both . {PSI(+)} is a conserved, protein-based genetic element that is formed by a change in the conformation and function of the translation termination factor Sup35p, and is transmitted from mother to progeny . Curing cells of {PSI(+)} alters their survival in different growth conditions and produces a spectrum of phenotypes in different genetic backgrounds . Here we show, by examining three plausible explanations for this phenotypic diversity, that all traits tested involved {PSI(+)}-mediated read-through of nonsense codons . Notably, the phenotypes analysed were genetically complex, and genetic re-assortment frequently converted {PSI(+)}-dependent phenotypes to stable traits that persisted in the absence of {PSI(+)} . Thus, {PSI(+)} provides a temporary survival advantage under diverse conditions, increasing the likelihood that new traits will become fixed by subsequent genetic change . As an epigenetic mechanism that globally affects the relationship between genotype and phenotype, {PSI(+)} expands the conceptual framework for phenotypic plasticity, provides a one-step mechanism for the acquisition of complex traits and affords a route to the genetic assimilation of initially transient epigenetic traits.

J Exp Bot, 2004 Sep, 55(405), 2015 - 27 Epub 2004 Aug 13.
Identification of ASK and clock-associated proteins as molecular partners of LKP2 (LOV kelch protein 2) in Arabidopsis; Yasuhara M et al.; The ADO/FKF/LKP/ZTL family of proteins of Arabidopsis thaliana Heynh . have a LOV domain, an F-box motif, and a kelch repeat region . LKP2 is a member of this family and functions either within or very close to the circadian oscillator in Arabidopsis . Promoter-GUS fusion studies revealed that the LKP2 gene was highly active in rosette leaves . In CaMV 35S:LKP2-GFP plants, GFP-associated fluorescence was detected in nuclei, suggesting that LKP2 is a nuclear protein . Yeast two-hybrid analysis demonstrated that LKP2 interacted with some Arabidopsis Skp1-like proteins (ASK), as do other ADO/FKF/LKP/ZTL family proteins, suggesting that LKP2 can form an SCF (Skp1-Cullin-F-box protein) complex that functions as a ubiquitin E3 ligase . LKP2 interacted not only with itself but also with other members of the family, LKP1 and FKF1 . The two-hybrid analysis also demonstrated that LKP2 interacted with TOC1, a clock component, but not with CCA1 or LHY, negative regulators of TOC1 gene expression . The LOV domain of LKP2 was shown to be necessary and sufficient for the interaction with TOC1 . An interaction between LKP2 and APRR5, a paralogue of TOC1, was also observed, but LKP2 did not interact with APRR3, APRR7, or APRR9, other paralogues of TOC1.

Digestion, 2004, 70(1), 49 - 54 Epub 2004 Aug 11.
Inflammatory bowel disease-specific autoantibodies in HLA-B27-associated spondyloarthropathies: increased prevalence of ASCA and pANCA; Torok HP et al.; AIMS: An association between inflammatory bowel disease (IBD) and spondyloarthropathies (SpA) has repeatedly been reported . The aim of the present study was to investigate whether serologic markers of IBD, e.g . antibodies against Saccharomyces cerevisiae (ASCA), antibodies against exocrine pancreas (PAB) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) are present in HLA-B27-associated SpA . METHODS: 87 patients with HLA-B27-positive SpA and 145 controls were tested for ASCA, PAB and pANCA employing ELISA or indirect immunofluorescence, respectively . Antibody-positive patients were interviewed regarding IBD-related symptoms using a standardized questionnaire . RESULTS/CONCLUSION: When compared to the controls, ASCA IgA but not ASCA IgG levels were significantly increased in patients with SpA, in particular in ankylosing spondylitis (AS) and undifferentiated SpA (uSpA) . pANCA were found in increased frequency in patients with SpA whereas PAB were not detected . The existence of autoantibodies was not associated with gastrointestinal symptoms but sustains the presence of a pathophysiological link between bowel inflammation and SpA.

J Biol Chem, 2004 Nov 5, 279(45), 47242 - 53 Epub 2004 Aug 11.
Proteomics of endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes from brefeldin A-treated HepG2 cells identifies ERGIC-32, a new cycling protein that interacts with human Erv46; Breuza L et al.; Cycling proteins play important roles in the organization and function of the early secretory pathway by participating in membrane traffic and selective transport of cargo between the endoplasmic reticulum (ER), the intermediate compartment (ERGIC), and the Golgi . To identify new cycling proteins, we have developed a novel procedure for the purification of ERGIC membranes from HepG2 cells treated with brefeldin A, a drug known to accumulate cycling proteins in the ERGIC . Membranes enriched 110-fold over the homogenate for ERGIC-53 were obtained and analyzed by mass spectrometry . Major proteins corresponded to established and putative cargo receptors and components mediating protein maturation and membrane traffic . Among the uncharacterized proteins, a 32-kDa protein termed ERGIC-32 is a novel cycling membrane protein with sequence homology to Erv41p and Erv46p, two proteins enriched in COPII vesicles of yeast . ERGIC-32 localizes to the ERGIC and partially colocalizes with the human homologs of Erv41p and Erv46p, which mainly localize to the cis-Golgi . ERGIC-32 interacts with human Erv46 (hErv46) as revealed by covalent cross-linking and mistargeting experiments, and silencing of ERGIC-32 by small interfering RNAs increases the turnover of hErv46 . We propose that ERGIC-32 functions as a modulator of the hErv41-hErv46 complex by stabilizing hErv46 . Our novel approach for the isolation of the ERGIC from BFA-treated cells may ultimately lead to the identification of all proteins rapidly cycling early in the secretory pathway.

DNA Cell Biol, 2004 Aug, 23(8), 475 - 89
The centrosome in normal and transformed cells; Wang Q et al.; The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells . During cell division, the centrosomes form the poles of the bipolar mitotic spindle . In addition, the centrosomes are also needed for cytokinesis . Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication . Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle . However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner . Intriguingly, amplification of the centrosome has been found in numerous forms of cancers . Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis . It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability . Here we review the current understanding of how the centrosome is assembled and duplicated . We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.

Anal Chem, 2004 Aug 15, 76(16), 4741 - 6
Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis; Gunasekera N et al.; Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells . In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae . Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question . In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins . The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed . As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus . To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed . While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed . The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells.

Philos Trans R Soc Lond B Biol Sci, 2004 Jun 29, 359(1446), 857 - 71
Metabolic profiles to define the genome: can we hear the phenotypes?
Griffin JL.
There is an increased reliance on genetically modified organisms as a functional genomic tool to elucidate the role of genes and their protein products . Despite this, many models do not express the expected phenotype thought to be associated with the gene or protein . There is thus an increased need to further define the phenotype resultant from a genetic modification to understand how the transcriptional or proteomic network may conspire to alter the expected phenotype . This is best typified by the description of the silent phenotype in genetic manipulations of yeast . High-resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy provides an ideal mechanism for the profiling of metabolites within biofluids, tissue extracts or, with recent advances, intact tissues . These metabolic datasets can be readily mined using a range of pattern recognition techniques, including hierarchical cluster analysis, principal components analysis, partial least squares and neural networks, with the combined approach being termed metabolomics . This review describes the application of NMR-based metabolomics or metabonomics to genetic and chemical interventions in a number of different species, demonstrating the versatility of such an approach, as well as suggesting how it may be integrated with other "omic" technologies.

Mol Microbiol, 2004 Aug, 53(4), 1243 - 9
Post-translational regulation of phosphatidylglycerolphosphate synthase in response to inositol; He Q et al.; Phosphatidylglycerolphosphate synthase (Pgs1p) catalyses the committed step in the synthesis of cardiolipin (CL) . This is the only step of CL synthesis that is regulated by inositol . We have shown previously that Pgs1p enzyme activity is decreased within minutes after supplementation with inositol, but PGS1 expression is unaltered . We utilized an epitope-tagged Pgs1p to determine if the rapid decrease in activity following inositol was because of degradation or inactivation of the protein . In this report, we show that, in response to inositol, the decrease in CL content and Pgs1p enzyme activity are associated with increased phosphorylation of Pgs1p, but not with degradation or mislocalization of the protein . This is the first evidence of phosphorylation of a phospholipid biosynthetic enzyme in response to inositol and identifies a new mechanism of inositol-mediated regulation.

Nucleic Acids Res, 2004 Aug 10, 32(14), 4257 - 68 Print 2004.
The DNA damage checkpoint pathways exert multiple controls on the efficiency and outcome of the repair of a double-stranded DNA gap; Haghnazari E et al.; A DNA gap repair assay was used to determine the effect of mutations in the DNA damage checkpoint system on the efficiency and outcome (crossover/non-crossover) of recombinational DNA repair . In Saccharomyces cerevisiae gap repair is largely achieved by homologous recombination . As a result the plasmid either integrates into the chromosome (indicative of a crossover outcome) or remains extrachromosomal (indicative of a non-crossover outcome) . Deletion mutants of the MEC1 and RAD53 checkpoint kinase genes exhibited a 5-fold decrease in gap repair efficiency, showing that 80% of the gap repair events depended on functional DNA damage checkpoints . Epistasis analysis suggests that the DNA damage checkpoints affect gap repair by modulating Rad51 protein-mediated homologous recombination . While in wild-type cells only approximately 25% of the gap repair events were associated with a crossover outcome, Mec1-deficient cells exhibited a >80% crossover association . Also mutations in the effector kinases Rad53, Chk1 and Dun1 were found to affect crossover association of DNA gap repair to various degrees . The data suggest that the DNA damage checkpoints are important for the optimal functioning of recombinational DNA repair with multiple terminal targets to modulate the efficiency and outcome of homologous recombination.

FEBS Lett, 2004 Aug 13, 572(1-3), 159 - 66
Isolation and characterisation of DOCK8, a member of the DOCK180-related regulators of cell morphology; Ruusala A et al.; In a yeast two-hybrid system screen for Cdc42-interacting proteins, we identified a protein with similarity to the CrkII-binding protein DOCK180 . A cDNA clone of this protein, designated DOCK8, encoded a gene-product of 1701 amino acid residues with a molecular mass of 190 kDa . Immunofluorescence staining showed that transiently transfected HA-tagged DOCK8, as well as endogenous DOCK8, was present at the cell edges in areas undergoing lamellipodia formation . Transient transfection of a C-terminal fragment of DOCK8 resulted in the formation of vesicular structures . Interestingly, these vesicles also contained filamentous actin . These data suggest an involvement of DOCK8 in processes that affect the organisation of filamentous actin.

FEBS Lett, 2004 Aug 13, 572(1-3), 147 - 53
Close head-to-head juxtaposition of genes favors their coordinate regulation in Drosophila melanogaster; Herr DR et al.; This report identifies a large number of gene-pairs in Drosophila melanogaster that share a common upstream region . 877 gene-pairs (approximately 12% of the genome) are separated by less than 350 bp in a head-to-head orientation . This positional relationship is more highly favored in flies than in other organisms . These gene pairs have a higher correlation of expression than similarly spaced genes that have head-to-tail or tail-to-tail orientations . Thus, the positional arrangement of genes appears to play a significant role in coordinating relative expression patterns and may provide clues for identifying the functions of unknown genes.

Free Radic Biol Med, 2004 Sep 15, 37(6), 859 - 68
Proteasome synthesis and assembly are required for survival during stationary phase; Chen Q et al.; We examined the alterations in 20S proteasome homeostasis, protein oxidation, and cell viability that occur during the stationary phase or chronological model of yeast aging . Data in this report demonstrate that proteasome subunit expression is increased, proteasome composition is altered, and levels of individual proteasome proteolytic activities are elevated during stationary phase-induced aging in Saccharomyces cerevisiae . Despite such alterations, a progressive loss of proteasome-mediated protein degradation and a significant increase in protein oxidation were observed in cells maintained under stationary phase conditions . Deletion of UMP1, a gene necessary for 20S proteasome biogenesis, had no effect on cellular viability under normal growth conditions, but impaired the ability of cells to survive under stationary phase conditions . During stationary phase, the levels of oxidized protein increased more rapidly and to higher levels in the mutant lacking UMP1 than in the wild-type cells . Taken together, these data implicate a role for proteasome synthesis and altered 20S proteasome composition in maintaining viability during stationary phase, and demonstrate that even with these modifications a gradual loss of proteasome-mediated protein degradation occurs during stationary phase-induced aging . These data also suggest a role for impaired proteasome-mediated protein degradation in increased protein oxidation and cell death observed during the aging of eukaryotic cells.

Mol Cell, 2004 Aug 13, 15(3), 399 - 407
Structure and mechanism of RNA polymerase II CTD phosphatases; Kamenski T et al.; Recycling of RNA polymerase II (Pol II) after transcription requires dephosphorylation of the polymerase C-terminal domain (CTD) by the phosphatase Fcp1 . We report the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain . The structure shows a core fold and an active center similar to those of phosphotransferases and phosphohydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1 . We demonstrate that the first aspartate in the signature motif undergoes metal-assisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride . Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1 . Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for binding of Fcp1 to the polymerase in vitro.

BMC Bioinformatics . 2004 Aug 10;5(1):108.
Linear fuzzy gene network models obtained from microarray data by exhaustive search; Sokhansanj BA et al.; BACKGROUND: Recent technological advances in high-throughput data collection allow for experimental study of increasingly complex systems on the scale of the whole cellular genome and proteome . Gene network models are needed to interpret the resulting large and complex data sets . Rationally designed perturbations (e.g., gene knock-outs) can be used to iteratively refine hypothetical models, suggesting an approach for high-throughput biological system analysis . We introduce an approach to gene network modeling based on a scalable linear variant of fuzzy logic: a framework with greater resolution than Boolean logic models, but which, while still semi-quantitative, does not require the precise parameter measurement needed for chemical kinetics-based modeling . RESULTS: We demonstrated our approach with exhaustive search for fuzzy gene interaction models that best fit transcription measurements by microarray of twelve selected genes regulating the yeast cell cycle . Applying an efficient, universally applicable data normalization and fuzzification scheme, the search converged to a small number of models that individually predict experimental data within an error tolerance . Because only gene transcription levels are used to develop the models, they include both direct and indirect regulation of genes . CONCLUSION: Biological relationships in the best-fitting fuzzy gene network models successfully recover direct and indirect interactions predicted from previous knowledge to result in transcriptional correlation . Fuzzy models fit on one yeast cell cycle data set robustly predict another experimental data set for the same system . Linear fuzzy gene networks and exhaustive rule search are the first steps towards a framework for an integrated modeling and experiment approach to high-throughput "reverse engineering" of complex biological systems.

J Inherit Metab Dis, 2004, 27(4), 455 - 63
GTP-cyclohydrolase I gene mutations in patients with autosomal dominant and recessive GTP-CH1 deficiency: identification and functional characterization of four novel mutations; Garavaglia B et al.; GTP-cyclohydrolase I (GTP-CH1, EC 3.5.4.16) is encoded by the GCH1 gene . Mutations in the GCH1 gene cause both dopa-responsive dystonia (McKusick 128230) and recessive GTP-CH1 deficiency (McKusick 600225) . The exact molecular mechanism resulting in decreased GTP-CH1 activity in the patients is still obscure . We report the clinical features and molecular and functional study of the GCH1 gene in eight Italian patients affected by dominant and recessive GTP-CH1 deficiency . All the studied patients had mutations in the GCH1 gene . Three missense mutations (V205G, K224R, P199A), a frameshift mutation (Delta G693), and a splice-site mutation (ivs5 + 1g > c) were found . Except for K224R these are all novel mutations . To analyse the defect caused by the novel mutations, an in vivo functional assay in a Saccharomyces cerevisiae strain lacking the endogenous gene encoding GTP-CH1 ( FOL2 ) was performed . Complementation analysis showed that the Delta G693 and V205G mutations abolish the enzymatic function, while the P199A mutation causes a conditional defect . In conclusion, the clinical phenotypes displayed by our patients confirm the wide clinical spectrum of the disease and further support the lack of correlation between a given mutation and a clinical phenotype . Complementation analysis in yeast is a useful tool for confirming the pathogenetic effect of GCH1 mutations.

Eukaryot Cell, 2004 Aug, 3(4), 976 - 83
Direct physical and functional interaction of the NuA4 complex components Yaf9p and Swc4p; Bittner CB et al.; Saccharomyces cerevisiae Yaf9p and the mammalian leukemia-associated protein ENL share a high degree of similarity . To investigate the biological function of Yaf9p, this protein was used to search for interacting proteins in a two-hybrid system . Here, we demonstrate that Yaf9p binds directly to Swc4p, the yeast homolog of the mammalian DNA-methyltransferase-associated protein 1 . Yaf9p and Swc4p associate through C-terminal domains, and both proteins coprecipitate in vitro in pull-down experiments and in vivo by immunoprecipitation . In living cells, Swc4p is present in a megadalton protein complex that shows a fractionation behavior in gel filtration similar to that of Esa1p, the histone acetyltransferase of the NuA4 complex . Recruitment of Yaf9p to DNA leads to promoter-specific transcriptional activation that can be inhibited by dominant negative Swc4p lacking the Yaf9p binding domain . Interference with Swc4p function also increases sensitivity to the microtubule toxin benomyl, a trait that corresponds to the known phenotype of a yaf9(-) knockout strain . In summary, the results suggest that Yaf9p and Swc4p form a protein pair that has a role in chromatin modification with possible implications also for the function of their mammalian counterparts.

Eukaryot Cell, 2004 Aug, 3(4), 893 - 9
Role of a 300-kilodalton nuclear complex in the maturation of Trypanosoma brucei initiator methionyl-tRNA; Arhin GK et al.; tRNAs are transcribed as precursors containing 5' leader and 3' extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs . Here, we examined the maturation pathway of tRNA(Met) in Trypanosoma brucei, an early divergent unicellular eukaryote . We identified an approximately 300-kDa complex located in the nucleus of T . brucei that is required for trimming the 5' leader of initiator tRNA(Met) precursors . One of the subunits of the complex (T . brucei MT40 {TbMT40}) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs . Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNA(Met) containing 5' extensions but processed 3' ends . In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNA(Met) molecules with 5' and 3' extensions in TbMT40-silenced cells, albeit at a much lower level . Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNA(Met) . Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNA(Met), 3' trimming precedes 5' processing.

Exp Cell Res, 2004 Sep 10, 299(1), 199 - 208
Nuclear localization properties of a conserved protuberance in the Sm core complex; Girard C et al.; The nuclear import signal of snRNPs is composed of two essential components, the m(3)G cap structure of the snRNA and the Sm core NLS carried by the Sm protein core complex . We have previously proposed that, in yeast, this last determinant is represented by a basic-rich protuberance formed by the C-terminal extensions of Sm proteins . In mammals, as well as in other organisms, this component has not yet been precisely defined . Using GFP-Sm fusion constructs and immunolocalization as well as biochemical experiments, we show here that the C-terminal domains of human SmD1 and SmD3 proteins possess nuclear localization properties . Deletions of these domains increase cytoplasmic fluorescence and cytoplasmic localization of GFP-Sm mutant fusion alleles . Our results are consistent with a model in which the Sm core NLS is evolutionarily conserved and composed of a basic-rich protuberance formed by C-terminal domains of different Sm subtypes.

Proc Natl Acad Sci U S A, 2004 Aug 17, 101(33), 12248 - 53 Epub 2004 Aug 06.
Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining; Ninomiya Y et al.; Gene disruption and overexpression play central roles in the analysis of gene function . Homologous recombination is, in principle, the most efficient method of disrupting, modifying, or replacing a target gene . Although homologous integration of exogenous DNA into the genome occurs readily in Saccharomyces cerevisiae, it is rare in many other organisms . We identified and disrupted Neurospora crassa genes homologous to human KU70 and KU80, which encode proteins that function in nonhomologous end-joining of double-stranded DNA breaks . The resulting mutants, named mus-51 and mus-52, showed higher sensitivity to methyl methanesulfonate, ethyl methanesulfonate, and bleomycin than wild type, but not to UV, 4-nitroquinoline 1-oxide, camptothecin, or hydroxyurea . Vegetative growth, conidiation, and ascospore production in homozygous crosses were normal . The frequency of integration of exogenous DNA into homologous sequences of the genome in the KU disruption strains of N . crassa was compared with that in wild type, mei-3, and mus-11 . In mei-3 and mus-11, which are defective in homologous recombination, none or few homologous integration events were observed under any conditions . When mtr target DNA with approximately 2-kb 5' and 3' flanking regions was used for transformation of the KU disruption strains, 100% of transformants exhibited integration at the homologous site, compared to 10 to 30% for a wild-type recipient . Similar results were obtained when the ad-3A gene was targeted for disruption . These results indicate that KU disruption strains are efficient recipients for gene targeting.

BMC Evol Biol . 2004 Aug 09;4(1):26.
Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III; Matsutani S; BACKGROUND: In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs) . The genes for these RNAs and SINEs have internal promoters that consist of two regions . These two regions are called the A and B blocks . The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter . Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously . RESULTS: Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches . Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions . In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region . The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif . Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related . CONCLUSION: Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants . Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

Genes Cells, 2004 Aug, 9(8), 709 - 21
In vivo synthesis of Taf1p lacking the TAF N-terminal domain using alternative transcription or translation initiation sites; Kasahara K et al.; The TAF N-terminal domain (TAND) of TAF1 includes two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively . Previous studies showed that the substitution of yeast TAND1 or TAND2 with the equivalent domain from a Drosophila homologue leads to accumulation of truncated Taf1p in yeast . This study demonstrates that these truncated Taf1p derivatives lack TAND . However, full-length Taf1p and untruncated derivatives are produced in yeast when several Met-to-Ala mutations are introduced in the carboxy-terminus of TAND . In contrast, mutations that reduce expression of full-length TAF1 do not reduce the amount of truncated Taf1p derivatives that are produced . These data suggest that TAND-deficient TAF1 derivatives are produced by initiating translation at alternative initiation sites . In addition, the TAF1 mRNA structure suggests that the TAND-deficient TAF1 derivatives may also be formed in yeast by use of (cryptic) alternative transcription initiation sites . Importantly, TAND-deficient truncated Taf1p appears to be produced at a low level in wild-type yeast as well . Finally, this study also demonstrates that Drosophila TAND2 substitutes functionally for yeast TAND2, but Drosophila TAND1 does not substitute for yeast TAND1.

Science, 2004 Sep 17, 305(5691), 1747 - 52 Epub 2004 Aug 05.
Mitochondrial fusion intermediates revealed in vitro; Meeusen S et al.; The events that occur during the fusion of double-membraned mitochondria are unknown . As an essential step toward determining the mechanism of mitochondrial fusion, we have captured this event in vitro . Mitochondrial outer and inner membrane fusion events were separable and mechanistically distinct, but both required guanosine 5'-triphosphate hydrolysis . Homotypic trans interactions of the ancient outer transmembrane guanosine triphosphatase, Fzo1, were required to promote the fusion of mitochondrial outer membranes, whereas electrical potential was also required for fusion of inner membranes . Our conclusions provide fundamental insights into the molecular events driving mitochondrial fusion and advance our understanding of the evolution of mitochondrial fusion in eukaryotic cells.

Dev Cell, 2004 Aug, 7(2), 263 - 74
Cdc28/Cdk1 regulates spindle pole body duplication through phosphorylation of Spc42 and Mps1; Jaspersen SL et al.; Duplication of the Saccharomyces cerevisiae spindle pole body (SPB) once per cell cycle is essential for bipolar spindle formation and accurate chromosome segregation during mitosis . We have investigated the role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, to better understand how SPB duplication is coordinated with cell cycle progression . Cdc28 is required for SPB duplication and Spc42 assembly, and we found that Cdc28 directly phosphorylates Spc42 to promote its assembly into the SPB . The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells . Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle.

Dev Cell, 2004 Aug, 7(2), 193 - 204
Convergence of signaling pathways in the control of differential cell growth in Arabidopsis; Li H et al.; Seedling apical hook development involves a complex interplay of hormones and light in the regulation of differential cell growth . However, the underlying molecular mechanisms that integrate these diverse signals to control bending of the embryonic stem are poorly understood . The Arabidopsis ethylene-regulated HOOKLESS1 (HLS1) gene is essential for apical hook formation . Herein, we identify two auxin response regulators that act downstream of HLS1 to control cell elongation in the hypocotyl . Extragenic suppressors of hls1 were identified as mutations in AUXIN RESPONSE FACTOR 2 (ARF2) . The level of ARF2 protein was decreased by ethylene, and this response required HLS1 . Exposure to light decreased HLS1 protein levels and evoked a concomitant increase in ARF2 accumulation . These studies demonstrate that both ethylene and light signals affect differential cell growth by acting through HLS1 to modulate the auxin response factors, pinpointing HLS1 as a key integrator of the signaling pathways that control hypocotyl bending.

Dev Cell, 2004 Aug, 7(2), 167 - 78
Role and regulation of starvation-induced autophagy in the Drosophila fat body; Scott RC et al.; In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation . Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear . Here, we show that signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body . In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation . Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells . Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival . Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor.

Nature, 2004 Aug 5, 430(7000), 640 - 5
Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template; Xiong Y et al.; Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3' end of tRNAs by means of the addition of the essential nucleotides CCA . However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template . Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA . We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5'-triphosphates . A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP . Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3' end of the tRNA.

J Biol Chem, 2004 Oct 8, 279(41), 42669 - 76 Epub 2004 Aug 04.
Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei; Uzcategui NL et al.; Trypanosoma brucei, causative for African sleeping sickness, relies exclusively on glycolysis for ATP production . Under anaerobic conditions, glucose is converted to equimolar amounts of glycerol and pyruvate, which are both secreted from the parasite . As we have shown previously, glycerol transport in T . brucei occurs via specific membrane proteins (Wille, U., Schade, B., and Duszenko, M . (1998) Eur . J . Biochem . 256, 245-250) . Here, we describe cloning and biochemical characterization of the three trypanosomal aquaglyceroporins (AQP; TbAQP1-3), which show a 40-45% identity to mammalian AQP3 and -9 . AQPs belong to the major intrinsic protein family and represent channels for small non-ionic molecules . Both TbAQP1 and TbAQP3 contain two highly conserved NPA motifs within the pore-forming region, whereas TbAQP2 contains NSA and NPS motifs instead, which are only occasionally found in AQPs . For functional characterization, all three proteins were heterologously expressed in yeast and Xenopus oocytes . In the yeast fps1Delta mutant, TbAQPs suppressed hypoosmosensitivity and rendered cells to a hyper-osmosensitive phenotype, as expected for unregulated glycerol channels . Under iso- and hyperosmotic conditions, these cells constitutively released glycerol, consistent with a glycerol efflux function of TbAQP proteins . TbAQP expression in Xenopus oocytes increased permeability for water, glycerol and, interestingly, dihydroxyacetone . Except for urea, TbAQPs were virtually impermeable for other polyols; only TbAQP3 transported erythritol and ribitol . Thus, TbAQPs represent mainly water/glycerol/dihydroxyacetone channels involved in osmoregulation and glycerol metabolism in T . brucei . This function and especially the so far not investigated transport of dihydroxyacetone may be pivotal for the survival of the parasite survival under non-aerobic or osmotic stress conditions.

J Biol Chem, 2004 Oct 15, 279(42), 43744 - 51 Epub 2004 Aug 04.
The role of Hot13p and redox chemistry in the mitochondrial TIM22 import pathway; Curran SP et al.; The small Tim proteins in the mitochondrial intermembrane space participate in the TIM22 import pathway for assembly of the inner membrane . Assembly of the small TIM complexes requires the conserved "twin CX3C" motif that forms juxtapositional intramolecular disulfide bonds . Here we identify a new intermembrane space protein, Hot13p, as the first component of a pathway that mediates assembly of the small TIM complexes . The small Tim proteins require Hot13p for assembly into a 70-kDa complex in the intermembrane space . Once assembled the small TIM complexes escort hydrophobic inner membrane proteins en route to the TIM22 complex . The mechanism by which the small Tim proteins bind and release substrate is not understood, and we investigated the affect of oxidant/reductant treatment on the TIM22 import pathway . With in organello import studies, oxidizing agents arrest the ADP/ATP carrier (AAC) bound to the Tim9p-Tim10p complex in the intermembrane space; this productive intermediate can be chased into the inner membrane upon subsequent treatment with reductant . Moreover, AAC import is markedly decreased by oxidant treatment in Deltahot13 mitochondria and improved when Hot13p is overexpressed, suggesting Hot13p may function to remodel the small TIM complexes during import . Together these results suggest that the small TIM complexes have a specialized assembly pathway in the intermembrane space and that the local redox state of the TIM complexes may mediate translocation of inner membrane proteins.

J Biol Chem, 2004 Oct 15, 279(42), 43530 - 9 Epub 2004 Aug 04.
The karyopherin Msn5/Kap142 requires Nup82 for nuclear export and performs a function distinct from translocation in RPA protein import; Belanger KD et al.; Protein transport between the nucleus and cytoplasm requires interactions between nuclear pore complex proteins (nucleoporins) and soluble nuclear transport factors (karyopherins, importins, and exportins) . Exactly how these interactions contribute to the nucleocytoplasmic transport of substrates remains unclear . Using a synthetic lethal screen with the nucleoporin NUP1, we have identified a conditional allele of NUP82, encoding an essential nuclear pore complex protein in Saccharomyces cerevisiae . This nup82-3 allele also exhibits synthetic genetic interactions with mutants of the karyopherin MSN5 . nup82-3 mutants accumulate the Msn5 export substrate Pho4 within the nucleus at non-permissive temperatures . The nuclear import of the RPA complex subunit Rfa2 is impaired in nup82-3 and in mutants of the karyopherin KAP95, but is not affected by the loss of MSN5 . Interestingly, deletion of MSN5 results in retention of Rfa2-GFP within the nucleus under conditions in which it normally diffuses out . These data provide evidence that Nup82 is important for Msn5-mediated nuclear protein export and Kap95-mediated protein import . In addition, Msn5 may play a role independent of import in the localization of Rfa2.

Cell, 2004 Aug 6, 118(3), 351 - 61
MAP kinase-mediated stress relief that precedes and regulates the timing of transcriptional induction; Proft M et al.; In yeast, hyperosmotic stress causes an immediate dissociation of most proteins from chromatin, presumably because cells are unprepared for, and initially unresponsive to, increased ion concentrations in the nucleus . Osmotic stress activates Hog1 MAP kinase, which phosphorylates at least two proteins located at the plasma membrane, the Nha1 Na+/H+ antiporter and the Tok1 potassium channel . Hog1 phosphorylation stimulates Nha1 activity, and this is crucial for the rapid reassociation of proteins with their target sites in chromatin . This initial response to hyperosmolarity precedes and temporally regulates the activation of stress-response genes that depends on Hog1 phosphorylation of transcription factors in the nucleus . Thus, a single MAP kinase coordinates temporally, spatially, and mechanistically distinct responses to stress, thereby providing very rapid stress relief that facilitates subsequent changes in gene expression that permit long-term adaptation to harsh environmental conditions.

J Biol Chem, 2004 Oct 8, 279(41), 42850 - 9 Epub 2004 Aug 02.
Regulation of Swi6/HP1-dependent heterochromatin assembly by cooperation of components of the mitogen-activated protein kinase pathway and a histone deacetylase Clr6; Kim HS et al.; A study of gene silencing within the mating-type region of fission yeast defines two distinct pathways responsible for the establishment of heterochromatin assembly . One is RNA interference-dependent and acts on centromere-homologous repeats (cenH) . The other is a stochastic Swi6 (the fission yeast HP1 homolog)-dependent mechanism that is not fully understood . Here we find that activating transcription factor (Atf1) and Pcr1, the fission yeast bZIP transcription factors homologous to human ATF-2, are crucial for proper histone deacetylation of both H3 and H4 . This deacetylation is a prerequisite for subsequent H3 lysine 9 methylation and Swi6-dependent heterochromatin assembly across the rest of the silent mating-type (mat) region lacking the RNA interference-dependent cenH repeat . Moreover, Atf1 and Pcr1 can form complexes with both a histone deacetylase, Clr6, and Swi6, and clr6 mutations affected the H3/H4 acetylation patterns, similar to the atf1 and pcr1 deletion mutant phenotypes at the endogenous mat loci and at the ctt1+ promoter region surrounding ATF/CRE-binding site . These data suggest that Atf1 and Pcr1 participate in an early step essential for heterochromatin assembly at the mat locus and silencing of transcriptional targets of Atf1 . Furthermore, a phosphorylation event catalyzed by the conserved mitogen-activated protein kinase pathway is important for regulation of heterochromatin silencing by Atf1 and Pcr1 . These findings suggest a role for the mitogen-activated protein kinase pathway and histone deacetylase in Swi6-based heterochromatin assembly.

J Biol Chem, 2004 Oct 15, 279(42), 43847 - 53 Epub 2004 Jul 30.
Interaction of the pacemaker channel HCN1 with filamin A; Gravante B et al.; Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons . Some of these functions require specific membrane localization . Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels . Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4 . Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton . Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4 . We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo . In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin-) indicating that interaction with filamin A affects membrane localization . Also, in filamin- cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells . The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.

J Biol Chem, 2004 Oct 8, 279(41), 42612 - 8 Epub 2004 Jul 29.
Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent; Gohil VM et al.; Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes . We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants . In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis . Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants . CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively . The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression . These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain . This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH.

J Biol Chem, 2004 Oct 8, 279(41), 43008 - 12 Epub 2004 Jul 29.
A CDC6-like factor from the archaea Sulfolobus solfataricus promotes binding of the mini-chromosome maintenance complex to DNA; De Felice M et al.; The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes . Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates . This function does not require the Cdc6 ATP and DNA binding activities . These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms.

J Agric Food Chem, 2004 Aug 11, 52(16), 5168 - 74
Analysis of volatiles in porcine liver pâtés with added sage and rosemary essential oils by using SPME-GC-MS; Estevez M et al.; The effect of the addition of two natural antioxidant extracts (sage and rosemary essential oils) and one synthetic (BHT) on the generation of volatile compounds in liver pates from Iberian and white pigs was analyzed using SPME-GC-MS . Lipid-derived volatiles such as aldehydes {hexanal, octanal, nonanal, hept-(Z)-4-enal, oct-(E)-2-enal, non-(Z)-2-enal, dec-(E)-2-enal, deca-(E,Z)-2,4-dienal} and alcohols (pentan-1-ol, hexan-1-ol, oct-1-en-3-ol) were the most abundant compounds in the headspace of porcine liver pates . Pates from different pig breeds presented different volatiles profiles due to their different oxidation susceptibilities as a probable result of their fatty acid profiles and vitamin E content . Regardless of the origin of the pates, the addition of BHT successfully reduced the amount of volatiles derived from PUFA oxidation . Added essential oils showed a different effect on the generation of volatiles whether they were added in pates from Iberian or white pigs because they inhibited lipid oxidation in the former and enhanced oxidative instability in the latter . SPME successfully allowed the isolation and analysis of 41 volatile terpenes from pates with added sage and rosemary essential oils including alpha-pinene, beta-myrcene, 1-limonene, (E)-caryophyllene, linalool, camphor, and 1,8-cineole, which might contribute to the aroma characteristics of liver pates.

Photochem Photobiol, 2004 Jun, 79(6), 506 - 9
Inhibition of DNA polymerization and antifungal specificity of furanocoumarins present in traditional medicines; Smith ML et al.; Antifungal activity is positively correlated to furanocoumarin content in extracts of the traditional phytomedicine northern prickly ash (Zanthoxylum americanum Mill . {Rutaceae}) . The specificity of these furanocoumarins in inhibiting replication of DNA was investigated with reference to significant base composition differences between fungal and mammalian mitochondrial DNA . We developed a polymerase chain reaction-based assay to investigate whether (1) furanocoumarins inhibit DNA polymerization and (2) distinct furanocoumarins specifically inhibit DNA replication depending on base composition . Specific inhibition of DNA polymerization by 5-methoxypsoralen and psoralen through high-adenine and thymine (AT) (84.3%) and low-AT (51.9%) DNA, respectively, suggests that furanocoumarins inhibit replicative functions of genomes or of regions within the genome that differ in base composition . Greater overall inhibition of DNA polymerization by Z . americanum husk extracts than with single or mixed furanocoumarins suggests that inhibitory compounds in addition to the major furanocoumarins are present in Z . americanum.

Inflamm Bowel Dis, 2004 May, 10(3), 240 - 4
Comparison of serological markers of inflammatory bowel disease with clinical diagnosis in children; Gupta SK et al.; BACKGROUND: Our purpose was to study the concordance of serological tests for inflammatory bowel disease with clinical diagnosis established by traditional testing in children . METHODS: All children seen in our division who had IBD Diagnostic System (ie, pANCA, ASCA IgA, and ASCA IgG) performed over a 21-month period (June 1998 to February 2000) were identified . Their medical records were reviewed for basic demographics, test results (endoscopy, histology, and radiology), IBD Diagnostic System results, and patient symptoms/medications . Results of the IBD Diagnostic System were compared with several patient characteristics including age, sex, absence/presence of symptoms, medication use, disease activity and duration . RESULTS: One hundred seven patients were divided into 6 groups based on clinical diagnosis and IBD Diagnostic System results . The sensitivity, specificity and +/- predictive values of the IBD Diagnostic System for ulcerative colitis were 69.2, 95.1, 90.0, and 87.1%, respectively, and for Crohn's disease were 54.1, 96.8, 90.9, and 80.8%, respectively . Overall, the results of the IBD Diagnostic System were concordant with the clinical diagnosis in 76 of the 107 (71%) patients . CONCLUSIONS: In our experience, the specificity of IBD Diagnostic System is better than the sensitivity; the sensitivity is better for ulcerative colitis than Crohn's disease (69.2% vs 54.1%) . The low sensitivity, especially for Crohn's disease, precludes the possibility that the IBD Diagnostic System can replace traditional studies when evaluating for inflammatory bowel disease . Though we do not exclude inflammatory bowel disease solely by IBD Diagnostic System results, it is reassuring to note that all patients without clinical evidence of inflammatory bowel disease also had negative IBD Diagnostic System results.

J Bioinform Comput Biol, 2003 Apr, 1(1), 21 - 40
CUBIC: identification of regulatory binding sites through data clustering; Olman V et al.; Transcription factor binding sites are short fragments in the upstream regions of genes, to which transcription factors bind to regulate the transcription of genes into mRNA . Computational identification of transcription factor binding sites remains an unsolved challenging problem though a great amount of effort has been put into the study of this problem . We have recently developed a novel technique for identification of binding sites from a set of upstream regions of genes, that could possibly be transcriptionally co-regulated and hence might share similar transcription factor binding sites . By utilizing two key features of such binding sites (i.e . their high sequence similarities and their relatively high frequencies compared to other sequence fragments), we have formulated this problem as a cluster identification problem . That is to identify and extract data clusters from a noisy background . While the classical data clustering problem (partitioning a data set into clusters sharing common or similar features) has been extensively studied, there is no general algorithm for solving the problem of identifying data clusters from a noisy background . In this paper, we present a novel algorithm for solving such a problem . We have proved that a cluster identification problem, under our definition, can be rigorously and efficiently solved through searching for substrings with special properties in a linear sequence . We have also developed a method for assessing the statistical significance of each identified cluster, which can be used to rule out accidental data clusters . We have implemented the cluster identification algorithm and the statistical significance analysis method as a computer software CUBIC . Extensive testing on CUBIC has been carried out . We present here a few applications of CUBIC on challenging cases of binding site identification.

J Bioinform Comput Biol, 2003 Jul, 1(2), 231 - 52
Bayesian network and nonparametric heteroscedastic regression for nonlinear modeling of genetic network; Imoto S et al.; We propose a new statistical method for constructing a genetic network from microarray gene expression data by using a Bayesian network . An essential point of Bayesian network construction is the estimation of the conditional distribution of each random variable . We consider fitting nonparametric regression models with heterogeneous error variances to the microarray gene expression data to capture the nonlinear structures between genes . Selecting the optimal graph, which gives the best representation of the system among genes, is still a problem to be solved . We theoretically derive a new graph selection criterion from Bayes approach in general situations . The proposed method includes previous methods based on Bayesian networks . We demonstrate the effectiveness of the proposed method through the analysis of Saccharomyces cerevisiae gene expression data newly obtained by disrupting 100 genes.

J Bioinform Comput Biol, 2003 Oct, 1(3), 459 - 74
Use of gene networks for identifying and validating drug targets; Imoto S et al.; We propose a new method for identifying and validating drug targets by using gene networks, which are estimated from cDNA microarray gene expression profile data . We created novel gene disruption and drug response microarray gene expression profile data libraries for the purpose of drug target elucidation . We use two types of microarray gene expression profile data for estimating gene networks and then identifying drug targets . The estimated gene networks play an essential role in understanding drug response data and this information is unattainable from clustering methods, which are the standard for gene expression analysis . In the construction of gene networks, we use the Bayesian network model . We use an actual example from analysis of the Saccharomyces cerevisiae gene expression profile data to express a concrete strategy for the application of gene network information to drug discovery.

J Bioinform Comput Biol, 2003 Oct, 1(3), 447 - 58
Cluster analysis of dynamic parameters of gene expression; Wu X et al.; Cluster analysis has proven to be a valuable statistical method for analyzing whole genome expression data . Although clustering methods have great utility, they do represent a lower level statistical analysis that is not directly tied to a specific model . To extend such methods and to allow for more sophisticated lines of inference, we use cluster analysis in conjunction with a specific model of gene expression dynamics . This model provides phenomenological dynamic parameters on both linear and non-linear responses of the system . This analysis determines the parameters of two different transition matrices (linear and nonlinear) that describe the influence of one gene expression level on another . Using yeast cell cycle microarray data as test set, we calculated the transition matrices and used these dynamic parameters as a metric for cluster analysis . Hierarchical cluster analysis of this transition matrix reveals how a set of genes influence the expression of other genes activated during different cell cycle phases . Most strikingly, genes in different stages of cell cycle preferentially activate or inactivate genes in other stages of cell cycle, and this relationship can be readily visualized in a two-way clustering image . The observation is prior to any knowledge of the chronological characteristics of the cell cycle process . This method shows the utility of using model parameters as a metric in cluster analysis.

Proc Natl Acad Sci U S A, 2004 Aug 10, 101(32), 11713 - 8 Epub 2004 Aug 02.
Recruitment of the ArgR/Mcm1p repressor is stimulated by the activator Gcn4p: a self-checking activation mechanism; Yoon S et al.; Transcription of the arginine biosynthetic gene ARG1 is repressed by the ArgR/Mcm1p complex in arginine-replete cells and activated by Gcn4p, a transcription factor induced by starvation for any amino acid . We show that all four subunits of the arginine repressor are recruited to ARG1 by Gcn4p in cells replete with arginine but starved for isoleucine/valine . None of these proteins is recruited to the Gcn4p target genes ARG4 and SNZ1, which are not regulated by ArgR/Mcm1p . Mcm1p and Arg80p were found in a soluble complex lacking Arg81p and Arg82p, and both Mcm1p and Arg80p were efficiently recruited to ARG1 in wild-type cells in the presence or absence of exogenous arginine, and also in arg81Delta cells . By contrast, the recruitment of Arg81p and Arg82p was stimulated by exogenous arginine . These findings suggest that Gcn4p constitutively recruits an Mcm1p/Arg80p heterodimer and that efficient assembly of a functional repressor also containing Arg81p and Arg82p occurs only in arginine excess . By recruiting an arginine-regulated repressor, Gcn4p can precisely modulate its activation function at ARG1 according to the availability of arginine.

Proc Natl Acad Sci U S A, 2004 Aug 10, 101(32), 11695 - 700 Epub 2004 Aug 02.
The mouse juvenile spermatogonial depletion (jsd) phenotype is due to a mutation in the X-derived retrogene, mUtp14b; Rohozinski J et al.; The recessive juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis, followed by failure of type A spermatogonia to differentiate, resulting in adult male sterility . We have identified a jsd-specific rearrangement in the mouse homologue of the Saccharomyces cerevisiae gene UTP14, termed mUtp14b . Confirmation that mUtp14b underlies the jsd phenotype was obtained by transgenic bacterial artificial chromosome (BAC) rescue . We also identified a homologous gene on the Mus musculus X chromosome (MMUX) (mUtp14a) that is the strict homologue of the yeast gene, from which the intronless mUtp14b has been derived by retrotransposition . Expression analysis showed that mUtp14b is predominantly expressed in the germ line of the testis from zygotene through round spermatids, whereas mUtp14a, although well expressed in all somatic tissues, could be detected only in the germ line in round spermatids . In yeast, depletion of the UTP proteins impedes production of 18S rRNA, leading to cell death . We propose that the retroposed autosomal copy mUtp14b, having acquired a testis-specific expression pattern, could have provided a mechanism for increasing the efficiency and/or numbers of germ cells produced by meeting the need for more 18S rRNA and protein . Such a mechanism would be of obvious reproductive advantage and be strongly selected for in evolution . Consistent with this hypothesis is the finding of a similar X-autosome retroposition of UTP14 in human which seems to have arisen independently of that in rodents . In jsd homozygotes, which lack a functional copy of Utp14b, insufficient production of rRNA quickly leads to a cessation of spermatogenesis.

J Biosci, 2004 Mar, 29(1), 33 - 43
Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0; Aruna K et al.; The ribosomal phosphoprotein P0 of the human malarial parasite Plasmodium falciparum (PfP0) has been identified as a protective surface protein . In Drosophila, P0 protein functions in the nucleus . The ribosomal function of P0 is mediated at the stalk of the large ribosomal subunit at the GTPase centre, where the elongation factor eEF2 binds . The multiple roles of the P0 protein presumably occur through interactions with other proteins . To identify such interacting protein domains, a yeast two-hybrid screen was carried out . Out of a set of sixty clones isolated, twelve clones that interacted strongly with both PfP0 and the Saccharomyces cerevisiae P0 (ScP0) protein were analysed . These belonged to three broad classes: namely (i) ribosomal proteins; (ii) proteins involved in nucleotide binding; and (iii) hypothetical integral membrane proteins . One of the strongest interactors (clone 67B) mapped to the gene YFL034W which codes for a hypothetical integral membrane protein, and is conserved amongst several eukaryotic organisms . The insert of clone 67B was expressed as a recombinant protein, and immunoprecipitaion (IP) reaction with anti-P0 antibodies pulled down this protein along with PfP0 as well as ScP0 protein . Using deletion constructions, the domain of ScP0, which interacted with clone 67B, was mapped to 60-148 amino acids . It is envisaged that the surface localization of P0 protein may be mediated through interactions with putative YFL034W-like proteins in P . falciparum.

Mol Biol (Mosk), 2004 May-Jun, 38(3), 420 - 8
{Editing of the mitochondrial cox3 transcript may yield a new site of protein-protein interactions in wild cereal Elymus sibiricus L.}; Tauson EL et al.; With PCR, RT-PCR, and direct sequencing, complete nucleotide sequences were established for the Elymus sibiricus mitochondrial cytochrome c oxidase subunit 3 gene (cox3) and its cDNA . The cox3 transcript was shown to have 12 editing sites with changes affecting the amino acid sequence of the protein product . The editing of the primary cox3 transcript was found to change the position of a site of protein-protein interactions . The results demonstrate again the important role of mRNA editing in posttranscriptional regulation of the expression of plant mitochondrial genes.

Plant Mol Biol, 2004 Feb, 54(3), 441 - 59
Sucrose regulates elongation of carrot somatic embryo radicles as a signal molecule; Yang Z et al.; Elongation of carrot somatic embryo radicles was inhibited by sucrose at or above 5% (145 mM) . This effect would not be released until the sucrose concentration was lowered again . Morphological and cytological studies as well as determination of ABA content and analysis of the expression mode of a Lea gene, all point to its similarity to natural dormancy and germination of seeds . Use of monosaccharides (glucose and fructose), other disaccharide (maltose), and isomolar concentration of osmotica (mannitol and sorbitol), did not show similar regulatory effect . It is thus clear that the regulatory effect is not a result of simple osmotic stress . Hexokinase inhibitors such as glucosamine and N -acetyl-glucosamine did not exert any influence on the regulation-deregulation effects of sucrose . Mannose, which inhibits germination of Arabidopsis seeds, did not prevent carrot somatic embryo radicles from elongating . It is thus inferred that this sucrose-signaling pathway may be independent of hexokinase . As a first step to understand the molecular mechanism of this process, a carrot sucrose transporter gene ( cSUT ) expressed in the embryos and roots specifically was isolated . Studies on transformed yeast mutant with cSUT cDNA identified its sucrose transport activity . Northern hybridization and gel retardation experiment revealed that there is a marked increase in expression of cSUT at the beginning of somatic embryo germination, and this is attributed to regulation on the level of transcription . This suggested the possibility that cSUT has an important role in this sucrose signal regulation system.

Bioinformatics, 2004 Dec 12, 20(18), 3508 - 15 Epub 2004 Jul 29.
Modeling interactome: scale-free or geometric?
Przulj N, Corneil DG, Jurisica I.
MOTIVATION: Networks have been used to model many real-world phenomena to better understand the phenomena and to guide experiments in order to predict their behavior . Since incorrect models lead to incorrect predictions, it is vital to have as accurate a model as possible . As a result, new techniques and models for analyzing and modeling real-world networks have recently been introduced . RESULTS: One example of large and complex networks involves protein-protein interaction (PPI) networks . We analyze PPI networks of yeast Saccharomyces cerevisiae and fruitfly Drosophila melanogaster using a newly introduced measure of local network structure as well as the standardly used measures of global network structure . We examine the fit of four different network models, including Erdos-Renyi, scale-free and geometric random network models, to these PPI networks with respect to the measures of local and global network structure . We demonstrate that the currently accepted scale-free model of PPI networks fails to fit the data in several respects and show that a random geometric model provides a much more accurate model of the PPI data . We hypothesize that only the noise in these networks is scale-free . Conclusions: We systematically evaluate how well-different network models fit the PPI networks . We show that the structure of PPI networks is better modeled by a geometric random graph than by a scale-free model . SUPPLEMENTARY INFORMATION: Supplementary information is available at http://www.cs.utoronto.ca/~juris/data/data/ppiGRG04/

Anal Chem, 2004 Aug 1, 76(15), 4472 - 83
Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry; Chang EJ et al.; We describe a strategy, which we term hypothesis-driven multiple-stage mass spectrometry (HMS-MS), for the sensitive detection and identification of phosphopeptides derived from enzymatic digests of phosphoproteins . In this strategy, we postulate that any or all of the potential sites of phosphorylation in a given protein may be phosphorylated . Using this assumption, we calculate the m/z values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the enzyme employed for proteolysis . We test ions at these m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry (MS(2)) in a vacuum MALDI ion trap mass spectrometer, where the neutral loss of the elements of H(3)PO(4) (98 Da) provides a sensitive assay for the presence of phosphopeptides . Subsequent MS(3) analysis of the (M + H - 98)(+) peaks allows us to confirm or reject the hypotheses that the putative phosphopeptides are present in the sample . HMS-MS was successfully applied to the detection and identification of phosphopeptides from substrates of the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6), basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro . The method was also used to find in vitro phosphopeptides from a domain of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr residues without assuming a consensus motif . Our results demonstrate that HMS-MS is a sensitive, highly specific tool for systematically surveying proteins for Ser/Thr phosphorylation, and represents a significant step toward our goal of comprehensive phosphorylation mapping.

Plant Physiol Biochem, 2004 Feb, 42(2), 111 - 6
SNF1-related protein kinase (snRK1) phosphorylates class I heat shock protein; Slocombe SP et al.; The nucleotide sequence of cBSnIP2, a cDNA that had been cloned from a barley (Hordeum vulgare) seed endosperm cDNA library by two-hybrid screening with barley SNF1-related protein kinase (SnRKI) was determined . It was found to contain a complete open reading frame encoding a class I heat shock protein . Transcripts corresponding to the cDNA (renamed cBHSP17) were detectable in RNA isolated from barley seeds harvested in mid-development but not RNA from roots or leaves . BHSP17 protein was expressed in Escherischia coli and shown to be phosphorylated by SnRKI from barley endosperm and spinach leaf . It was found to be a less effective substrate than 3-hydroxy-3-methylglutaryl-Coenzyme A, a previously identified substrate of SnRK1 . However, a specific phosphorylation site at serine-35 was identified by solid phase sequencing of RP-HPLC-purified peptides after phosphorylation by spinach SnRK1.

Yeast, 2004 Jul 15, 21(9), 793 - 800
Localization of proteins that are coordinately expressed with Cln2 during the cell cycle; Sundin BA et al.; The localization of proteins can give important clues about their function and help sort data from large-scale proteomic screens . Forty-five proteins were tagged with the GFP variant YFP . These proteins were chosen because they are encoded by genes that display strong cell cycle-dependent expression that peaks in G(1) . Most of these proteins localize to either the nucleus or to sites of cell growth . We are able to assign new cellular component GO terms to ASF2, TOS4, RTT109, YBR070C, YKR090W, YOL007C, YOL019W and YPR174C . We also have localization data for 21 other proteins . Noteworthy localizations were found for Rfa1p, a member of the DNA replication A complex, and Pri2p and Pol12p, subunits of the alpha-DNA polymerase : primase complex . In addition to its nuclear localization, Rfa1p assembled into cytoplasmic foci adjacent to the nucleus in cells during the G(1)-S phase transition of the cell cycle . Pri2 and Pol12 took on a beaded appearance at the G(1)-S transition and later in the cell cycle were enriched in the nuclear envelope . A new spindle pole body/nuclear envelope component encoded by YPR174 was identified . The cell cycle-dependent abundance of Tos4p mirrored Yox1p and these two proteins were the only proteins that were found exclusively at the G(1)-S phase of the cell cycle . A complete list of localizations, along with images, can be found at our website .

Mol Cell Biol, 2004 Aug, 24(16), 7284 - 97
A plant snoRNP complex containing snoRNAs, fibrillarin, and nucleolin-like proteins is competent for both rRNA gene binding and pre-rRNA processing in vitro; Saez-Vasquez J et al.; In eukaryotes the primary cleavage of the precursor rRNA (pre-rRNA) occurs in the 5' external transcribed spacer (5'ETS) . In Saccharomyces cerevisiae and animals this cleavage depends on a conserved U3 small nucleolar ribonucleoprotein particle (snoRNP), including fibrillarin, and on other transiently associated proteins such as nucleolin . This large complex can be visualized by electron microscopy bound to the nascent pre-rRNA soon after initiation of transcription . Our group previously described a radish rRNA gene binding activity, NF D, that specifically binds to a cluster of conserved motifs preceding the primary cleavage site in the 5'ETS of crucifer plants including radish, cauliflower, and Arabidopsis thaliana (D . Caparros-Ruiz, S . Lahmy, S . Piersanti, and M . Echeverria, Eur . J . Biochem . 247:981-989, 1997) . Here we report the purification and functional characterization of NF D from cauliflower inflorescences . Remarkably NF D also binds to 5'ETS RNA and accurately cleaves it at the primary cleavage site mapped in vivo . NF D is a multiprotein factor of 600 kDa that dissociates into smaller complexes . Two polypeptides of NF D identified by microsequencing are homologues of nucleolin and fibrillarin . The conserved U3 and U14 snoRNAs associated with fibrillarin and required for early pre-rRNA cleavages are also found in NF D . Based on this it is proposed that NF D is a processing complex that assembles on the rDNA prior to its interaction with the nascent pre-rRNA.

Mol Cell Biol, 2004 Aug, 24(16), 7249 - 59
Cluster analysis of mass spectrometry data reveals a novel component of SAGA; Powell DW et al.; The SAGA histone acetyltransferase and TFIID complexes play key roles in eukaryotic transcription . Using hierarchical cluster analysis of mass spectrometry data to identify proteins that copurify with components of the budding yeast TFIID transcription complex, we discovered that an uncharacterized protein corresponding to the YPL047W open reading frame significantly associated with shared components of the TFIID and SAGA complexes . Using mass spectrometry and biochemical assays, we show that YPL047W (SGF11, 11-kDa SAGA-associated factor) is an integral subunit of SAGA . However, SGF11 does not appear to play a role in SAGA-mediated histone acetylation . DNA microarray analysis showed that SGF11 mediates transcription of a subset of SAGA-dependent genes, as well as SAGA-independent genes . SAGA purified from a sgf11 Delta deletion strain has reduced amounts of Ubp8p, and a ubp8 Delta deletion strain shows changes in transcription similar to those seen with the sgf11 Delta deletion strain . Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes . Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA.

Mol Cell Biol, 2004 Aug, 24(16), 7206 - 13
Scrambled prion domains form prions and amyloid; Ross ED et al.; The {URE3} prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p . The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content . Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions . To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged . Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro . Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that {URE3} formation is driven primarily by amino acid composition, largely independent of primary sequence.

Mol Cell Biol, 2004 Aug, 24(16), 7082 - 90
Mrc1 is required for sister chromatid cohesion to aid in recombination repair of spontaneous damage; Xu H et al.; The SRS2 gene of Saccharomyces cerevisiae encoding a 3'-->5' DNA helicase is part of the postreplication repair pathway and functions to ensure proper repair of DNA damage arising during DNA replication through pathways that do not involve homologous recombination . Through a synthetic gene array analysis, genes that are essential when Srs2 is absent have been identified . Among these are MRC1, TOF1, and CSM3, which mediate the intra-S checkpoint response . srs2 Delta mrc1 Delta synthetic lethality is due to inappropriate recombination, as the lethality can be suppressed by genetic elimination of homologous recombination . srs2 Delta mrc1 Delta synthetic lethality is dependent on the role of Mrc1 in DNA replication but independent of the role of Mrc1 in a DNA damage checkpoint response . mrc1 Delta, tof1 Delta and csm3 Delta mutants have sister chromatid cohesion defects, implicating sister chromatid cohesion established at the replication fork as an important factor in promoting repair of stalled replication forks through gap repair.

Mol Cell Biol, 2004 Aug, 24(16), 7043 - 58
RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits; Jeronimo C et al.; We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags . Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1) . The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo . A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11 . We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Biochim Biophys Acta, 2004 Jul 23, 1658(1-2), 157 - 64
Activation by retinoids of the uncoupling protein UCP1; Tomas P et al.; The uncoupling protein from brown adipose tissue (UCP1) is a transporter that catalyzes a regulated discharged of the mitochondrial proton gradient . The proton conductance in UCP1 is inhibited by nucleotides and activated by fatty acids . We have recently shown that all-trans-retinoic acid (ATRA) is a high-affinity activator of UCP1 . In the present report, we have set to analyze the structural requirements for the ligands that activate UCP1 and particularly the specificity for different retinoids . For this purpose, we have developed a new protocol to determine the activity of UCP1 in respiring yeast mitochondria that can be adapted for high-throughput screenings . Our results evidence differences between the structural requirements for the activation by fatty acids and retinoids . Thus, although all active retinoids must possess a carboxylate, the introduction of additional polar groups renders them inactive . The linear and rigid structure of these molecules suggests the existence of a long hydrophobic binding pocket . We postulate that the access to the retinoid binding site must occur from the lipid bilayer and this could be at the interface between two transmembrane alpha-helices.

Biotechnol Bioeng, 2004 Aug 5, 87(3), 347 - 53
Use of dimensionless residence time to study variations in breakthrough behaviour in expanded beds formed from varied particle size distributions; Gardner PJ et al.; This work demonstrates an experimental method for studying breakthrough behaviour in expanded beds . The behaviour of beds made with differently sized particles were studied at varying flowrates . The use of a dimensionless residence time measurement allowed a more valid comparison of breakthrough characteristics in expanded bed operation by compensating for the changes in bed volume that occur during expansion . We demonstrate that bed breakthrough behaviour can be compared directly even when the beds contain different-sized particles and hence have different expanded volumes . By utilising this concept we demonstrate that, in the case of the Alcohol Dehydrogenase (ADH) / STREAMLINE Phenyl system used here, there was little or no variation in ADH breakthrough behaviour between beds of differently sized particles operating at flowrates above 100 cm/h . This suggests that the higher specific surface area and hence binding capacity of smaller particles is negated in this case due to mass transfer limitations and the increase in system void volume even at normal operating flowrates of 200-300 cm/h.

Biotechnol Bioeng, 2004 Aug 5, 87(3), 337 - 46
The influence of biomass on the hydrodynamic behavior and stability of expanded beds; Lin DQ et al.; Expanded bed adsorption is an innovative chromatographic technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed . Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feedstock and the biomass is not influencing protein transport towards the adsorbent surface, a sorption performance comparable to packed beds is found . The influence of biomass on the hydrodynamic stability of expanded beds is essential and was investigated systematically in this article . Residence-time distribution analyses were performed using model systems and a yeast suspension under various fluid-phase conditions . It is demonstrated that three factors (biomass/adsorbent interactions, biomass concentration, and flow rate) play an interdependent role disturbing the classified fluidization of an expanded bed . A clear correlation between the degree of aggregative fluidization--obtained by PDE modeling of RTD data--and the expansion behavior of the fluidized bed has been found . Thus, combining three analytical methods, namely cell transmission index analysis, expansion analysis, and RTD analysis provides a solid base for understanding and control of the fluidization behavior and thus further process design during the initial phase of process development.

Cell Cycle, 2004 Aug, 3(8), 1011 - 3 Epub 2004 Aug 07.
Ubiquitination of PCNA and the polymerase switch in human cells; Kannouche PL et al.; Replicative DNA polymerases are blocked by damage in the template DNA . To get past this damage, the cell employs specialized translesion synthesis (TLS) polymerases, which have reduced stringency and are able to bypass different lesions . For example, DNA polymerase eta (poleta) is able to carry out TLS past UV-induced cyclobutane pyrimidine dimers . How does the cell bring about the switch from replicative to TLS polymerase? We have shown that, in human cells, when the replication machinery is blocked at DNA damage, PCNA, the sliding clamp required for DNA replication, is mono-ubiquitinated and that this modified form of PCNA has increased affinity for poleta . This provides a mechanism for the polymerase switch . In this Extra-View, we discuss the possible signals that might trigger ubiquitination of PCNA, whether PCNA becomes de-ubiquitinated after TLS has been accomplished and the role of the hREV1 protein in TLS . We point out some apparent differences between mechanisms in Saccharomyces cerevisiae and human cells.

Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11380 - 5 Epub 2004 Jul 27.
Rad6-Bre1-mediated histone H2B ubiquitylation modulates the formation of double-strand breaks during meiosis; Yamashita K et al.; An E2 ubiquitin-conjugating enzyme, Rad6, working with an E3 ubiquitin ligase Bre1, catalyzes monoubiquitylation of histone H2B on a C-terminal lysine residue . The rad6 mutant of Saccharomyces cerevisiae shows a meiotic prophase arrest . Here, we analyzed meiotic defects of a rad6 null mutant of budding yeast . The rad6 mutant exhibits pleiotropic phenotypes during meiosis . RAD6 is required for efficient formation of double-strand breaks (DSBs) at meiotic recombination hotspots, which is catalyzed by Spo11 . The mutation decreases overall frequencies of DSBs in a cell . The effect of the rad6 mutation is local along chromosomes; levels of DSBs at stronger hotspots are particularly reduced in the mutant . The absence of RAD6 has little effect on the formation of ectopic DSBs targeted by Spo11 fusion protein with a Gal4 DNA-binding domain . Furthermore, the disruption of the BRE1 as well as substitution of the ubiquitylation site of histone H2B also reduces some DSB formation similar to the rad6 . These results suggest that Rad6-Bre1, through ubiquitylation of histone H2B, is necessary for efficient recruitment and/or stabilization of a DSB-forming machinery containing Spo11 . Histone tail modifications might play a role in DSB formation during meiosis.

Nucleic Acids Res, 2004 Jul 27, 32(13), 3921 - 9 Print 2004.
SMC1 coordinates DNA double-strand break repair pathways; Schar P et al.; The SMC1/SMC3 heterodimer acts in sister chromatid cohesion, and recent data indicate a function in DNA double-strand break repair (DSBR) . Since this role of SMC proteins has remained largely elusive, we explored interactions between SMC1 and the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways for DSBR in Saccharomyces cerevisiae . Analysis of conditional single- and double mutants of smc1-2 with rad52Delta, rad54Delta, rad50Delta or dnl4Delta illustrates a significant contribution of SMC1 to the overall capacity of cells to repair DSBs . smc1 but not smc2 mutants show increased hypersensitivity of HR mutants to ionizing irradiation and to the DNA crosslinking agent cis-platin . Haploid, but not diploid smc1-2 mutants were severely affected in repairing multiple genomic DNA breaks, suggesting a selective role of SMC1 in sister chromatid recombination . smc1-2 mutants were also 15-fold less efficient and highly error-prone in plasmid end-joining through the NHEJ pathway . Strikingly, inactivation of RAD52 or RAD54 fully rescued efficiency and accuracy of NHEJ in the smc1 background . Therefore, we propose coordination of HR and NHEJ processes by Smc1p through interaction with the RAD52 pathway.

J Biol Chem, 2004 Nov 12, 279(46), 47506 - 12 Epub 2004 Jul 27.
Methylation of H3 lysine 4 at euchromatin promotes Sir3p association with heterochromatin; Santos-Rosa H et al.; Set1p methylates lysine 4 of histone H3 and can activate transcription by recruiting the chromatin-remodeling factor Isw1p . In addition, Lys-4-methylated H3 is required for maintenance of silencing at the telomeres, rDNA, and HML locus in Saccharomyces cerevisiae . The molecular mechanism underlying the role of Set1p in silencing is not known . Here we report that euchromatic methylation of H3 Lys-4 is necessary to maintain silencing at specific heterochromatic sites . Inactivation of Set1p catalytic activity or mutation of H3 Lys-4 leads to decreased binding of the silent information regulator Sir3p at heterochromatic sites . Concomitantly, there is an increase in the amount of Sir3p bound to genes located in subtelomeric regions . Consistent with this result is the finding that in vitro, Sir3p preferentially binds histone H3 tails when methylation is absent at H3 Lys-4, a situation found in heterochromatin . The inability of Sir3p to bind methylated H3 Lys-4 tails suggests a model whereby H3 Lys-4 methylation prevents Sir3p association at euchromatic sites and therefore concentrates Sir3p at unmodified, heterochromatic regions of the genome.

Proc Natl Acad Sci U S A, 2004 Aug 10, 101(32), 11903 - 8 Epub 2004 Jul 26.
Specific and differential inhibition of very-long-chain fatty acid elongases from Arabidopsis thaliana by different herbicides; Trenkamp S et al.; In higher plants, very-long-chain fatty acids (VLCFAs) are the main constituents of hydrophobic polymers that prevent dessication at the leaf surface and provide stability to pollen grains . Of the 21 genes encoding VLCFA elongases (VLCFAEs) from Arabidopsis thaliana, 17 were expressed heterologously in Saccharomyces cerevisiae . Six VLCFAEs, including three known elongases (FAE1, KCS1, and KCS2) and three previously uncharacterized gene products (encoded by At5g43760, At1g04220, and At1g25450) were found to be enzymatically active with endogenous yeast fatty acid substrates and to some extent with externally supplied unsaturated substrates . The spectrum of VLCFAs accumulated in expressing yeast strains was determined by gas chromatography/mass spectrometry . Marked specificity was found among elongases tested with respect to their elongation products, which encompassed saturated and monounsaturated fatty acids 20-30 carbon atoms in length . The active VLCFAEs revealed highly distinct patterns of differential sensitivity to oxyacetamides, chloroacetanilides, and other compounds tested, whereas yeast endogenous VLCFA production, which involves its unrelated elongase (ELO) in sphingolipid synthesis, was unaffected . Several compounds inhibited more than one VLCFAE, and some inhibited all six active enzymes . These findings pinpoint VLCFAEs as the target of the widely used K(3) class herbicides, which have been in commercial use for 50 years, provide important clues as to why spontaneous resistance to this class is rare, and point to complex patterns of substrate specificity and product spectrum among members of the Arabidopsis VLCFAE family.

Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11269 - 74 Epub 2004 Jul 26.
Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells; Vattem KM et al.; During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance . Central to this stress response is the transcriptional regulator activating transcription factor, ATF4 . Here we describe the mechanism regulating ATF4 expression involving the differential contribution of two upstream ORFs (uORFs) in the 5' leader of the mouse ATF4 mRNA . The 5' proximal uORF1 is a positive-acting element that facilitates ribosome scanning and reinitiation at downstream coding regions in the ATF4 mRNA . When eIF2-GTP is abundant in nonstressed cells, ribosomes scanning downstream of uORF1 reinitiate at the next coding region, uORF2, an inhibitory element that blocks ATF4 expression . During stress conditions, phosphorylation of eIF2 and the accompanying reduction in the levels of eIF2-GTP increase the time required for the scanning ribosomes to become competent to reinitiate translation . This delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead reinitiate at the ATF4-coding region . Increased expression of ATF4 would contribute to the expression of genes involved in remediation of cellular stress damage . These results suggest that the mechanism of translation reinitiation involving uORFs is conserved from yeast to mammals.

J Cell Biol, 2004 Aug 2, 166(3), 325 - 35 Epub 2004 Jul 26.
Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function; Ellis CD et al.; In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae . Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins . Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis . Our results indicate that Msc2 is one route of zinc entry into the ER . Msc2 localizes to the ER when expressed at normal levels . UPR induction in low zinc is exacerbated in an msc2 mutant . Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction . Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants . We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition . Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.

J Cell Biol, 2004 Aug 2, 166(3), 337 - 45 Epub 2004 Jul 26.
Monitoring disulfide bond formation in the eukaryotic cytosol; Ostergaard H et al.; Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol . The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined . Here, we present a glutathione-specific green fluorescent protein-based redox probe termed redox sensitive YFP (rxYFP) . Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer . Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases . For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione . We find it to be -289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range . Even under these conditions a significant fraction of rxYFP is oxidized.

J Biol Chem, 2004 Oct 1, 279(40), 42157 - 68 Epub 2004 Jul 23.
The essential ATP-binding cassette protein RLI1 functions in translation by promoting preinitiation complex assembly; Dong J et al.; RLI1 is an essential yeast protein closely related in sequence to two soluble members of the ATP-binding cassette family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20) . We show that affinity-tagged RLI1 co-purifies with eukaryotic translation initiation factor 3 (eIF3), eIF5, and eIF2, but not with other translation initiation factors or with translation elongation or termination factors . RLI1 is associated with 40 S ribosomal subunits in vivo, but it can interact with eIF3 and -5 independently of ribosomes . Depletion of RLI1 in vivo leads to cessation of growth, a lower polysome content, and decreased average polysome size . There was also a marked reduction in 40 S-bound eIF2 and eIF1, consistent with an important role for RLI1 in assembly of 43 S preinitiation complexes in vivo . Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal . A mutation in the second ATP-binding cassette domain of RLI1 had a dominant negative phenotype, decreasing the rate of translation initiation in vivo, and the mutant protein inhibited translation of a luciferase mRNA reporter in wild-type cell extracts . These findings are consistent with a direct role for the ATP-binding cassettes of RLI1 in translation initiation . RLI1-depleted cells exhibit a deficit in free 60 S ribosomal subunits, and RLI1-green fluorescent protein was found in both the nucleus and cytoplasm of living cells . Thus, RLI1 may have dual functions in translation initiation and ribosome biogenesis.

Biochemistry, 2004 Aug 3, 43(30), 9877 - 87
Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases; Borra MT et al.; The Silent information regulator 2 (Sir2) family of enzymes consists of NAD(+)-dependent histone/protein deacetylases that tightly couple the hydrolysis of NAD(+) and the deacetylation of an acetylated substrate to form nicotinamide, the deacetylated product, and the novel metabolite O-acetyl-ADP-ribose (OAADPR) . In this paper, we analyzed the substrate specificity of the yeast Sir2 (ySir2), the yeast HST2, and the human SIRT2 homologues toward various monoacetylated histone H3 and H4 peptides, determined the basic kinetic mechanism, and resolved individual chemical steps of the Sir2 reaction . Using steady-state kinetic analysis, we have shown that ySir2, HST2, and SIRT2 exhibit varying catalytic efficiencies and display a preference among the monoacetylated peptide substrates . Bisubstrate kinetic analysis indicates that Sir2 enzymes follow a sequential mechanism, where both the acetylated substrate and NAD(+) must bind to form a ternary complex, prior to any catalytic step . Using rapid-kinetic analysis, we have shown that after ternary complex formation, nicotinamide cleavage occurs first, followed by the transfer of the acetyl group from the donor substrate to the ADP-ribose portion of NAD(+) to form OAADPr and the deacetylated product . Product and dead-end inhibition analyses revealed that nicotinamide is the first product released followed by random release of OAADPr and the deacetylated product.

Science, 2004 Jul 23, 305(5683), 516 - 9
Phosphorylation by cyclin B-Cdk underlies release of mitotic exit activator Cdc14 from the nucleolus; Azzam R et al.; Budding yeast protein phosphatase Cdc14 is sequestered in the nucleolus in an inactive state during interphase by the anchor protein Net1 . Upon entry into anaphase, the Cdc14 early anaphase release (FEAR) network initiates dispersal of active Cdc14 throughout the cell . We report that the FEARnetwork promotes phosphorylation of Net1 by cyclin-dependent kinase (Cdk) complexed with cyclin B1 or cyclin B2 . These phosphorylations appear to be required for FEAR and sustain the proper timing of late mitotic events . Thus, a regulatory circuit exists to ensure that the arbiter of the mitotic state, Cdk, sets in motion events that culminate in exit from mitosis.

BMC Bioinformatics . 2004 Jul 23;5(1):100.
Graph-based iterative Group Analysis enhances microarray interpretation; Breitling R et al.; BACKGROUND: One of the most time-consuming tasks after performing a gene expression experiment is the biological interpretation of the results by identifying physiologically important associations between the differentially expressed genes . A large part of the relevant functional evidence can be represented in the form of graphs, e.g . metabolic and signaling pathways, protein interaction maps, shared GeneOntology annotations, or literature co-citation relations . Such graphs are easily constructed from available genome annotation data . The problem of biological interpretation can then be described as identifying the subgraphs showing the most significant patterns of gene expression . We applied a graph-based extension of our iterative Group Analysis (iGA) approach to obtain a statistically rigorous identification of the subgraphs of interest in any evidence graph . RESULTS: We validated the Graph-based iterative Group Analysis (GiGA) by applying it to the classic yeast diauxic shift experiment of DeRisi et al., using GeneOntology and metabolic network information . GiGA reliably identified and summarized all the biological processes discussed in the original publication . Visualization of the detected subgraphs allowed the convenient exploration of the results . The method also identified several processes that were not presented in the original paper but are of obvious relevance to the yeast starvation response . CONCLUSIONS: GiGA provides a fast and flexible delimitation of the most interesting areas in a microarray experiment, and leads to a considerable speed-up and improvement of the interpretation process.

Plant J, 2004 Aug, 39(4), 513 - 26
Functional interactions between a glutamine synthetase promoter and MYB proteins; Gomez-Maldonado J et al.; In Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) {EC 6.3.1.2}, which is encoded by two genes, PsGS1a and PsGS1b . PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism . The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored . The PsGS1b promoter contains a region that is enriched in previously characterized cis-acting elements, known as AC elements . Pine nuclear proteins bound these AC element-rich regions in a tissue-specific manner . As previous experiments had shown that R2R3-MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined . Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells . Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts . Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter . Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co-regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.

J Bioinform Comput Biol, 2004 Mar, 2(1), 77 - 98
Combining microarrays and biological knowledge for estimating gene networks via bayesian networks; Imoto S et al.; We propose a statistical method for estimating a gene network based on Bayesian networks from microarray gene expression data together with biological knowledge including protein-protein interactions, protein-DNA interactions, binding site information, existing literature and so on . Microarray data do not contain enough information for constructing gene networks accurately in many cases . Our method adds biological knowledge to the estimation method of gene networks under a Bayesian statistical framework, and also controls the trade-off between microarray information and biological knowledge automatically . We conduct Monte Carlo simulations to show the effectiveness of the proposed method . We analyze Saccharomyces cerevisiae gene expression data as an application . Copyright Imperial College Press

J Biol Chem, 2004 Oct 8, 279(41), 42638 - 47 Epub 2004 Jul 22.
The twisted abdomen phenotype of Drosophila POMT1 and POMT2 mutants coincides with their heterophilic protein O-mannosyltransferase activity; Ichimiya T et al.; Walker-Warburg syndrome, caused by mutations in protein O-mannosyltransferase-1 (POMT1), is an autosomal recessive disorder characterized by severe brain malformation, muscular dystrophy, and structural eye abnormalities . As humans have a second POMT, POMT2, we cloned each Drosophila ortholog of the human POMT genes and carried out RNA interference (RNAi) knock-down to investigate the function of these proteins in vivo . Drosophila POMT2 (dPOMT2) RNAi mutant flies showed a "twisted abdomen phenotype," in which the abdomen is twisted 30-60 degrees , similar to the dPOMT1 mutant . Moreover, dPOMT2 interacted genetically with dPOMT1, suggesting that the dPOMTs function in collaboration with each other in vivo . We expressed dPOMTs in Sf21 cells and measured POMT activity . dPOMT2 transferred a mannose to the dystroglycan protein only when it was coexpressed with dPOMT1 . Likewise, dPOMT1 showed POMT activity only when coexpressed with dPOMT2, and neither dPOMT showed any activity by itself . Each dPOMT RNAi fly totally reduced POMT activity, despite the specific reduction in the level of each dPOMT mRNA . The expression pattern of dPOMT2 mRNA was found to be similar to that of dPOMT1 mRNA using whole mount in situ hybridization . These results demonstrate that the two dPOMTs function as a protein O-mannosyltransferase in association with each other, in vitro and in vivo, to generate and maintain normal muscle development.

J Biol Chem, 2004 Oct 22, 279(43), 44683 - 9 Epub 2004 Jul 22.
Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p; Roy A et al.; Phosphatidylinositol (PtdIns) phosphate (PtdInsP) lipids are used as intracellular signposts for the recruitment and activation of peripheral membrane proteins . Whereas the distribution of most PtdInsPs is restricted to a single organelle, PtdIns(4)P is unique in that it exists in several discrete pools, and so proteins that bind PtdIns(4)P must use extra receptors to achieve a restricted localization . Here we compare the two highly related pleckstrin homology (PH) domains from Osh1p and Osh2p, yeast homologues of oxysterol-binding protein (OSBP), that target membranes using PtdIns(4)P, and in vitro bind both PtdIns(4)P and PtdIns(4,5)P2 . We show that Golgi targeting is specified by an additional site on PH(Osh1), which lies on a face of the domain not previously known to interact with receptors . In contrast, PH(Osh2) does not have a demonstrable second site, and targets multiple pools of PtdInsPs, each dependent on a different PtdIns 4-kinase . This lack of a second site in PH(Osh2) allows it to be used as an unbiased reporter for altered distribution of 4-phosphorylated PtdIns . For example, in cells with excess PtdIns(4)P caused by inactivation of the phosphatase Sac1p, PH(Osh2) indicates that PtdIns(4)P accumulates on the plasma membrane, whereas other Golgi-targeted PH domains fail to detect this change.

J Nat Prod, 2004 Jul, 67(7), 1162 - 4
A DNA-damaging oxoaporphine alkaloid from Piper caninum; Ma J et al.; Bioassay-guided fractionation of an active organic extract of Piper caninum, using a sensitive yeast assay to monitor putative double-strand DNA-damaging activity, resulted in the isolation of the 4,5-dioxoaporphine alkaloid cepharadione A (1) . Compound 1 exhibited potent inhibitory activity in a yeast cytotoxicity assay with IC(50) values of 50.2 nM toward RS321NpRAD52 grown on glucose versus 293 nM toward the same yeast strain grown on galactose.

J Gen Virol, 2004 Aug, 85(Pt 8), 2429 - 33
The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA; Pantaleo V et al.; The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA . Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other . DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity . Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA . DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone . By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an 'all-or-none' behaviour . Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined . Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

Curr Biol, 2004 Jul 27, 14(14), R565 - 7
Telomerase RNA: a flexible RNA scaffold for telomerase biosynthesis; Lustig AJ; Determination of the structure of the yeast telomerase RNA component TLC1 has been hampered by its large size and high rate of evolutionary divergence . But detailed phylogenetic comparisons have now revealed the unusually flexible and modular architecture of this important RNA molecule.

Plant Physiol, 2004 Jul, 135(3), 1206 - 20
Identification of genes required for embryo development in Arabidopsis; Tzafrir I et al.; A long-term goal of Arabidopsis research is to define the minimal gene set needed to produce a viable plant with a normal phenotype under diverse conditions . This will require both forward and reverse genetics along with novel strategies to characterize multigene families and redundant biochemical pathways . Here we describe an initial dataset of 250 EMB genes required for normal embryo development in Arabidopsis . This represents the first large-scale dataset of essential genes in a flowering plant . When compared with 550 genes with other knockout phenotypes, EMB genes are enriched for basal cellular functions, deficient in transcription factors and signaling components, have fewer paralogs, and are more likely to have counterparts among essential genes of yeast (Saccharomyces cerevisiae) and worm (Caenorhabditis elegans) . EMB genes also represent a valuable source of plant-specific proteins with unknown functions required for growth and development . Analyzing such unknowns is a central objective of genomics efforts worldwide . We focus here on 34 confirmed EMB genes with unknown functions, demonstrate that expression of these genes is not embryo-specific, validate a strategy for identifying interacting proteins through complementation with epitope-tagged proteins, and discuss the value of EMB genes in identifying novel proteins associated with important plant processes . Based on sequence comparison with essential genes in other model eukaryotes, we identify 244 candidate EMB genes without paralogs that represent promising targets for reverse genetics . These candidates should facilitate the recovery of additional genes required for seed development.

J Biol Chem, 2004 Sep 17, 279(38), 39604 - 10 Epub 2004 Jul 20.
Negative regulation of phosphatidylinositol 4,5-bisphosphate levels by the INP51-associated proteins TAX4 and IRS4; Morales-Johansson H et al.; Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an important second messenger in signaling pathways in organisms ranging from yeast to mammals, but the regulation of PI(4,5)P(2) levels remains unclear . Here we present evidence that PI(4,5)P(2) levels in Saccharomyces cerevisiae are down-regulated by the homologous and functionally redundant proteins TAX4 and IRS4 . The EPS15 homology domain-containing proteins TAX4 and IRS4 bind and activate the PI(4,5)P 5-phosphatase INP51 via an Asn-Pro-Phe motif in INP51 . Furthermore, the INP51-TAX4/IRS4 complex negatively regulates the cell integrity pathway . Thus, TAX4 and IRS4 are novel regulators of PI(4,5)P(2) and PI(4,5)P(2)-dependent signaling . The interaction between TAX4/IRS4 and INP51 is analogous to the association of EPS15 with the 5-phosphatase synaptojanin 1 in mammalian cells, suggesting that EPS15 is an activator of synaptojanin 1.

J Am Chem Soc, 2004 Jul 28, 126(29), 8940 - 7
Interfacial nano-structuring of designed peptides regulated by solution pH; Lu JR et al.; The in-situ conformations of peptide layers formed from the adsorption of two different synthetic 15-mer peptides at the hydrophilic silicon oxide/aqueous solution interface have been determined using neutron reflectivity (NR) . The first peptide is based on the native sequence of a protein-binding domain within a heteromeric transcriptional activator, HAP2, identified from yeast Saccharomyces cerevisiae, with tyrosine (Y) present at the 1st, 8th and 15th amino acid positions, hence we denote this YYY15 . Substitution of tryptophan (W) at the same locations gives WWW15 . Both peptides have alpha-helical structure in phosphate buffer, as determined by circular dichroism (CD) spectra . D(2)O was used as solvent in the NR experiments to highlight structural heterogeneity across the hydrogenated peptide layers . At pH 7, YYY15 was found to form a weakly adsorbed interfacial monolayer . However, the mutant WWW15 showed strong interfacial adsorption, with the interfacial layer characterized by a middle hydrophobic sublayer of 7-8 A with lower scattering length density and two almost symmetrical hydrophilic outer sublayers of 6-8 A with higher scattering length density, suggesting the formation of a "sideways-on" helical conformation . An increase in pH to 9 resulted in the improved packing within the interfacial layer with similar structure . However, decrease in pH to 5 reduced the interfacial adsorption, mainly due to the enhanced solubility of the peptides associated with the protonation of arginine (R) and lysine (K) groups and the decreasing concentration of divalent HPO(4)(2-) in the phosphate buffer . Subsequent assessment of the reversibility of adsorption showed that once the peptide layers were formed they did not desorb . These interfacial structures may provide feasible routes to interfacial nano-templating.

Proc Natl Acad Sci U S A, 2004 Jul 27, 101(30), 10907 - 12 Epub 2004 Jul 19.
Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711; Pitula JS et al.; Iron-regulatory protein 1 (IRP1) is a dual-function protein with mutually exclusive roles as a posttranscriptional regulator of animal-cell iron metabolism or as the cytosolic isoform of the iron-sulfur enzyme aconitase (c-acon) . Much effort has focused on the role of IRP1 in posttranscriptional gene regulation and in factors that influence its interconversion with c-acon, but little is known about the metabolic function and regulation of c-acon . The role of PKC-dependent phosphorylation of S711 on IRP1/c-acon function was examined . Phosphorylation state-specific antibodies revealed that S711 is phosphorylated by PKC in vitro and in human embryonic kidney cells treated with a PKC activator . In aco1 yeast, the phosphomimetic mutants S711D and S711E exhibited severely impaired aconitase function, whereas S711A and S711T were unaffected relative to the WT protein . Aconitase activity in yeast extracts displayed a similar pattern when assayed for capacity to convert citrate to isocitrate: WT, S711A, and S711T were active, but S711D and S711E activity was undetectable . In contrast, when measured by the conversion of isocitrate to cis-aconitate, S711D and S711E displayed substantial activity, indicating that phosphorylation impairs the citrate but not isocitrate mode of aconitase function . This possibility was confirmed in vivo by demonstrating that S711D and S711E specifically antagonized the requirement for isocitrate in two metabolic scenarios . Iron-responsive element RNA-binding affinity was unaffected by S711 mutations . Our results show that S711 is a target of phosphorylation capable of conferring distinct effects on c-acon function potentially dictating changes in cytosolic citrate/isocitrate metabolism.

Bioinformatics, 2004 Aug 4, 20 Suppl 1, I363 - I370
Protein network inference from multiple genomic data: a supervised approach; Yamanishi Y et al.; MOTIVATION: An increasing number of observations support the hypothesis that most biological functions involve the interactions between many proteins, and that the complexity of living systems arises as a result of such interactions . In this context, the problem of inferring a global protein network for a given organism, using all available genomic data about the organism, is quickly becoming one of the main challenges in current computational biology . RESULTS: This paper presents a new method to infer protein networks from multiple types of genomic data . Based on a variant of kernel canonical correlation analysis, its originality is in the formalization of the protein network inference problem as a supervised learning problem, and in the integration of heterogeneous genomic data within this framework . We present promising results on the prediction of the protein network for the yeast Saccharomyces cerevisiae from four types of widely available data: gene expressions, protein interactions measured by yeast two-hybrid systems, protein localizations in the cell and protein phylogenetic profiles . The method is shown to outperform other unsupervised protein network inference methods . We finally conduct a comprehensive prediction of the protein network for all proteins of the yeast, which enables us to propose protein candidates for missing enzymes in a biosynthesis pathway . AVAILABILITY: Softwares are available upon request.

Bioinformatics, 2004 Aug 4, 20 Suppl 1, I257 - I264
HyBrow: a prototype system for computer-aided hypothesis evaluation; Racunas SA et al.; MOTIVATION: Experimental design, hypothesis-testing and model-building in the current data-rich environment require the biologists' to collect, evaluate and integrate large amounts of information of many disparate kinds . Developing a unified framework for the representation and conceptual integration of biological data and processes is a major challenge in bioinformatics because of the variety of available data and the different levels of detail at which biological processes can be considered . RESULTS: We have developed the HyBrow (Hypothesis Browser) system as a prototype bioinformatics tool for designing hypotheses and evaluating them for consistency with existing knowledge . HyBrow consists of a modeling framework with the ability to accommodate diverse biological information sources, an event-based ontology for representing biological processes at different levels of detail, a database to query information in the ontology and programs to perform hypothesis design and evaluation . We demonstrate the HyBrow prototype using the galactose gene network in Saccharomyces cerevisiae as our test system, and evaluate alternative hypotheses for consistency with stored information . AVAILABILITY: www.hybrow.org

Bioinformatics, 2004 Aug 4, 20 Suppl 1, I178 - I185
Filling gaps in a metabolic network using expression information; Kharchenko P et al.; MOTIVATION: The metabolic models of both newly sequenced and well-studied organisms contain reactions for which the enzymes have not been identified yet . We present a computational approach for identifying genes encoding such missing metabolic enzymes in a partially reconstructed metabolic network . RESULTS: The metabolic expression placement (MEP) method relies on the coexpression properties of the metabolic network and is complementary to the sequence homology and genome context methods that are currently being used to identify missing metabolic genes . The MEP algorithm predicts over 20% of all known Saccharomyces cerevisiae metabolic enzyme-encoding genes within the top 50 out of 5594 candidates for their enzymatic function, and 70% of metabolic genes whose expression level has been significantly perturbed across the conditions of the expression dataset used . AVAILABILITY: Freely available (in Supplementary information) . SUPPLEMENTARY INFORMATION: Available at the following URL http://arep.med.harvard.edu/kharchenko/mep/supplements.html

Trends Genet, 2004 Aug, 20(8), 340 - 4
Comparative genomics for reliable protein-function prediction from genomic data; Huynen MA et al.; Genomic data provide invaluable, yet unreliable information about protein function . However, if the overlap in information among various genomic datasets is taken into account, one observes an increase in the reliability of the protein-function predictions that can be made . Recently published approaches achieved this either by comparing the same type of data from multiple species (horizontal comparative genomics) or by using subtle, Bayesian methods to compare different types of genomic data from a single species (vertical comparative genomics) . In this article, we discuss these methods, illustrating horizontal comparative genomics by comparing yeast two-hybrid (Y2H) data from Saccharomyces cerevisiae with Y2H data from Drosophila melanogaster, and illustrating vertical comparative genomics by comparing RNA expression data with proteomic data from Plasmodium falciparum.

Mol Cell, 2004 Jul 23, 15(2), 295 - 301
A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits; Nissan TA et al.; Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm . Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm . A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle . Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure . Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits . Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.

Mol Cell, 2004 Jul 23, 15(2), 287 - 93
Expanded CAG repeats activate the DNA damage checkpoint pathway; Lahiri M et al.; Trinucleotide repeats (TNRs) are sequences whose expansion causes several genetic diseases and chromosome breakage . We report a novel finding that expanded CAG repeats activate the DNA damage response . Mutations in yeast MEC1, RAD9, or RAD53 genes result in increased rates of fragility of a CAG repeat tract while single or double deletions of RAD17 or RAD24 have only a modest effect on TNR fragility, indicating that signaling down the Rad9 pathway and not the Rad17-Rad24 pathway plays a major role in sensing and repairing CAG-tract breaks . Deletion of CHK1 had no effect on CAG fragility, suggesting that a Chk1-mediated G2 arrest is not required for TNR repair . Absence of Mec1, Ddc2, Rad17, Rad24, or Rad53 also gives rise to increased frequency of CAG repeat contractions, indicating that components of the checkpoint machinery play an active role in the maintenance of both chromosomal integrity and repeat stability at expanded CAG sequences.

Mol Cell, 2004 Jul 23, 15(2), 173 - 83
AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets; Souret FF et al.; Messenger RNA degradation is an essential step in gene expression that can be regulated by siRNAs or miRNAs . However, most of our knowledge of in vivo eukaryotic mRNA degradation mechanisms derives from Saccharomyces cerevisiae, which lacks miRNAs and RNAi capability . Using reverse genetic and microarray analyses, we have identified multiple substrates of AtXRN4, the Arabidopsis homolog of the major yeast mRNA degrading exoribonuclease, Xrn1p . Insertional mutation of AtXRN4 leads to accumulation of the 3' end of several mRNAs, in a manner that correlates with increased stability of the 3' end, and is reversed following complementation with AtXRN4 . Moreover, 3' products of miRNA-mediated cleavage of SCARECROW-LIKE transcripts and several other miRNA target transcripts are among those that accumulate in xrn4 mutants . The demonstration that an Xrn1p homolog degrades mRNA in a multicellular eukaryote and contributes to the miRNA-mediated decay pathway of selected targets has implications for XRNs in other organisms.

Mol Cell, 2004 Jul 23, 15(2), 166 - 8
Necessity's sharp pinch; Chun KT et al.; In a recent issue of Cell, Hughes and coworkers (Mnaimneh et al., 2004) provide a great leap forward in the analysis of essential yeast genes by constructing a strain set that expresses each essential gene from a tetracycline-regulatable promoter.

Biochemistry, 2004 Jul 27, 43(29), 9519 - 26
Altered substrate specificity in flavocytochrome b2: structural insights into the mechanism of L-lactate dehydrogenation; Mowat CG et al.; Flavocytochrome b(2) from Saccharomyces cerevisiae is a l-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes . The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined {Xia, Z.-X., and Mathews, F . S . (1990) J . Mol . Biol . 212, 837-863} . The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230 . These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases . Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively . Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids . In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme {Daff, S., Manson, F . D . C., Reid, G . A., and Chapman, S . K . (1994) Biochem . J . 301, 829-834}, and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate l-mandelate than it is with l-lactate {Sinclair, R., Reid, G . A., and Chapman, S . K . (1998) Biochem . J . 333, 117-120} . To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 A resolution) . We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 A resolution . These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained . In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.

EMBO Rep, 2004 Aug, 5(8), 818 - 24 Epub 2004 Jul 16.
Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation; Schuberth C et al.; Known activities of the ubiquitin-selective AAA ATPase Cdc48 (p97) require one of the mutually exclusive cofactors Ufd1/Npl4 and Shp1 (p47) . Whereas Ufd1/Npl4 recruits Cdc48 to ubiquitylated proteins destined for degradation by the 26S proteasome, the UBX domain protein p47 has so far been linked exclusively to nondegradative Cdc48 functions in membrane fusion processes . Here, we show that all seven UBX domain proteins of Saccharomyces cerevisiae bind to Cdc48, thus constituting an entire new family of Cdc48 cofactors . The two major yeast UBX domain proteins, Shp1 and Ubx2, possess a ubiquitin-binding UBA domain and interact with ubiquitylated proteins in vivo . Deltashp1 and Deltaubx2 strains display defects in the degradation of a ubiquitylated model substrate, are sensitive to various stress conditions and are genetically linked to the 26S proteasome . Our data suggest that Shp1 and Ubx2 are adaptors for Cdc48-dependent protein degradation through the ubiquitin/proteasome pathway.

Science, 2004 Jul 16, 305(5682), 393 - 6
Spatial coordination of cytokinetic events by compartmentalization of the cell cortex; Dobbelaere J et al.; During cytokinesis, furrow ingression and plasma membrane fission irreversibly separate daughter cells . How actomyosin ring assembly and contraction, vesicle fusion, and abscission are spatially coordinated was unknown . We found that during cytokinesis septin rings, located on both sides of the actomyosin ring, acted as barriers to compartmentalize the cortex around the cleavage site . Compartmentalization maintained diffusible cortical factors, such as the exocyst and the polarizome, to the site of cleavage . In turn, such factors were required for actomyosin ring function and membrane abscission . Thus, a specialized cortical compartment ensures the spatial coordination of cytokinetic events.

Proc Natl Acad Sci U S A, 2004 Aug 31, 101(35), 12792 - 7 Epub 2004 Jul 15.
Identification of 315 genes essential for early zebrafish development; Amsterdam A et al.; We completed a large insertional mutagenesis screen in zebrafish to identify genes essential for embryonic and early larval development . We isolated 525 mutants, representing lesions in approximately 390 different genes, and we cloned the majority of these . Here we describe 315 mutants and the corresponding genes . Our data suggest that there are roughly 1,400 embryonic-essential genes in the fish . Thus, we have mutations in approximately 25% of these genes and have cloned approximately 22% of them . Re-screens of our collection to identify mutants with specific developmental defects suggest that approximately 50 genes are essential for the development of some individual organs or cell types . Seventy-two percent of the embryonic-essential fish genes have homologues in yeast, 93% have homologues in invertebrates (fly or worm), and 99% have homologues in human . Yeast and worm orthologues of genes that are essential for early zebrafish development have a strong tendency to be essential for viability in yeast and for embryonic development in the worm . Thus, the trait of being a genetically essential gene is conserved in evolution . This mutant collection should be a valuable resource for diverse studies of cell and developmental biology .

Genes Dev, 2004 Jul 15, 18(14), 1709 - 24
Cdk1-Clb4 controls the interaction of astral microtubule plus ends with subdomains of the daughter cell cortex; Maekawa H et al.; As in many polarized cells, spindle alignment in yeast is essential and cell cycle regulated . A key step that governs spindle alignment is the selective binding of the Kar9 protein to only one of the two spindle pole bodies (SPBs) . It has been suggested that cyclin-dependent kinase Cdc28, in complex with cyclin Clb4, associates only with the SPB in the mother cell and so prevents Kar9 binding to this SPB . However, here we show that the nonoverexpressed Clb4 associates with the budward-directed SPB through Kar9 . Cdc28-Clb4 then uses Kar9 as a carrier to move from this SPB to the plus ends of astral microtubules, where Cdc28-Clb4 regulates the interactions between microtubule ends and subdomains of the bud cortex . In the absence of Cdc28-Clb4 activity (G1/S phase), astral microtubules interact with the bud tip in a manner dependent on actin, Myo2, and Kar9 . Coincidentally with reaching the bud cortex in S phase, Cdc28-Clb4 facilitates the dissociation of the microtubule bud tip interaction and their capture by the bud neck . This transition prevents the preanaphase spindle from becoming prematurely pulled into the bud . Thus, Cdc28-Clb4 facilitates spindle alignment by regulating the interaction of astral microtubules with subdomains of the bud cortex .

Bioinformatics, 2004 Dec 12, 20(18), 3302 - 7 Epub 2004 Jul 15.
Preferred in vivo ubiquitination sites; Catic A et al.; MOTIVATION: The conjugation of ubiquitin to target molecules involves several enzymatic steps . Little is known about the specificity of ubiquitination . How E3 ligases select their substrate and which lysines are targeted for ubiquitin conjugation is largely an enigma . The object of this study is to identify preferred ubiquitination sites . Genetic approaches to study this question have proven difficult, because of the redundancy of ligases and the lack of strictly required motifs . However, a better understanding of acceptor site selection could help to predict ubiquitination sites and clarify yet unsolved structure-function relationships of the transfer reaction . RESULTS: In an effort to define preferences for ubiquitination, we systematically analyzed structure and sequence of 135 known ubiquitination sites in 95 proteins in Saccharomyces cerevisiae . The results show clear structural preferences for ubiquitin ligation to target proteins, and compartment-specific amino acid patterns in close proximity to the modified side chain . SUPPLEMENTARY INFORMATION: http://www.people.fas.harvard.edu/~catic.

Plant J, 2004 Aug, 39(3), 425 - 39
Two genes encoding Arabidopsis halleri MTP1 metal transport proteins co-segregate with zinc tolerance and account for high MTP1 transcript levels; Drager DB et al.; The zinc hyperaccumulator plant Arabidopsis halleri is able to naturally accumulate 100-fold higher leaf zinc concentrations when compared with non-accumulator species such as the closely related A . lyrata and A . thaliana, without showing toxicity symptoms . A novel member of the cation diffusion facilitator (CDF) protein family, an A . halleri metal tolerance protein 1 (MTP1), and the homologous A . thaliana Zn transporter (ZAT)/AtMTP1 metal-specifically complement the zinc hypersensitivity of a Saccharomyces cerevisiae zrc1 cot1 mutant strain . A fusion of the AhMTP1 protein to green fluorescent protein (GFP) localizes to the vacuolar membrane of A . thaliana protoplasts . When compared with A . lyrata and A . thaliana, the total MTP1 transcript levels are substantially higher in the leaves and upregulated upon exposure to high zinc concentrations in the roots of A . halleri . The high MTP1 transcript levels in A . halleri can be primarily attributed to two genetically unlinked genomic AhMTP1 gene copies . The two corresponding loci co-segregate with zinc tolerance in the back-cross 1 generation of a cross between the zinc-tolerant species A . halleri and the zinc-sensitive species A . lyrata . In contrast, a third MTP1 gene in the genome of A . halleri generates only minor amounts of MTP1 transcripts and does not co-segregate with zinc tolerance . Our data suggests that zinc tolerance in A . halleri involves an expanded copy number of an ancestral MTP1 gene, encoding functional proteins that mediate the detoxification of zinc in the cell vacuole . At the transcript level, MTP1 gene copies of A . halleri are regulated differentially and in response to changes in zinc supply.

Nature, 2004 Aug 5, 430(7000), 686 - 9 Epub 2004 Jul 14.
Sirtuin activators mimic caloric restriction and delay ageing in metazoans; Wood JG et al.; Caloric restriction extends lifespan in numerous species . In the budding yeast Saccharomyces cerevisiae this effect requires Sir2 (ref . 1), a member of the sirtuin family of NAD+-dependent deacetylases . Sirtuin activating compounds (STACs) can promote the survival of human cells and extend the replicative lifespan of yeast . Here we show that resveratrol and other STACs activate sirtuins from Caenorhabditis elegans and Drosophila melanogaster, and extend the lifespan of these animals without reducing fecundity . Lifespan extension is dependent on functional Sir2, and is not observed when nutrients are restricted . Together these data indicate that STACs slow metazoan ageing by mechanisms that may be related to caloric restriction.

Mol Cell Biol, 2004 Aug, 24(15), 6710 - 8
mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells; Murakami M et al.; TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes . In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control . However, the in vivo functions of mammalian TOR (mTOR) remain unclear . In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination . While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments . Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro . Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells . These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells.

J Proteome Res, 2004 May-Jun, 3(3), 417 - 25
Toward a general chemical method for rapidly mapping multi-protein complexes; Denison C et al.; Ru(II)(bpy2)32+Cl2, ammonium persulfate, and visible light irradiation has been shown to rapidly and efficiently cross-link several interacting proteins . However, this methodology has not yet been used to map the architecture of large multi-protein complexes . In this study, this chemistry is applied to the crystallographically characterized yeast proteasome . The data obtained demonstrate both the method's increased generality and fidelity in comparison to traditional bifunctional cross-linking reagents, while also highlighting the future need for developing better analytical techniques to separate cross-linked products.

Arch Environ Contam Toxicol, 2004 May, 46(4), 502 - 10
Comparison of two sites in Mobile Bay using in vivo biomarkers in largemouth bass, sediment bioassays, and sediment contaminant analysis; Annavarapu S et al.; This research addresses whether chemical contaminants are having a biologically measurable effect on largemouth bass (Micropterus salmoides), an important fish species in the Mobile Bay National Estuary . Bass and sediment samples were collected in September 2001 from Big Bateau and D'Olive Bays; the latter has been suggested to be an environmentally impacted site . There was a significant difference in the age distribution of bass collected from the two sites . However, none of the animals showed any evidence of reproductive activity or had mature gametes . The age and gonadosomatic index were greater in animals collected from the Bateau site . Fish liver microsome ethoxyresorufin-O-deethylase (EROD) activities (elevated in the presence of CYP1A-inducing contaminants) were not significantly different between sites but were significantly higher in older bass . Plasma steroid concentrations in younger animals (less than 1 year old) tended to be higher and more variable than the concentrations found in older animals . Once the significant effects of age on plasma steroid concentrations were removed, an analysis of the residual change in steroids revealed no differences in testosterone or estradiol between animals collected at the two sites . Sediments were collected for chemical extraction and analysis in two bioassays: the H4IIE rat hepatoma EROD assay and the yeast estrogen screen (YES assay) . The H4IIE bioassay indicated the presence of sediment BaP induction equivalents between 72 and 320 ng/g, whereas the YES assay indicated that potent estrogenic substances were not present in the sediment samples . The H4IIE bioassay results were higher for D'Olive samples, which was consistent with higher PAH concentrations in those sediments . Taken together the biomarkers and chemical analysis suggested generally low organic contamination at these two sites.

J Biol Chem, 2004 Sep 10, 279(37), 38673 - 82 Epub 2004 Jul 12.
A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi; Montalvetti A et al.; We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic . The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer . TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated . Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl(2) and AgNO(3) . glycerol transport was detected . No Immunofluorescence microscopy of T . cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H(+)-PPase), a marker of acidocalcisomes . This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP . In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex . Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T . cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.

BMC Bioinformatics . 2004 Jul 13;5(1):95.
Clustering proteins from interaction networks for the prediction of cellular functions; Brun C et al.; BACKGROUND: Developing reliable and efficient strategies allowing to infer a function to yet uncharacterized proteins based on interaction networks is of crucial interest in the current context of high-throughput data generation . In this paper, we develop a new algorithm for clustering vertices of a protein-protein interaction network using a density function, providing disjoint classes . RESULTS: Applied to the yeast interaction network, the classes obtained appear to be biological significant . The partitions are then used to make functional predictions for uncharacterized yeast proteins, using an annotation procedure that takes into account the binary interactions between proteins inside the classes . We show that this procedure is able to enhance the performances with respect to previous approaches . Finally, we propose a new annotation for 37 previously uncharacterized yeast proteins . CONCLUSION: We believe that our results represent a significant improvement for the inference of cellular functions, that can be applied to other organism as well as to other type of interaction graph, such as genetic interactions.

Proc Natl Acad Sci U S A, 2004 Jul 20, 101(29), 10572 - 7 Epub 2004 Jul 12.
Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation; Chen YK et al.; Saccharomyces cerevisiae Hop2 and Mnd1 are abundant meiosisspecific chromosomal proteins, and mutations in the corresponding genes lead to defects in meiotic recombination and in homologous chromosome interactions during mid-prophase . Analysis of various double mutants suggests that HOP2, MND1, and DMC1 act in the same genetic pathway for the establishment of close juxtaposition between homologous meiotic chromosomes . Biochemical studies indicate that Hop2 and Mnd1 proteins form a stable heterodimer with a higher affinity for double-stranded than single-stranded DNA, and that this heterodimer stimulates the strand assimilation activity of Dmc1 in vitro . Together, the genetic and biochemical results suggest that Hop2, Mnd1, and Dmc1 are functionally interdependent during meiotic DNA recombination.

Biochem Biophys Res Commun, 2004 Aug 6, 320(4), 1271 - 6
Oligomeric Aip2p/Dld2p forms a novel grapple-like structure and has an ATP-dependent F-actin conformation modifying activity in vitro; Hachiya NS et al.; In order to investigate the molecular mechanism of the F-actin conformation modifying activity {Biochem . Biophys . Res . Commun . 319 (2004) 78} of actin-interacting protein 2 (Aip2p) {Nat . Struct . Biol . 2 (1995) 28}/D-lactate dehydrogenase protein 2 (Dld2p) {Yeast 15 (1999) 1377; Biochem . Biophys . Res . Commun . 295 (2002) 910}, the ultrastructure and the regulatory mechanism of the activity were further examined . Interestingly, a novel oligomeric grapple-like structure of 10-12 subunits with an ATP-dependent opening was observed . ATP regulates the opening and closing of the "gate" that forms the opening within oligomeric Aip2p/Dld2p, where binding to the substrate occurs while in the open form . In the presence of ATP (open state of oligomeric Aip2p/Dld2p), oligomeric Aip2p/Dld2p bound the F-actin fiber within the opening, whereas in the absence of ATP (closed state of oligomeric Aip2p/Dld2p), no binding was observed . Simultaneously, the oligomeric Aip2p/Dld2p increased the trypsin susceptibility of F-actin in an ATP-dependent manner . Use of the non-hydrolyzable ATP analogue AMP-PNP yielded similar results to those observed with ATP, suggesting that ATP binding rather than ATP hydrolysis is required for the protein conformation modifying reaction of oligomeric Aip2p/Dld2p . Endogenous Aip2p/Dld2p purified from Saccharomyces cerevisiae also exhibited such protein conformation modifying activity, but monomeric Aip2p/Dld2p with a C-terminal coiled-coil region-truncation failed to exhibit the activity . These data suggest that the oligomerization of Aip2p/Dld2p, which exhibits the unique grapple-like structure with an ATP-dependent opening, is required for the F-actin conformation modifying activity.

Nat Genet, 2004 Aug, 36(8), 900 - 5 Epub 2004 Jul 11.
Evidence for nucleosome depletion at active regulatory regions genome-wide; Lee CK et al.; The identification of nuclease-hypersensitive sites in an active globin gene and in the 5' regions of fruit fly heat shock genes first suggested that chromatin changes accompany gene regulation in vivo . Here we present evidence that the basic repeating units of eukaryotic chromatin, nucleosomes, are depleted from active regulatory elements throughout the Saccharomyces cerevisiae genome in vivo . We found that during rapid mitotic growth, the level of nucleosome occupancy is inversely proportional to the transcriptional initiation rate at the promoter . We also observed a partial loss of histone H3 and H4 tetramers from the coding regions of the most heavily transcribed genes . Alterations in the global transcriptional program caused by heat shock or a change in carbon source resulted in an increased nucleosome occupancy at repressed promoters, and a decreased nucleosome occupancy at promoters that became active . Nuclease-hypersensitive sites occur in species from yeast to humans and result from chromatin perturbation . Given the conservation of sequence and function among components of both chromatin and the transcriptional machinery, nucleosome depletion at promoters may be a fundamental feature of eukaryotic transcriptional regulation.

Science, 2004 Jul 9, 305(5681), 242 - 5
Frataxin acts as an iron chaperone protein to modulate mitochondrial aconitase activity; Bulteau AL et al.; Numerous degenerative disorders are associated with elevated levels of prooxidants and declines in mitochondrial aconitase activity . Deficiency in the mitochondrial iron-binding protein frataxin results in diminished activity of various mitochondrial iron-sulfur proteins including aconitase . We found that aconitase can undergo reversible citrate-dependent modulation in activity in response to pro-oxidants . Frataxin interacted with aconitase in a citrate-dependent fashion, reduced the level of oxidant-induced inactivation, and converted inactive {3Fe-4S}1+ enzyme to the active {4Fe-4S}2+ form of the protein . Thus, frataxin is an iron chaperone protein that protects the aconitase {4Fe-4S}2+ cluster from disassembly and promotes enzyme reactivation.

Plant Physiol, 2004 Jul, 135(3), 1324 - 35 Epub 2004 Jul 09.
Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis; Stahl U et al.; A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, {2000} Proc Natl Acad Sci USA 97: 6487-6492) . The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase . A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity) . The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter . Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT) . The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22 . The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups . The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position . The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type . Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols . However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.

J Biol Chem, 2004 Sep 10, 279(37), 38861 - 70 Epub 2004 Jul 06.
Mechanistic analysis of the mitotic kinesin Eg5; Cochran JC et al.; Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division . We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration . The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm . 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1) . ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1) . Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt) . These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.

J Biol Chem, 2004 Sep 10, 279(37), 39068 - 74 Epub 2004 Jul 09.
Uth1p is involved in the autophagic degradation of mitochondria; Kissova I et al.; The absence of the outer mitochondrial membrane protein Uth1p was found to induce resistance to rapamycin treatment and starvation, two conditions that induce the autophagic process . Biochemical studies showed the onset of a fully active autophagic activity both in wild-type and Deltauth1 strains . On the other hand, the disorganization of the mitochondrial network induced by rapamycin treatment or 15 h of nitrogen starvation was followed in cells expressing mitochondria-targeted green fluorescent protein; a rapid colocalization of green fluorescent protein fluorescence with vacuole-selective FM4-64 labeling was observed in the wild-type but not in the Deltauth1 strain . Degradation of mitochondrial proteins, followed by Western blot analysis, did not occur in mutant strains carrying null mutations of the vacuolar protease Pep4p, the autophagy-specific protein Atg5p, and Uth1p . These data show that, although the autophagic machinery was fully functional in the absence of Uth1p, this protein is involved in the autophagic degradation of mitochondria.

J Biol Chem, 2004 Sep 10, 279(37), 38338 - 45 Epub 2004 Jul 06.
Reversible assembly of the ATP-binding cassette transporter Mdl1 with the F1F0-ATP synthase in mitochondria; Galluhn D et al.; The half-ABC transporter Mdl1 is localized in the inner membrane of mitochondria and mediates the export of peptides generated upon proteolysis of mitochondrial proteins . The physiological role of the peptides released from mitochondria is currently not understood . Here, we have analyzed the oligomeric state of Mdl1 in the inner membrane and demonstrate nucleotide-dependent binding to the F(1)F(0)-ATP synthase . Mdl1 forms homo-oligomeric, presumably dimeric complexes in the presence of ATP, but was found in association with the F(1)F(0)-ATP synthase at low ATP levels . Mdl1 binds membrane-embedded parts of the ATP synthase complex after the assembly of the F(1) and F(0) moieties . Although independent of Mdl1 activity, complex formation is impaired upon inhibition of the F(1)F(0)-ATP synthase with oligomycin or N,N'-dicyclohexylcarbodiimide . These results are consistent with an activation of Mdl1 upon dissociation from the ATP synthase and suggest a link of peptide export from mitochondria to the activity of the F(1)F(0)-ATP synthase and the cellular energy metabolism.

Ann N Y Acad Sci, 2004 Jun, 1019, 260 - 4
Mitochondrial dysfunction is a common phenotype in aging and cancer; Singh KK; An interesting clue with regard to molecular mechanisms underlying age-associated cancers is the apparent defect in mitochondrial function . Recent studies demonstrate a progressive decline in mitochondrial function during aging . Studies have established that the decline in mitochondrial function is due to the accumulation of mutations in mitochondrial DNA . These observations suggest that the mitochondrial dysfunction that accompanies aging may exert a major influence on carcinogenesis.

Arch Biochem Biophys, 2004 Aug 15, 428(2), 165 - 9
Interaction of Doppel with the full-length laminin receptor precursor protein; Yin SM et al.; Doppel (Dpl) is a homolog of normal cellular prion protein (PrPc) with unknown functions . Ectopic expression of Dpl in the central nervous system (CNS) causes neurotoxicity and this effect is rescued by the expression of PrPc . However, the molecular basis for the protective effect of PrPc remains unclear . Using a yeast two-hybrid system, we showed that Dpl binds the full-length 37-kDa laminin receptor precursor protein (LRP), one of the receptors of PrPc . The interaction was also validated by immunoprecipitation and immunoblotting using transfected cell lines and in vivo derived tissues . Further mapping experiments showed that although the middle fragment containing residues 100-220 of LRP was able to interact with Dpl, deletion of the N-terminal domain of the full-length LRP abolished its interaction with Dpl . These results suggest that while both PrPc and Dpl interact with LRP, the domains that are involved in the binding are not the same . Our results may have implications for the molecular mechanisms of Dpl-PrPc antagonism and physiological roles of Dpl.

Vaccine, 2004 Jul 29, 22(21-22), 2800 - 5
Insertional mutagenesis of ricin A chain: a novel route to an anti-ricin vaccine; Marsden CJ et al.; The insertion of a specific 25-residue internal peptide into ricin toxin A chain (RTA) reduced the catalytic activity of this protein approximately 300-fold . Directed proteolytic cleavage of the peptide insert essentially restored catalytic activity of the resulting two peptide A chain to normal levels . Ricin holotoxin containing unprocessed mutant A chain was not toxic to cultured mammalian cells, due to enhanced proteasomal degradation, nor was it toxic when injected into rats at a concentration that is lethal in the case of native ricin . Rats treated in this way were completely resistant to native ricin when subsequently challenged with a potentially lethal dose of the toxin . These ricin-resistant animals had a significant anti-ricin antibody titer, indicating that this approach has potential for developing an effective vaccine against this toxin.

Biosystems, 2004 Jul, 75(1-3), 57 - 65
Dynamic Bayesian network and nonparametric regression for nonlinear modeling of gene networks from time series gene expression data; Kim S et al.; We propose a dynamic Bayesian network and nonparametric regression model for constructing a gene network from time series microarray gene expression data . The proposed method can overcome a shortcoming of the Bayesian network model in the sense of the construction of cyclic regulations . The proposed method can analyze the microarray data as a continuous data and can capture even nonlinear relations among genes . It can be expected that this model will give a deeper insight into complicated biological systems . We also derive a new criterion for evaluating an estimated network from Bayes approach . We conduct Monte Carlo experiments to examine the effectiveness of the proposed method . We also demonstrate the proposed method through the analysis of the Saccharomyces cerevisiae gene expression data.

Cell, 2004 Jul 9, 118(1), 99 - 110
Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system; Verma R et al.; Recruitment of ubiquitinated proteins to the 26S proteasome lies at the heart of the ubiquitin-proteasome system (UPS) . Genetic studies suggest a role for the multiubiquitin chain binding proteins (MCBPs) Rad23 and Rpn10 in recruitment, but biochemical studies implicate the Rpt5 ATPase . We addressed this issue by analyzing degradation of the ubiquitinated Cdk inhibitor Sic1 (UbSic1) in vitro . Mutant rpn10Delta and rad23Delta proteasomes failed to bind or degrade UbSic1 . Although Rpn10 or Rad23 restored UbSic1 recruitment to either mutant, rescue of degradation by Rad23 uncovered a requirement for the VWA domain of Rpn10 . In vivo analyses confirmed that Rad23 and the multiubiquitin binding domain of Rpn10 contribute to Sic1 degradation . Turnover studies of multiple UPS substrates uncovered an unexpected degree of specificity in their requirements for MCBPs . We propose that recruitment of substrates to the proteasome by MCBPs provides an additional layer of substrate selectivity in the UPS.

Cell, 2004 Jul 9, 118(1), 31 - 44
Exploration of essential gene functions via titratable promoter alleles; Mnaimneh S et al.; Nearly 20% of yeast genes are required for viability, hindering genetic analysis with knockouts . We created promoter-shutoff strains for over two-thirds of all essential yeast genes and subjected them to morphological analysis, size profiling, drug sensitivity screening, and microarray expression profiling . We then used this compendium of data to ask which phenotypic features characterized different functional classes and used these to infer potential functions for uncharacterized genes . We identified genes involved in ribosome biogenesis (HAS1, URB1, and URB2), protein secretion (SEC39), mitochondrial import (MIM1), and tRNA charging (GSN1) . In addition, apparent negative feedback transcriptional regulation of both ribosome biogenesis and the proteasome was observed . We furthermore show that these strains are compatible with automated genetic analysis . This study underscores the importance of analyzing mutant phenotypes and provides a resource to complement the yeast knockout collection.

Nature, 2004 Jul 8, 430(6996), 223 - 6
Recognition of RNA polymerase II carboxy-terminal domain by 3'-RNA-processing factors; Meinhart A et al.; During transcription, RNA polymerase (Pol) II synthesizes eukaryotic messenger RNA . Transcription is coupled to RNA processing by the carboxy-terminal domain (CTD) of Pol II, which consists of up to 52 repeats of the sequence Tyr 1-Ser 2-Pro 3-Thr 4-Ser 5-Pro 6-Ser 7 (refs 1, 2) . After phosphorylation, the CTD binds tightly to a conserved CTD-interacting domain (CID) present in the proteins Pcf11 and Nrd1, which are essential and evolutionarily conserved factors for polyadenylation-dependent and -independent 3'-RNA processing, respectively . Here we describe the structure of a Ser 2-phosphorylated CTD peptide bound to the CID domain of Pcf11 . The CTD motif Ser 2-Pro 3-Thr 4-Ser 5 forms a beta-turn that binds to a conserved groove in the CID domain . The Ser 2 phosphate group does not make direct contact with the CID domain, but may be recognized indirectly because it stabilizes the beta-turn with an additional hydrogen bond . Iteration of the peptide structure results in a compact beta-spiral model of the CTD . The model suggests that, during the mRNA transcription-processing cycle, compact spiral regions in the CTD are unravelled and regenerated in a phosphorylation-dependent manner.

Nucleic Acids Res, 2004 Jul 07, 32(12), 3581 - 9 Print 2004.
New strategy for the representation and the integration of biomolecular knowledge at a cellular scale; Barriot R et al.; The combination of sequencing and post-sequencing experimental approaches produces huge collections of data that are highly heterogeneous both in structure and in semantics . We propose a new strategy for the integration of such data . This strategy uses structured sets of sequences as a unified representation of biological information and defines a probabilistic measure of similarity between the sets . Sets can be composed of sequences that are known to have a biological relationship (e.g . proteins involved in a complex or a pathway) or that share similar values for a particular attribute (e.g . expression profile) . We have developed a software, BlastSets, which implements this strategy . It exploits a database where the sets derived from diverse biological information can be deposited using a standard XML format . For a given query set, BlastSets returns target sets found in the database whose similarity to the query is statistically significant . The tool allowed us to automatically identify verified relationships between correlated expression profiles and biological pathways using publicly available data for Saccharomyces cerevisiae . It was also used to retrieve the members of a complex (ribosome) based on the mining of expression profiles . These first results validate the relevance of the strategy and demonstrate the promising potential of BlastSets.

Biochem Biophys Res Commun, 2004 Jul 30, 320(3), 840 - 5
Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage; Saeki Y et al.; Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance . A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear . To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast . Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage.

Biochem Biophys Res Commun, 2004 Jul 30, 320(3), 648 - 55
Initiation sites for human DNA replication at a putative ribulose-5-phosphate 3-epimerase gene; Hu L et al.; Replication of the human genome requires the activation of thousands of replicons distributed along each one of the chromosomes . Each replicon contains an initiation, or origin, site, at which DNA synthesis begins . However, very little information is known about the nature and positioning of these initiation sites along human chromosomes . We have recently focused our attention to a 1.1 kb region of human chromosome 2 which functioned as an episomal origin in the yeast Saccharomyces cerevisiae . This region corresponded to the largest exon of a putative ribulose-5-phosphate-3-epimerase gene (RPE) . In the present study we have used a real-time PCR-based nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 13.4 kb region encompassing the putative RPE gene . By applying this analysis to a 1-1.4 kb nascent strand DNA fraction isolated from both normal skin fibroblasts, and the breast cell line MCF10; we have identified five initiation sites within the 13.4 kb region of chromosome 2 . The initiation sites appear to map to similar positions in both cell lines and occur outside the coding regions of the putative RPE gene.

Dev Cell, 2004 Jul, 7(1), 61 - 71
The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane; Meisinger C et al.; The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex) . The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40 . We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology . Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected . Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology . We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology.

BMC Bioinformatics . 2004 Jul 07;5(1):91.
SPOC: a widely distributed domain associated with cancer, apoptosis and transcription; Sanchez-Pulido L et al.; BACKGROUND: The Split ends (Spen) family are large proteins characterised by N-terminal RNA recognition motifs (RRMs) and a conserved SPOC (Spen paralog and ortholog C-terminal) domain . The aim of this study is to characterize the family at the sequence level . RESULTS: We describe undetected members of the Spen family in other lineages (Plasmodium and Plants) and localise SPOC in a new domain context, in a family that is common to all eukaryotes using profile-based sequence searches and structural prediction methods . CONCLUSIONS: The widely distributed DIO (Death inducer-obliterator) family is related to cancer and apoptosis and offers new clues about SPOC domain functionality.

Genome Biol . 2004;5(7):R50 . Epub 2004 Jun 24.
Detecting DNA regulatory motifs by incorporating positional trends in information content; Kechris KJ et al.; On the basis of the observation that conserved positions in transcription factor binding sites are often clustered together, we propose a simple extension to the model-based motif discovery methods . We assign position-specific prior distributions to the frequency parameters of the model, penalizing deviations from a specified conservation profile . Examples with both simulated and real data show that this extension helps discover motifs as the data become noisier or when there is a competing false motif.

Genome Biol . 2004;5(7):R49 . Epub 2004 Jun 29.
Systematic quantification of gene interactions by phenotypic array analysis; Hartman JL 4th et al.; A phenotypic array method, developed for quantifying cell growth, was applied to the haploid and homozygous diploid yeast deletion strain sets . A growth index was developed to screen for non-additive interacting effects between gene deletion and induced perturbations . From a genome screen for hydroxyurea (HU) chemical-genetic interactions, 298 haploid deletion strains were selected for further analysis . The strength of interactions was quantified using a wide range of HU concentrations affecting reference strain growth . The selectivity of interaction was determined by comparison with drugs targeting other cellular processes . Bio-modules were defined as gene clusters with shared strength and selectivity of interaction profiles . The functions and connectivity of modules involved in processes such as DNA repair, protein secretion and metabolic control were inferred from their respective gene composition . The work provides an example of, and a general experimental framework for, quantitative analysis of gene interaction networks that buffer cell growth.

Genome Biol . 2004;5(7):R48 . Epub 2004 Jun 28.
From co-expression to co-regulation: how many microarray experiments do we need?
Yeung KY, Medvedovic M, Bumgarner RE.
BACKGROUND: Cluster analysis is often used to infer regulatory modules or biological function by associating unknown genes with other genes that have similar expression patterns and known regulatory elements or functions . However, clustering results may not have any biological relevance . RESULTS: We applied various clustering algorithms to microarray datasets with different sizes, and we evaluated the clustering results by determining the fraction of gene pairs from the same clusters that share at least one known common transcription factor . We used both yeast transcription factor databases (SCPD, YPD) and chromatin immunoprecipitation (ChIP) data to evaluate our clustering results . We showed that the ability to identify co-regulated genes from clustering results is strongly dependent on the number of microarray experiments used in cluster analysis and the accuracy of these associations plateaus at between 50 and 100 experiments on yeast data . Moreover, the model-based clustering algorithm MCLUST consistently outperforms more traditional methods in accurately assigning co-regulated genes to the same clusters on standardized data . CONCLUSIONS: Our results are consistent with respect to independent evaluation criteria that strengthen our confidence in our results . However, when one compares ChIP data to YPD, the false-negative rate is approximately 80% using the recommended p-value of 0.001 . In addition, we showed that even with large numbers of experiments, the false-positive rate may exceed the true-positive rate . In particular, even when all experiments are included, the best results produce clusters with only a 28% true-positive rate using known gene transcription factor interactions.

J Mol Biol, 2004 Jul 23, 340(5), 1197 - 206
Pairwise interactions of the six human MCM protein subunits; Yu Z et al.; The eukaryotic minichromosome maintenance (MCM) proteins have six subunits, Mcm2 to 7p . Together they play essential roles in the initiation and elongation of DNA replication, and the human MCM proteins present attractive targets for potential anticancer drugs . The six MCM subunits interact and form a ring-shaped heterohexameric complex containing one of each subunit in a variety of eukaryotes, and subcomplexes have also been observed . However, the architecture of the human MCM heterohexameric complex is still unknown . We systematically studied pairwise interactions of individual human MCM subunits by using the yeast two-hybrid system and in vivo protein-protein crosslinking with a non-cleavable crosslinker in human cells followed by co-immunoprecipitation . In the yeast two-hybrid assays, we revealed multiple binary interactions among the six human MCM proteins, and a subset of these interactions was also detected as direct interactions in human cells . Based on our results, we propose a model for the architecture of the human MCM protein heterohexameric complex . We also propose models for the structures of subcomplexes . Thus, this study may serve as a foundation for understanding the overall architecture and function of eukaryotic MCM protein complexes and as clues for developing anticancer drugs targeted to the human MCM proteins.

Nat Biotechnol, 2004 Aug, 22(8), 1013 - 6 Epub 2004 Jul 04.
High-resolution genome-wide mapping of histone modifications; Roh TY et al.; The expression patterns of eukaryotic genomes are controlled by their chromatin structure, consisting of nucleosome subunits in which DNA of approximately 146 bp is wrapped around a core of 8 histone molecules . Post-translational histone modifications play an essential role in modifying chromatin structure . Here we apply a combination of SAGE and chromatin immunoprecipitation (ChIP) protocols to determine the distribution of hyperacetylated histones H3 and H4 in the Saccharomyces cerevisiae genome . We call this approach genome-wide mapping technique (GMAT) . Using GMAT, we find that the highest acetylation levels are detected in the 5' end of a gene's coding region, but not in the promoter . Furthermore, we show that the histone acetyltransferase, GCN5p, regulates H3 acetylation in the promoter and 5' end of the coding regions . These findings indicate that GMAT should find valuable applications in mapping target sites of chromatin-modifying enzymes.

Nat Struct Mol Biol, 2004 Aug, 11(8), 738 - 46 Epub 2004 Jul 04.
HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations; Swanson KA et al.; Recruitment of the histone deacetylase (HDAC)-associated Sin3 corepressor is an obligatory step in many eukaryotic gene silencing pathways . Here we show that HBP1, a cell cycle inhibitor and regulator of differentiation, represses transcription in a HDAC/Sin3-dependent manner by targeting the mammalian Sin3A (mSin3A) PAH2 domain . HBP1 is unrelated to the Mad1 repressor for which high-resolution structures in complex with PAH2 have been described . We show that like Mad1, the HBP1 transrepression domain binds through a helical structure to the hydrophobic cleft of mSin3A PAH2 . Notably, the HBP1 helix binds PAH2 in a reversed orientation relative to Mad1 and, equally unexpectedly, this is correlated with a chain reversal of the minimal Sin3 interaction motifs . These results not only provide insights into how multiple, unrelated transcription factors recruit the same coregulator, but also have implications for how sequence similarity searches are conducted.

Oncogene, 2004 Aug 26, 23(39), 6590 - 602
Paired-like homeodomain protein ESXR1 possesses a cleavable C-terminal region that inhibits cyclin degradation; Ozawa H et al.; The eukaryotic cell cycle is regulated by sequential activation and inactivation of cyclin-cyclin-dependent kinase (Cdk) complexes . In this work, we screened human cDNAs that can rescue yeast Saccharomyces cerevisiae from lethality caused by ectopic expression of human cyclin E and isolated a cDNA encoding ESXR1, a paired-like homeodomain-containing protein with a unique C-terminal proline-rich repeat region . In adult tissues, ESXR1 is primarily expressed in the testis . We demonstrate that ESXR1 prevents degradation of ubiquitinated cyclins in human cells . Accordingly, elevation of ESXR1 level results in accumulation of cyclin A and cyclin B1 and thereby provokes M-phase arrest . In human cells, the 65-kDa full-length ESXR1 protein is capable of proteolytically processing into N-terminal 45-kDa and C-terminal 20-kDa fragments . The C-terminal fragment, containing a proline-rich repeat region, is localized to the cytoplasm and displays the ability to inhibit cyclin degradation . In contrast, the N-terminal fragment, containing a paired-like homeodomain, is localized exclusively in the nucleus, suggesting that it plays a role in transcription . Our results indicate that proteolytic processing of ESXR1 plays a role in concerted regulation of the cell cycle and transcription in human cells.

J Am Soc Mass Spectrom, 2004 Jul, 15(7), 1099 - 108
Construction of a hybrid quadrupole/Fourier transform ion cyclotron resonance mass spectrometer for versatile MS/MS above 10 kDa; Patrie SM et al.; Technological advancements including an open-cylindrical Penning trap with capacitively coupled ICR cell, selective ion accumulation with a resolving quadrupole, and a voltage gradient used during ion extraction from an octopole ion trap, have individually improved dynamic range and sensitivity in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) . Documented here is a new instrument utilizing these technologies toward the robust detection and fragmentation of biomolecules >10 kDa . Up to 55-fold enhancement in ion population by selective ion accumulation combined with 10- to 20- fold signal-to-noise improvement by application of a DC voltage gradient to an accumulation octopole during the ion transfer event offers improved signal-to-noise (or speed) of MS/MS experiments, for proteins from Methanococcus jannaschii and Saccharomyces cerevisiae whole cell lysates . After external quadrupole filtering with a 40 m/z window, three proteins were fragmented (and identified) in parallel from the database of Methanococcus jannaschii . Electron capture dissociation (ECD) of an intact yeast protein provides extensive sequence information resulting in a high degree of localization for an N-terminal acetylation . Hybrid fragmentation, infrared multiphoton dissociation (IRMPD) followed by low energy electrons (ECD), with the electron source located laterally off the z-axis and external to the magnet bore, presents a strategy for identification of proteins by means of the sequence tag approach . Automated implementation of diverse MS(n) approaches in a Q-FTMS instrument promises to help realize "top-down" proteomics in the future.

Neurochem Int, 2004 Oct, 45(5), 765 - 72
JAB1 participates in unfolded protein responses by association and dissociation with IRE1; Oono K et al.; Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER) . IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER . IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death . However, little is known about the regulatory mechanisms that select between the UPR and apoptosis . We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system . We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers . Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha . These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.

Eur J Biochem, 2004 Jul, 271(14), 2991 - 7
Properties of two multifunctional plant fatty acid acetylenase/desaturase enzymes; Carlsson AS et al.; The properties of the Delta6 desaturase/acetylenase from the moss Ceratodon purpureus and the Delta12 acetylenase from the dicot Crepis alpina were studied by expressing the encoding genes in Arabidopsis thaliana and Saccharomyces cerevisiae . The acetylenase from C . alpinaDelta12 desaturated both oleate and linoleate with about equal efficiency . The desaturation of oleate gave rise to 9(Z),12(E)- and 9(Z),12(Z)-octadecadienoates in a ratio of approximately 3 : 1 . Experiments using stereospecifically deuterated oleates showed that the pro-R hydrogen atoms were removed from C-12 and C-13 in the introduction of the 12(Z) double bond, whereas the pro-R and pro-S hydrogen atoms were removed from these carbons during the formation of the 12(E) double bond . The results suggested that the Delta12 acetylenase could accommodate oleate having either a cisoid or transoid conformation of the C(12)-C(13) single bond, and that these conformers served as precursors of the 12(Z) and 12(E) double bonds, respectively . However, only the 9(Z),12(Z)-octadecadienoate isomer could be further desaturated to 9(Z)-octadecen-12-ynoate (crepenynate) by the enzyme . The evolutionarily closely related Delta12 epoxygenase from Crepis palaestina had only weak desaturase activity but could also produce 9(Z),12(E)-octadecadienoate from oleate . The Delta6 acetylenase/desaturase from C . purpureus, on the other hand, produced only the 6(Z) isomers using C16 and C18 acyl groups possessing a Delta9 double bond as substrates . The Delta6 double bond was efficiently further converted to an acetylenic bond by a second round of desaturation but only if the acyl substrate had a Delta12 double bond and that this was in the Z configuration.

Eur J Biochem, 2004 Jul, 271(14), 2923 - 36
Two functionally redundant isoforms of Drosophila melanogaster eukaryotic initiation factor 4B are involved in cap-dependent translation, cell survival, and proliferation; Hernandez G et al.; Eukaryotic initiation factor (eIF) 4B is part of the protein complex involved in the recognition and binding of mRNA to the ribosome . DrosophilaeIF4B is a single-copy gene that encodes two isoforms, termed eIF4B-L (52.2 kDa) and eIF4B-S (44.2 kDa), generated as a result of the alternative recognition of two polyadeynlation signals during transcription termination and subsequent alternative splicing of the two pre-mRNAs . Both eIF4B mRNAs and proteins are expressed during the entire embryogenesis and life cycle . The proteins are cytoplasmic with polarized distribution . The two isoforms bind RNA with the same affinity . eIF4B-L and eIF4B-S preferentially enhance cap-dependent over IRES-dependent translation initiation in a Drosophila cell-free translation system . RNA interference experiments suggest that eIF4B is required for cell survival, although only a modest reduction in rate of protein synthesis is observed . Overexpression of eIF4B in Drosophila cells in culture and in developing eye imaginal discs promotes cell proliferation.

J Biol Chem, 2004 Sep 3, 279(36), 37385 - 97 Epub 2004 Jun 30.
Functional characterization of Pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle; Dosil M et al.; Here we report the functional characterization of Pwp2, an evolutionary conserved component of the 90 S pre-ribosome . Conditional depletion of the Pwp2 protein in yeast specifically impairs pre-rRNA proccessing at sites A(0), A(1), and A(2), leading to a strong decrease in 18 S rRNA and 40 S ribosomal subunit levels . Pre-ribosomal particle sedimentation analysis indicated that these defects are caused by a block in the formation of 90 S pre-ribosomes . We demonstrate that in Pwp2-depleted cells the U3 small nucleolar ribonucleoprotein is not able to interact with the 35 S pre-rRNA and accumulates as a free complex . Similarly, other 90 S particle components such as Imp3 and Imp4 do not associate with the pre-rRNA precursor in the absence of Pwp2 . In addition, we have found that after blocking U3 ribonucleoprotein assembly, Pwp2 predominantly accumulates as a complex in association with five proteins: Dip2, Utp6, Utp13, Utp18, and Utp21 . Immunoprecipitation and gradient sedimentation analysis revealed that this Pwp2 small subcomplex is capable of interacting directly with the 35 S pre-rRNA 5' end . Taken together, these results indicate that Pwp2 forms part of a stable particle subunit independent of the U3 small nucleolar ribonucleoprotein that is essential for the initial assembly steps of the 90 S pre-ribosome.

Genome Res, 2004 Jul, 14(7), 1315 - 23
A protein interaction framework for human mRNA degradation; Lehner B et al.; The degradation of mRNA is an important regulatory step in the control of gene expression . However, mammalian RNA decay pathways remain poorly characterized . To provide a framework for studying mammalian RNA decay, a two-hybrid protein interaction map was generated using 54 constructs from 38 human proteins predicted to function in mRNA decay . The results provide evidence for interactions between many different proteins required for mRNA decay . Of particular interest are interactions between the poly(A) ribonuclease and the exosome and between the Lsm complex, decapping factors, and 5'-->3' exonucleases . Moreover, multiple interactions connect 5'-->3' and 3'-->5' decay proteins to each other and to nonsense-mediated decay factors, providing the opportunity for coordination between decay pathways . The interaction network also predicts the internal organization of the exosome and Lsm complexes . Additional interactions connect mRNA decay factors to many novel proteins and to proteins required for other steps in gene expression . These results provide an experimental insight into the organization of proteins required for mRNA decay and their coupling to other cellular processes, and the physiological relevance of many of these interactions are supported by their evolutionary conservation . The interactions also provide a wealth of hypotheses to guide future research on mRNA degradation and demonstrate the power of exhaustive protein interaction mapping in aiding understanding of uncharacterized protein complexes and pathways .

Nature, 2004 Jul 29, 430(6999), 573 - 8 Epub 2004 Jun 30.
Cohesin relocation from sites of chromosomal loading to places of convergent transcription; Lengronne A et al.; Sister chromatids, the products of eukaryotic DNA replication, are held together by the chromosomal cohesin complex after their synthesis . This allows the spindle in mitosis to recognize pairs of replication products for segregation into opposite directions . Cohesin forms large protein rings that may bind DNA strands by encircling them, but the characterization of cohesin binding to chromosomes in vivo has remained vague . We have performed high resolution analysis of cohesin association along budding yeast chromosomes III-VI . Cohesin localizes almost exclusively between genes that are transcribed in converging directions . We find that active transcription positions cohesin at these sites, not the underlying DNA sequence . Cohesin is initially loaded onto chromosomes at separate places, marked by the Scc2/Scc4 cohesin loading complex, from where it appears to slide to its more permanent locations . But even after sister chromatid cohesion is established, changes in transcription lead to repositioning of cohesin . Thus the sites of cohesin binding and therefore probably sister chromatid cohesion, a key architectural feature of mitotic chromosomes, display surprising flexibility . Cohesin localization to places of convergent transcription is conserved in fission yeast, suggesting that it is a common feature of eukaryotic chromosomes.

J Biol Chem, 2004 Sep 10, 279(37), 38532 - 43 Epub 2004 Jun 30.
The stress-induced Tfs1p requires NatB-mediated acetylation to inhibit carboxypeptidase Y and to regulate the protein kinase A pathway; Caesar R et al.; The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p . We found by two-dimensional PAGE analysis that nat3Delta exhibited protein expression during growth in basal medium resembling protein expression in salt-adapted wild-type cells . The stress-induced carboxypeptidase Y (CPY) inhibitor and phosphatidylethanolamine-binding protein family member Tfs1p was identified as a novel NatB substrate . The N-terminal acetylation status of Tfs1p, Act1p, and Rnr4p in both wild type and nat3Delta was confirmed by tandem mass spectrometry . Furthermore it was found that unacetylated Tfs1p expressed in nat3Delta showed an approximately 100-fold decrease in CPY inhibition compared with the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfs1p . Phosphatidylethanolamine-binding proteins in other organisms have been reported to be involved in the regulation of cell signaling . Here we report that a number of proteins, whose expression has been shown previously to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found to be regulated in line with low PKA activity in the nat3Delta strain . The involvement of Nat3p and Tfs1p in PKA signaling was supported by caffeine growth inhibition studies . First, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs1Delta . Second, this suppression by tfs1Delta was abolished in the nat3Delta background, indicating that Tfs1p was not functional in the nat3Delta strain possibly because of a lack of N-terminal acetylation . We conclude that the NatB-dependent acetylation of Tfs1p appears to be essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway.

J Biol Chem, 2004 Sep 17, 279(38), 39814 - 23 Epub 2004 Jun 30.
Retrograde transport of the mannosyltransferase Och1p to the early Golgi requires a component of the COG transport complex; Bruinsma P et al.; The yeast COG complex has been proposed to function as a vesicle-tethering complex on an early Golgi compartment, but its role is not fully understood . COG complex mutants exhibit a dramatic reduction in Golgi-specific glycosylation and other defects . Here we show that a strain carrying a COG3 temperature-sensitive allele, cog3-202, clearly exhibited the glycosylation defect while exhibiting nearly normal secretion kinetics . Two Golgi mannosyltransferases, Och1p and Mnn1p, were mislocalized in cog3-202 cells . In cog3-202 cells Och1-HA was found in lighter density membranes than in wild type cells . In sed5(ts) and sft1(ts) strains, Och1p rapidly accumulated in vesicle-like structures consistent with the delivery of Och1p back to the cis-Golgi on retrograde vesicles via a Sed5p/Sft1p-containing SNARE complex . In contrast to cog3-202 cells, the membranes in sed5(ts) cells that contained Och1p were denser than in wild type . Together these results indicate that Och1p does not accumulate in retrograde vesicles in the cog3-202 mutant and are consistent with the COG complex playing a role in sorting of Och1p into retrograde vesicles . In wild type cells Och1p has been shown previously to cycle between the cis-Golgi and minimally as far as the late Golgi . We find that Och1p does not cycle via endosomes during its normal itinerary suggesting that Och1p engages in intra-Golgi cycling only . However, Och1p does use a post-Golgi pathway for degradation because a portion of Och1p was degraded in the vacuole . Most surprisingly, Och1p can use either the carboxypeptidase Y or AP-3 pathways to reach the vacuole for degradation.

Mol Cell Biol, 2004 Jul, 24(14), 6514 - 24
Mcm1 promotes replication initiation by binding specific elements at replication origins; Chang VK et al.; Minichromosome maintenance protein 1 (Mcm1) is required for efficient replication of autonomously replicating sequence (ARS)-containing plasmids in yeast cells . Reduced DNA binding activity in the Mcm1-1 mutant protein (P97L) results in selective initiation of a subset of replication origins and causes instability of ARS-containing plasmids . This plasmid instability in the mcm1-1 mutant can be overcome for a subset of ARSs by the inclusion of flanking sequences . Previous work showed that Mcm1 binds sequences flanking the minimal functional domains of ARSs . Here, we dissected two conserved telomeric X ARSs, ARS120 (XARS6L) and ARS131a (XARS7R), that replicate with different efficiencies in the mcm1-1 mutant . We found that additional Mcm1 binding sites in the C domain of ARS120 that are missing in ARS131a are responsible for efficient replication of ARS120 in the mcm1-1 mutant . Mutating a conserved Mcm1 binding site in the C domain diminished replication efficiency in ARS120 in wild-type cells, and increasing the number of Mcm1 binding sites stimulated replication efficiency . Our results suggest that threshold occupancy of Mcm1 in the C domain of telomeric ARSs is required for efficient initiation . We propose that origin usage in Saccharomyces cerevisiae may be regulated by the occupancy of Mcm1 at replication origins.

Mol Cell Biol, 2004 Jul, 24(14), 6419 - 29
TATA-binding protein mutants that are lethal in the absence of the Nhp6 high-mobility-group protein; Eriksson P et al.; The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply . Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III . Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6 . The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB) . The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1 . Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation . It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA . Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant . Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU) . However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1 . Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity . These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.

Mol Cell Biol, 2004 Jul, 24(14), 6362 - 78
The NEF4 complex regulates Rad4 levels and utilizes Snf2/Swi2-related ATPase activity for nucleotide excision repair; Ramsey KL et al.; Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae . Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits . We report previously unrecognized similarity between Rad7 and F-box proteins . Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase . Mutational analysis provides strong support for this model . The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function . Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4 . Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria . Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23 . As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4.

Mol Cell Biol, 2004 Jul, 24(14), 6324 - 37
Npa1p, a component of very early pre-60S ribosomal particles, associates with a subset of small nucleolar RNPs required for peptidyl transferase center modification; Dez C et al.; We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p . In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely . Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles . By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor . Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center . Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.

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