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 experimen