S. cerevisiae

Anal Bioanal Chem, 2004 Feb, 378(3), 684 - 7 Epub 2003 Dec 05.
Luminescent enzyme-linked receptor assay for estrogenic compounds; Seifert M; The analytics of endocrine-disrupting compounds has become a major issue during recent years . Several test systems have been developed for endocrine-disrupting chemicals . Yeast reporter gene assays and MCF-7 cell-based proliferation assays (E-screen) are particularly popular . A correlation of an enzyme-linked receptor assay (ELRA) with a yeast reporter gene assay is shown . In addition, the development of an ultra-sensitive luminescent ELRA with a detection limit of 20 ng/L for 17 beta-estradiol in the sample is reported . Data for real sample analysis are shown in this paper . ELRA characteristics are compared with cell-based assays, and the issue of detection limits is addressed . In this context, the detection limits of the cell-based assays have been claimed to be below the ELRA detection limits . However, it is clarified that the given detection limits for the yeast estrogen screen and the E-screen are usually based on concentrations of 17 beta-estradiol in the well, not in the sample, whereas ELRA detection limits are concentrations in the sample.

Cell Cycle, 2004 Jan, 3(1), 61 - 3
Why do cells require heat shock proteins to survive heat stress?
Riezman H.
The cellular response to heat stress includes the induction of a group of proteins called the Heat Shock Proteins, whose functions include the synthesis of the thermoprotectant trehalose, refolding of denatured proteins, and ubiquitin- and proteasome-dependent degradation . Recent studies show that simply increasing the activity of ubiquitin- and proteasome-dependent degradation can replace the essential functions played by the induction of heat shock proteins during a heat stress . These results suggest that accumulation of denatured or aggregated proteins is the reason for the loss of cell viability due to heat stress.

Science, 2003 Dec 5, 302(5651), 1765 - 8
Spatiotemporal rescue of memory dysfunction in Drosophila; McGuire SE et al.; We have developed a method for temporal and regional gene expression targeting (TARGET) in Drosophila and show the simultaneous spatial and temporal rescue of a memory defect . The transient expression of the rutabaga-encoded adenylyl cyclase in the mushroom bodies of the adult brain was necessary and sufficient to rescue the rutabaga memory deficit, which rules out a developmental brain defect in the etiology of this deficit and demonstrates an acute role for rutabaga in memory formation in these neurons . The TARGET system offers general utility in simultaneously addressing issues of when and where gene products are required.

Mol Biol Cell, 2004 Feb, 15(2), 761 - 73 Epub 2003 Dec 02.
Exchangeable chaperone modules contribute to specification of type I and type II Hsp40 cellular function; Fan CY et al.; Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology . Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions . The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear . Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function . To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS . Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively . In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1 Delta and facilitate maintenance of the prion {RNQ+} . Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function.

Mol Biol Cell, 2004 Feb, 15(2), 468 - 80 Epub 2003 Dec 02.
Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S; Katzmann DJ et al.; The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole . Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway . Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes . This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326) . At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2 . A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS . However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants . We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2 . However, our analyses reveal that fab1 mutants do not exhibit a defect in ubiquitination of pCPS . Thus, both Rsp5 and Fab1 play distinct and essential roles in the targeting of biosynthetic MVB cargoes . However, whereas Rsp5 seems to be responsible for cargo ubiquitination, the precise role for Fab1 remains to be elucidated.

Mol Biol Cell, 2004 Feb, 15(2), 896 - 907 Epub 2003 Dec 02.
A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin; Moseley JB et al.; Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain . Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes . Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains . The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent association during elongation . This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1 . Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs . In addition, we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin . Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo . From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly.

EMBO J, 2003 Dec 15, 22(24), 6621 - 30
Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis; Guo C et al.; Pol kappa and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass {translesion DNA synthesis (TLS)} . We demonstrate that mouse Rev1 protein physically associates with Pol kappa . We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Pol zeta) and with two other Y-family polymerases, Pol iota and Pol eta . Mouse Pol kappa, Rev7, Pol iota and Pol eta each bind to the same approximately 100 amino acid C-terminal region of Rev1 . Furthermore, Rev7 competes directly with Pol kappa for binding to the Rev1 C-terminus . Notwithstanding the physical interaction between Rev1 and Pol kappa, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini . Our observations suggest that Rev1 plays a role(s) in mediating protein-protein interactions among DNA polymerases required for TLS . The precise function(s) of these interactions during TLS remains to be determined.

EMBO J, 2003 Dec 15, 22(24), 6584 - 97
Nsl1p is essential for the establishment of bipolarity and the localization of the Dam-Duo complex; Scharfenberger M et al.; We identified a physical complex consisting of Mtw1p, an established kinetochore protein, with Nnf1p, Nsl1p and Dsn1p and have demonstrated that Nnf1p, Nsl1p and Dsn1p localize to the Saccharomyces cerevisiae kinetochore . When challenged prior to metaphase, the temperature-sensitive mutants nsl1-16 and nsl1-42 as well as Nsl1p-depleted cells failed to establish a bipolar spindle-kinetochore interaction and executed monopolar segregation of sister chromatids . In contrast, an nsl1-16 defect could not be evoked after the establishment of bipolarity . The observed phenotype is characteristic of that of mutants with defects in the protein kinase Ipl1p or components of the Dam-Duo kinetochore complex . However nsl1 mutants did not exhibit a defect in microtubule-kinetochore untethering as the ipl1-321 mutant does . Instead, they exhibited a severe defect in the kinetochore localization of the Dam-Duo complex suggesting this to be the cause for the failure of nsl1 cells to establish bipolarity . Moreover the analysis of Nsl1p-depleted cells indicated that Nsl1p is required for the spindle checkpoint and kinetochore integrity.

EMBO J, 2003 Dec 15, 22(24), 6562 - 72
A La protein requirement for efficient pre-tRNA folding; Chakshusmathi G et al.; The La protein protects the 3' ends of many nascent small RNAs from exonucleases . Here we report that La is required for efficient folding of certain pre-tRNAs . A mutation in pre-tRNA(Arg)(CCG) causes yeast cells to be cold-sensitive and to require the La protein Lhp1p for efficient growth . When the mutant cells are grown at low temperature, or when Lhp1p is depleted, mature tRNA(Arg)(CCG) is not efficiently aminoacylated . The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to misfold into an alternative helix in vitro . Intragenic suppressor mutations that disrupt the misfolded helix or strengthen the correct helix alleviate the requirement for Lhp1p, providing evidence that the anticodon stem misfolds in vivo . Chemical and enzymatic footprinting experiments suggest a model in which Lhp1p stabilizes the correctly folded stem . Lhp1p is also required for efficient aminoacylation of two wild-type tRNAs when yeast are grown at low temperature . These experiments reveal that pre-tRNAs can require protein assistance for efficient folding in vivo.

EMBO J, 2003 Dec 15, 22(24), 6448 - 57
Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria; Szyrach G et al.; The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane . Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes . The molecular mechanism of this co-translational insertion process is unknown . Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes . Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase . This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase.

Genome Res, 2003 Dec, 13(12), 2568 - 76
From gene networks to gene function; Schlitt T et al.; We propose a novel method to identify functionally related genes based on comparisons of neighborhoods in gene networks . This method does not rely on gene sequence or protein structure homologies, and it can be applied to any organism and a wide variety of experimental data sets . The character of the predicted gene relationships depends on the underlying networks;they concern biological processes rather than the molecular function . We used the method to analyze gene networks derived from genome-wide chromatin immunoprecipitation experiments, a large-scale gene deletion study, and from the genomic positions of consensus binding sites for transcription factors of the yeast Saccharomyces cerevisiae . We identified 816 functional relationships between 159 genes and show that these relationships correspond to protein-protein interactions, co-occurrence in the same protein complexes, and/or co-occurrence in abstracts of scientific articles . Our results suggest functions for seven previously uncharacterized yeast genes: KIN3 and YMR269W may be involved in biological processes related to cell growth and/or maintenance, whereas IES6, YEL008W, YEL033W, YHL029C, YMR010W, and YMR031W-A are likely to have metabolic functions.

Int J Oncol, 2004 Jan, 24(1), 115 - 25
The role of p53 in the chemotherapeutic responses to cisplatin, doxorubicin and 5-fluorouracil treatment; Dart DA et al.; A panel of tumour models used extensively for in vivo evaluation of new drugs was characterised for their p53 status . Basal p53 protein levels were measured by immunodetection on both formalin-fixed tumour tissue and from protein extracts of fresh tumours . High levels of nuclear-specific staining, indicative of p53 mutation, was seen in 15/25 tumours, with the remainder showing intermittent or no staining . The functional status of p53 cDNA from these tumours was assayed within the functional analysis of separated alleles in yeast (F.A.S.A.Y.) reporter system . The cDNA from those tumours with high levels of p53 protein showed 14/15 failing to activate the reporter gene . The cDNA from tumours with low or non-detectable p53 levels showed 8/10 with wild-type p53 . Tumours were grown subcutaneously in mice (n=10) . Each mouse was given maximum tolerated doses for either doxorubicin, 5-fluorouracil or cisplatin . Tumour volumes were measured daily, alongside untreated controls . The specific growth delay values for each tumour were separated into two groups, those with functional p53 (wild-type) and those without (mutant and null status) . The Mann-Whitney U test was performed on the groups of data, to evaluate differences in their response on the basis of p53 status . Cisplatin was moderately active against tumours with wild-type and mutant p53 genes with no significant difference seen between both groups . However, a significant difference in specific growth delay was seen between the two groups when treated with doxorubicin or 5-fluorouracil (P=0.05), indicating a role for p53 protein in modulating the in vivo efficacy of these agents.

FEMS Yeast Res, 2003 Dec, 4(3), 323 - 7
Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system; Hansen J et al.; Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance . We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S . carlsbergensis (syn . of S . pastorianus) brewing yeast as well as in Saccharomyces kluyveri . As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option . Furthermore, the lacZ system for analyzing gene expression was included in the system.

FEMS Yeast Res, 2003 Dec, 4(3), 315 - 21
The delivery of ADP/ATP carrier protein to mitochondria probed by fusions with green fluorescent protein and beta-galactosidase; Polcicova K et al.; The import of proteins into mitochondria is an essential process, largely investigated in vitro with isolated mitochondria and radioactively labeled precursors . In this study, we used intact cells and fusions with genes encoding two reporter proteins, green fluorescent protein (GFP) and beta-galactosidase (lacZ), to probe the import of the ADP/ATP carrier (AAC) . Typical mitochondrial fluorescence was observed with AAC-GFP fusions containing at least one complete transmembrane loop . This confirms the results of in vitro analysis demonstrating that an internal targeting signal was present in each one of the three transmembrane loops of the carrier . The fusions of AAC fragments to beta-galactosidase demonstrated that the targeting signal was capable of delivering the reporter molecule to the mitochondrial surface, but not to internalize it to a protease-inaccessible location . The delivery to a protease-inaccessible location required the presence of more distal sequences present within the third (C-terminal) transmembrane loop of the carrier molecule . The results of our study provide an alternative for investigation in a natural context of mitochondrial protein import in cells when the isolation of intact, functional mitochondria is not achievable.

Biochem J, 2004 Mar 15, 378(Pt 3), 889 - 98
Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases; Darnet S et al.; In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO) . We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively . SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO . The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation . To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana . SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N . benthamiana inoculated with the viral transcripts . After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9beta,19-cyclopropylsterols (i.e . 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4alpha-methylsterols were not affected . In the case of silencing with SMO2, a large accumulation of 4alpha-methyl-Delta7-sterols (i.e . 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols . These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4alpha-methylsterol precursors respectively.

Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 1266 - 72
WD dipeptide motifs and LXXLL motif of chicken HIRA are necessary for transcription repression and the latter motif is essential for interaction with histone deacetylase-2 in vivo; Ahmad A et al.; We previously reported not only that chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional corepressors Hir1p and Hir2p, possesses seven WD dipeptide motifs and a LXXLL motif in its N-terminal half and C-terminal half, respectively, but also that the N-terminal and C-terminal halves, respectively, bind to CAF-1p48 and HDAC-1 and -2 in vitro . Seven WD dipeptide motifs in the N-terminal half of HIRA are required for the in vitro interaction with CAF-1p48 . The LXXLL motif at positions 993-997 of HIRA is necessary for the in vitro interaction with HDAC-2 . Here we revealed not only that the N-terminal and C-terminal halves of HIRA mediate individually transcription repressions but also that even one of the seven WD dipeptide motifs and the LXXLL motif of HIRA are essential for the mediations in vivo . Moreover, the LXXLL motif is essential for the interaction with endogenous or recombinant HDAC-2 in vivo, probably resulting in formation of the active complex, harboring the HDAC activity . Taken together, these results indicate that HIRA should participate differentially in a number of DNA-utilizing processes, including transcription repressions, through interactions of its distinct regions with CAF-1p48 and HDAC-2, respectively.

Cell, 2003 Nov 26, 115(5), 508 - 10
Rad53: a controller ensuring the fine-tuning of histone levels; Quivy JP et al.; Checkpoint proteins are activated in response to genotoxic insults or replication stress to maintain genome integrity . Their function is believed to depend largely on the detection of the DNA damage or defects occurring during replication fork progression.

Biochem J, 2004 Mar 1, 378(Pt 2), 665 - 71
A novel omega3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid; Pereira SL et al.; Long-chain n-3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5 n-3) have important therapeutic and nutritional benefits in humans . In plants, cyanobacteria and nematodes, omega3-desaturases catalyse the formation of these n-3 fatty acids from n-6 fatty acid precursors . Here we describe the isolation and characterization of a gene ( sdd17 ) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel omega3-desaturase . This gene was isolated by PCR amplification of an S . diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known omega3-desaturases . Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n-6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4 n-6), converting it into EPA . This activity differs from that of the known omega3-desaturases from any organism . Plant and cyanobacterial omega3-desaturases exclusively desaturate 18-carbon n-6 PUFAs, and a Caenorhabditis elegans omega3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast . The sdd17 -encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2140 - 9 Epub 2003 Nov 27.
Refined structure of Pyrococcus furiosus ornithine carbamoyltransferase at 1.87 A; Massant J et al.; Using synchrotron radiation, X-ray data have been collected from Pyrococcus furiosus ornithine carbamoyltransferase (Pfu OTCase) to a maximal resolution of 1.87 A, allowing the refinement of a previous structure at 2.7 A {Villeret et al . (1998), Proc . Natl Acad . Sci . USA, 95, 2801-2806} . Thanks to the high resolution of this refined structure, two sulfate ions and 191 water molecules could be localized directly from the electron-density maps . The identification of these molecules allowed a more rigorous description of the active site and the identification of residues involved in binding carbamoyl phosphate . The improved quality of the model resulted in a better definition of several loops and the various interfaces . The dodecameric protein is composed of four catalytic trimers disposed in a tetrahedral manner . The extreme thermal stability of Pfu OTCase is mainly the result of the strengthening of the intersubunit interactions in a trimer and oligomerization of the trimers into a dodecamer . Interfaces between monomers in a catalytic trimer are characterized by an increase in ion-pair networks compared with mesophilic OTCases . However, the interfaces between catalytic trimers in the dodecameric oligomer are mainly hydrophobic and also involve aromatic-aromatic and cation-pi interactions.

Mol Pharmacol, 2003 Dec, 64(6), 1549 - 56
Pharmacological and genetic analysis of 90-kDa heat shock isoprotein-aryl hydrocarbon receptor complexes; Cox MB et al.; The 90-kDa heat shock protein (Hsp90) is an abundant chaperone that regulates a diverse set of intracellular signaling proteins . Drugs that inhibit Hsp90 activity have been useful in the identification of novel Hsp90-dependent signaling pathways . One class of inhibitory compounds disrupts Hsp90-dependent processes by binding to the N-terminal ATPase/p23-binding domain of Hsp90, whereas a second inhibitor class binds within the C-terminal domain . We used signaling by aryl hydrocarbon receptor (AhR), an Hsp90-dependent transcription factor, as a functional probe to study the effects of Hsp90 inhibitors in yeast strains with deletion mutations of individual Hsp90 and p23 cochaperone genes . The more abundant and constitutively expressed Hsp90 isoform, Hsc82, functioned best in supporting AhR signaling . Deletion of the more inducible isoform, Hsp82, had no effect on signaling . AhR complexes containing Hsc82 were preferentially sensitive to the effects of low concentrations of the N-terminal inhibitors radicicol and herbimycin A . However, both Hsp90 isoforms were equally sensitive to the AhR-specific effects of novobiocin, which binds to the C terminus . Hsp90 inhibitors had no preferential effects on AhR signaling in strains that lacked p23, suggesting that the inhibitors exert their effects through a p23-independent mechanism . In contrast, overexpression of p23 buffered the effects of radicicol and herbimycin A, but not novobiocin, on AhR signaling . The data collectively suggest preferential use or function of the Hsc82 isoprotein in AhR signaling and provide new insight into the effects of three structurally unrelated Hsp90 inhibitors.

Mol Cell Biol, 2003 Dec, 23(24), 9283 - 92
Novel methyltransferase for modified uridine residues at the wobble position of tRNA; Kalhor HR et al.; We have identified a novel tRNA methyltransferase in Saccharomyces cerevisiae that we designate Trm9 . This enzyme, the product of the YML014w gene, catalyzes the esterification of modified uridine nucleotides, resulting in the formation of 5-methylcarbonylmethyluridine in tRNA(Arg3) and 5-methylcarbonylmethyl-2-thiouridine in tRNA(Glu) . In intact yeast cells, disruption of the TRM9 gene results in the complete loss of these modified wobble bases and increased sensitivity at 37 degrees C to paromomycin, a translational inhibitor . These results suggest a role for this potentially reversible methyl esterification reaction when cells are under stress.

Mol Cell Biol, 2003 Dec, 23(24), 9178 - 88
The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork; Kobayashi T; The replication fork barrier site (RFB) is an approximately 100-bp DNA sequence located near the 3' end of the rRNA genes in the yeast Saccharomyces cerevisiae . The gene FOB1 is required for this RFB activity . FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats . Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined . Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB . Fob1p binds to two separated sequences in the RFB . A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities . The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.

Mol Cell Biol, 2003 Dec, 23(24), 9251 - 61
Ubiquitin depletion as a key mediator of toxicity by translational inhibitors; Hanna J et al.; Cycloheximide acts at the large subunit of the ribosome to inhibit translation . Here we report that ubiquitin levels are critical for the survival of Saccharomyces cerevisiae cells in the presence of cycloheximide: ubiquitin overexpression confers resistance to cycloheximide, while a reduced ubiquitin level confers sensitivity . Consistent with these findings, ubiquitin is unstable in yeast (t(1/2) = 2 h) and is rapidly depleted upon cycloheximide treatment . Cycloheximide does not noticeably enhance ubiquitin turnover, but serves principally to block ubiquitin synthesis . Cycloheximide also induces UBI4, the polyubiquitin gene . The cycloheximide-resistant phenotype of ubiquitin overexpressors is also characteristic of partial-loss-of-function proteasome mutants . Ubiquitin is stabilized in these mutants, which may account for their cycloheximide resistance . Previous studies have reported that ubiquitin is destabilized in the absence of Ubp6, a proteasome-associated deubiquitinating enzyme, and that ubp6 mutants are hypersensitive to cycloheximide . Consistent with the model that cycloheximide-treated cells are ubiquitin deficient, the cycloheximide sensitivity of ubp6 mutants can be rescued either by ubiquitin overexpression or by mutations in proteasome subunit genes . These results also show that ubiquitin wasting in ubp6 mutants is proteasome mediated . Ubiquitin overexpression rescued cells from additional translational inhibitors such as anisomycin and hygromycin B, suggesting that ubiquitin depletion may constitute a widespread mechanism for the toxicity of translational inhibitors.

Mol Cell Biol, 2003 Dec, 23(24), 9136 - 49
Global control of histone modification by the anaphase-promoting complex; Ramaswamy V et al.; Acetylation and phosphorylation of the amino-terminal tails of the core histones fluctuate on a global scale in concert with other major events in chromosome metabolism . A ubiquitin ligase, the anaphase-promoting complex (APC), controls events in chromosome metabolism such as sister chromatid cohesion and may regulate H3 phosphorylation by targeting Aurora A, one of several S10-directed H3 kinases in vertebrate cells, for destruction by the proteasome . Our analysis of apc10Delta and apc11(ts) loss-of-function mutants reveals that the APC controls the global level of H3 S10 phosphorylation in cycling yeast cells . Surprisingly, it also regulates dephosphorylation of H3 and global deacetylation of H2B, H3, and H4 during exit from the cell cycle into G(0) . Genetic, biochemical, and microarray analyses suggest that APC-dependent cell cycle control of H3 phosphorylation is exerted at the level of an Aurora H3 kinase, Ipl1p, while APC-dependent transcriptional induction of GLC7, an essential H3 phosphatase, contributes to sustained H3 dephosphorylation upon cell cycle withdrawal . Collectively, our results establish that core histone acetylation state and H3 phosphorylation are physiologically regulated by the APC and suggest a model in which global reconfiguration of H3 phosphorylation state involves APC-dependent control of both an H3 kinase and a conserved phosphatase.

Biosystems, 2003 Dec, 72(3), 229 - 39
Genetic network inference: the effects of preprocessing; Lindlof A et al.; Clustering of gene expression data and gene network inference from such data has been a major research topic in recent years . In clustering, pairwise measurements are performed when calculating the distance matrix upon which the clustering is based . Pairwise measurements can also be used for gene network inference, by deriving potential interactions above a certain correlation or distance threshold . Our experiments show how interaction networks derived by this simple approach exhibit low-but significant-sensitivity and specificity . We also explore the effects that normalization and prefiltering have on the results of methods for identifying interactions from expression data . Before derivation of interactions or clustering, preprocessing is often performed by applying normalization to rescale the expression profiles and prefiltering where genes that do not appear to contribute to regulation are removed . In this paper, different ways of normalizing in combination with different distance measurements are tested on both unfiltered and prefiltered data, different prefiltering criteria are considered.

Mutat Res, 2003 Nov 27, 532(1-2), 245 - 53
G2 and spindle assembly checkpoint adaptation, and tetraploidy arrest: implications for intrinsic and chemically induced genomic instability; Andreassen PR et al.; While checkpoints that act in S-phase are essential to the maintenance of genomic stability, these checkpoints do not act alone . Additionally, G2 DNA damage checkpoints, the spindle assembly checkpoint, and a post-mitotic G1 tetraploidy checkpoint act subsequent to DNA replication to ensure genetic fidelity in cell division . In this review, we will examine how these checkpoints cooperate in the maintenance of genomic stability in response to either DNA damage or cytoskeletal disruption . Since the G2 and spindle assembly checkpoints are subject to adaptation, we will discuss how the G1 tetraploidy checkpoint acts in concert with these checkpoints to mediate stable arrest . We will also probe the relationship of these checkpoints by exploring common features of their regulation . Finally, the consequences of malfunction of these checkpoints for both intrinsic and chemically induced genomic instability will be examined . Among these consequences are aneuploidization, extranumerary centrosomes, and micronucleation.

Mutat Res, 2003 Nov 27, 532(1-2), 117 - 35
Role of the error-free damage bypass postreplication repair pathway in the maintenance of genomic stability; Smirnova M et al.; The postreplication repair pathway (PRR) is composed of error-free and error-prone sub-pathways that allow bypass of DNA damage-induced replication-blocking lesions . The error-free sub-pathway is also used for bypass of spontaneous DNA damage and functions in cooperation with recombination pathways . In diploid yeast cells, error-free PRR is needed to prevent genomic instability, which is manifest as loss of heterozygosity (LOH) events of increased chromosome loss and recombination . Homologous recombination acts synergistically with the error-free damage avoidance branch of PRR to prevent chromosome loss . The DNA damage checkpoint gene MEC1 acts synergistically with the PRR pathway in maintaining genomic stability . Integration of the PRR pathway with other cellular pathways for preventing genomic instability is discussed . In diploid strains, the most dramatic increase is in the abnormality of chromosome loss when a repair or damage detection pathway is defective.

Mutat Res, 2003 Nov 27, 532(1-2), 29 - 40
Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development; Kim JM et al.; Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes . Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases . Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication . In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis . Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase) . Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis . Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals . Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells . Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.

Fungal Genet Biol, 2004 Jan, 41(1), 75 - 88
Aspergillus nidulans hypA regulates morphogenesis through the secretion pathway; Shi X et al.; Aspergillus nidulans hypA encodes a predicted 1474 amino acid, 161.9 kDa cytoplasmic peptide . Strains with hypA1 and hypA6 alleles are wild type at 28 degrees C but have wide, slow-growing hyphae and thick walls at 42 degrees C . hypA1 and hypA6 have identical genetic lesions . hypA1 and hypA6 restrictive phenotypes have statistically similar morphometry, and strains with either allele can conidiate at 42 degrees C . hypA deletion strains require osmotic support and have aberrant morphology, but produce viable spores at 28 degrees C . hypA has full-length orthologs in filamentous fungi and yeasts and a 200 amino acid region with similarity to sequences in plants and animals . The Saccharomyces cerevisiae hypA ortholog is TRS120, a regulatory subunit in the TRAPP II complex that mediates traffic through the Golgi equivalent . Enzyme secretion is reduced in hypA1 cells at 42 degrees C . Endomembranes and cytoplasmic actin arrays in hypA1 have weak polarity at 42 degrees C and cytoplasmic microtubules have reduced number and normal distribution.

Fungal Genet Biol, 2004 Jan, 41(1), 13 - 22
Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis; Guest GM et al.; Growth of the filamentous fungus Aspergillus nidulans begins when the conidium breaks dormancy and grows isotropically . Eventually a germ tube emerges and the axis of growth remains fixed in the primary hypha while new growth axes are established basally to form secondary germ tubes and lateral branches . Rho1 is a Rho family GTPase that has been shown to be involved in polarity establishment and cell wall deposition in Saccharomyces cerevisiae . A gene predicted to encode a Rho1 homolog was cloned from A . nidulans and named rhoA . Strains carrying ectopic copies of the constitutively active rhoA(G14V) allele or the dominant rhoA(E40I) allele were created and characterized . The constitutively active rhoA(G14V) strain grew slowly relative to wild type and showed an abnormal clustered pattern of branch emergence . The rhoA(G14V) strain also labeled intensely with calcofluor, showed elevated levels of cell wall N-acetylglucosamine and had unusually thick cell walls . The dominant rhoA(E40I)strain was accelerated in the emergence of secondary and tertiary germ tubes, and lateral branches relative to wild type and showed lysis with prolonged incubation . The rhoA(E40I) strain also was hypersensitive to the cell wall disrupting agents calcofluor and caspofungin acetate and showed an increase in cell wall N-acetylglucosamine levels . Our results suggest that rhoA plays a role in polarity, proper branching pattern, and cell wall deposition.

Biochem Soc Trans, 2003 Dec, 31(Pt 6), 1140 - 2
Mechanism of glucose sensing in the small intestine; Dyer J et al.; Sensing nutrients is a fundamental task for all living cells . For most eukaryotic cells glucose is a major source of energy, having significant and varied effects on cell function . Interest in identifying mechanisms by which cells sense and respond to variations in glucose concentration has increased recently . The epithelial cells lining the intestinal tract are exposed, from the luminal domain, to an environment with continuous and massive fluctuations in the levels of dietary monosaccharides . Enterocytes therefore have to sense and respond to the significant changes in the levels of luminal sugars, and regulate the expression of the intestinal glucose transporter (Na+/glucose co-transporter, SGLT1) accordingly . Our data, using a combination of in vivo and in vitro model systems, suggest that glucose in the lumen of the intestine is sensed by a glucose sensor residing on the external face of the enterocyte luminal membrane . Glucose binds to the sensor and generates an intracellular signal leading to enhancement in the expression of SGLT1 . The generated signal is independent of glucose metabolism and is likely to operate via a G-protein-coupled receptor and cAMP/protein kinase A signalling cascade.

Arzneimittelforschung, 2003, 53(10), 738 - 43
Synthesis, in vitro/in vivo antifungal evaluation and structure-activity relationship study of 3(2H)-pyridazinones; Karolyhazy L et al.; The synthesis, in vitro/in vivo antifungal evaluation and a structure-activity relationship (SAR) study of 3(2H)-pyridazinones was carried out . The results reported here may be helpful in the structural identification and understanding of the minimum structural requirements for these molecules acting as antifungal agents . In addition, the most active structure in this series was tested for its capacity of inhibiting Saccharomyces cerevisiae beta 1,3-glucan synthase and chitin synthase, enzymes that catalyze the synthesis of the major polymers of the fungal cell wall.

Planta, 2004 Mar, 218(5), 784 - 92 Epub 2003 Nov 26.
Kinetic properties of a micronutrient transporter from Pisum sativum indicate a primary function in Fe uptake from the soil; Cohen CK et al.; Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe(2+) ions, which are then absorbed via a metal ion transporter . We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn(2+) and Cd(2+) into pea ( Pisum sativum L.) roots {Cohen et al . (1998) Plant Physiol 116:1063-1072) . To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings . This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport . Chelate buffer techniques were used to control Fe(2+) in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity (59)Fe(2+) uptake system ( K(m) =54-93 nM) . Additionally, radiotracer ((65)Zn, (109)Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx ( K(m) of 4 and 100 microM, for Zn(2+) and Cd(2+), respectively) . These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe(2+) transporter that mediates root Fe acquisition . This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition . However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain . Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil.

Curr Genet, 2004 Feb, 45(2), 96 - 103 Epub 2003 Nov 26.
A novel beta-glucosidase in Uromyces fabae: feast or fight?
Haerter AC, Voegele RT.
Efficient nutrient mobilization is a key element for biotrophic plant parasites such as the rust fungi . In the course of a cDNA library screen for elements involved in sugar utilization in Uromyces fabae, we identified a sequence with homology to beta-glucosidases . Full-length genomic and cDNA clones of the gene, termed BGL1, were isolated and sequenced . The BGL1 gene comprises 3,372 nucleotides, including nine introns . The open reading frame encompasses 2,532 bases and codes for a polypeptide of 843 amino acids with an apparent molecular mass of 92.4 kDa . Analysis of the polypeptide revealed a potential secretion signal, indicating an extracellular localization of mature BGL1p (89.8 kDa) . BGL1 seems to be expressed in all stages of growth, including haustoria, the feeding structures of rust fungi . In the course of immunolocalization studies, the gene product BGL1p was localized in the periphery of intercellular hyphae and haustoria . On the basis of sequence homology, the BGL1 gene was identified as a fungal beta-glucosidase.

Oncogene, 2004 Feb 12, 23(6), 1206 - 13
Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing; Lucca C et al.; The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability . Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities . It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks . We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses . We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase . Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA) . rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta . Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2 . We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.

Nat Genet, 2004 Jan, 36(1), 69 - 76 Epub 2003 Nov 30.
Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex; Sheen VL et al.; Disruption of human neural precursor proliferation can give rise to a small brain (microcephaly), and failure of neurons to migrate properly can lead to an abnormal arrest of cerebral cortical neurons in proliferative zones near the lateral ventricles (periventricular heterotopia) . Here we show that an autosomal recessive condition characterized by microcephaly and periventricular heterotopia maps to chromosome 20 and is caused by mutations in the gene ADP-ribosylation factor guanine nucleotide-exchange factor-2 (ARFGEF2) . By northern-blot analysis, we found that mouse Arfgef2 mRNA levels are highest during embryonic periods of ongoing neuronal proliferation and migration, and by in situ hybridization, we found that the mRNA is widely distributed throughout the embryonic central nervous system (CNS) . ARFGEF2 encodes the large (>200 kDa) brefeldin A (BFA)-inhibited GEF2 protein (BIG2), which is required for vesicle and membrane trafficking from the trans-Golgi network (TGN) . Inhibition of BIG2 by BFA, or by a dominant negative ARFGEF2 cDNA, decreases cell proliferation in vitro, suggesting a cell-autonomous regulation of neural expansion . Inhibition of BIG2 also disturbed the intracellular localization of such molecules as E-cadherin and beta-catenin by preventing their transport from the Golgi apparatus to the cell surface . Our findings show that vesicle trafficking is an important regulator of proliferation and migration during human cerebral cortical development.

Science, 2004 Jan 16, 303(5656), 343 - 8 Epub 2003 Nov 26.
ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex; Mizuguchi G et al.; The conserved histone variant H2AZ has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin . How histone variants such as H2AZ are incorporated into nucleosomes has been obscure . We have found that Swr1, a Swi2/Snf2-related adenosine triphosphatase, is the catalytic core of a multisubunit, histone-variant exchanger that efficiently replaces conventional histone H2A with histone H2AZ in nucleosome arrays . Swr1 is required for the deposition of histone H2AZ at specific chromosome locations in vivo, and Swr1 and H2AZ commonly regulate a subset of yeast genes . These findings define a previously unknown role for the adenosine triphosphate-dependent chromatin remodeling machinery.

Plant Physiol, 2003 Dec, 133(4), 1630 - 42 Epub 2003 Nov 26.
Overexpression of a mutant basic helix-loop-helix protein HFR1, HFR1-deltaN105, activates a branch pathway of light signaling in Arabidopsis; Yang KY et al.; The HFR1, a basic helix-loop-helix protein, is required for a subset of phytochrome A-mediated photoresponses in Arabidopsis . Here, we show that overexpression of the HFR1-deltaN105 mutant, which lacks the N-terminal 105 amino acids, confers exaggerated photoresponses even in darkness . Physiological analysis implied that overexpression of HFR1-deltaN105 activated constitutively a branch pathway of light signaling that mediates a subset of photomorphogenic responses, including germination, de-etiolation, gravitropic hypocotyl growth, blocking of greening, and expression of some light-regulated genes such as CAB, DRT112, PSAE, PSBL, PORA, and XTR7, without affecting the light-responsiveness of anthocyanin accumulation and expression of other light-regulated genes such as CHS and PSBS . Although the end-of-day far-red light response and petiole elongation were suppressed in the HFR1-deltaN105-overexpressing plants, flowering time was not affected by HFR1-deltaN105 . In addition, the HFR1-deltaN105-overexpressing plants showed hypersensitive photoresponses in the inhibition of hypocotyl elongation, dependently on phytochrome A, FHY1, and FHY3 under FR light or phyB under R light, respectively . Moreover, our double mutant analysis suggested that the hypersensitive photoresponse is due to functional cooperation between HFR1-deltaN105 and other light-signaling components including HY5, a basic leucine zipper protein . Taken together, our results of gain-of-function approach with HFR1-deltaN105 suggest the existence of a complex and important basic helix-loop-helix protein-mediated transcriptional network controlling a branch pathway of light signaling and provide a useful framework for further genetic dissection of light-signaling network in Arabidopsis.

J Neurosci, 2003 Nov 26, 23(34), 10756 - 64
Mechanism of toxicity in rotenone models of Parkinson's disease; Sherer TB et al.; Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson's disease, including selective nigrostriatal dopaminergic degeneration and alpha-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003) . Here, we examined mechanisms of rotenone toxicity using three model systems . In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 microm) caused dose-dependent ATP depletion, oxidative damage, and death . To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a "replacement" for endogenous complex I . In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I . Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death . In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death . To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model . In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by alpha-tocopherol . Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinson's disease . These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinson's disease.

J Biol Chem, 2004 Feb 20, 279(8), 7055 - 63 Epub 2003 Nov 25.
Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling; Varadan R et al.; Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules . The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains . Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes . Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking . Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain . The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units . This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W . J., Jeffrey, L . C., Carson, M., Zhijian, C., and Pickart, C . M . (1992) J . Biol . Chem . 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D . (2002) J . Mol . Biol . 324, 637-647) . Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin . In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined . This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations.

Adv Food Nutr Res, 2003, 47, 73 - 112
The nutritional significance, metabolism and toxicology of selenomethionine; Schrauzer GN; SeMet is a naturally occurring toxic amino acid but at the same time represents the major nutritional source of selenium for higher animals and humans . The ability of SeMet to be incorporated into the body proteins in place of Met furthermore provides a means of reversible Se storage in organs and tissues . This property is not shared by any other naturally occurring selenoamino acid and thus could be associated with a specific physiological function of SeMet . Since higher animals cannot synthesize SeMet, yet from it all needed forms of Se are produced, SeMet meets the criteria of an essential amino acid . Accordingly, SeMet, or enriched food sources thereof, are appropriate forms of Se for human nutritional Se supplementation . However, while SeMet or Se yeast are already widely used in over-the-counter nutritional supplements, infant formulas and parenteral feeding mixtures still contain Se in the form of sodium selenate or sodium selenite, even though these are not the normal nutritional forms of Se . In animal nutrition, these inorganic selenium salts are increasingly replaced by food sources of SeMet such as Se yeast . Synthetic SeMet could also be employed as a feed additive, but its regulatory status is as yet undetermined . The optimal nutritional levels of SeMet for different animal species still need to be determined . The expectation is that lower additions to feedstock of equivalent levels of SeMet will suffice to achieve adequacy than currently approved maximum levels of Se in the form of inorganic Se salts.

J Cell Biol, 2003 Nov 24, 163(4), 707 - 13
A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria; Truscott KN et al.; Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM) . Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1 . We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70 . The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix . We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.

Infect Immun, 2003 Dec, 71(12), 7109 - 18
Phenotypic switching and mating type switching of Candida glabrata at sites of colonization; Brockert PJ et al.; Candida glabrata switches spontaneously at high frequency among the following four graded phenotypes discriminated on agar containing 1 mM CuSO(4): white, light brown, dark brown (DB), and very dark brown . C . glabrata also contains three mating type loci with a configuration similar to that of the Saccharomyces cerevisiae mating type cassette system, suggesting it may also undergo cassette switching at the expression locus MTL1 . To analyze both reversible, high-frequency phenotypic switching and mating type switching at sites of colonization, primary samples from the oral cavities and vaginal canals of three patients suffering from C . glabrata vaginitis were clonally plated on agar containing CuSO(4) . It was demonstrated that (i) in each vaginitis patient, there was only one colonizing strain; (ii) an individual could have vaginal colonization without oral colonization; (iii) phenotypic switching occurred at sites of colonization; (iv) the DB phenotype predominated at the site of infection in all three patients; (v) genetically unrelated strains switched in similar, but not identical, fashions and caused vaginal infection; (vi) different switch phenotypes of the same strain could simultaneously dominate different body locations in the same host; (vii) pathogenesis could be caused by cells in different mating type classes; and (viii) mating type switching demonstrated at both the genetic and transcription levels occurred in one host.

Curr Opin Genet Dev, 2003 Dec, 13(6), 636 - 43
Turning the clock back on ancient genome duplication; Seoighe C; Complete genome sequence data led rapidly to the conclusion that ancient genome duplications had shaped the genomes of the model organisms Saccharomyces cerevisiae and Arabidopsis thaliana . Recent contributions have gone on to refine date estimates for these duplications and, in the case of Arabidopsis, to infer additional, more ancient, rounds of duplication by reconstructing gene order before the most recent duplication event . It is becoming widely accepted that an ancient duplication occurred before the radiation of the ray-finned fish . However, despite methodological advances and the availability of complete genome sequence data the debate over whether very ancient genome duplications have occurred early in the vertebrate lineage has not yet been fully resolved.

Gene, 2003 Dec 4, 321, 173 - 83
MdAP, a novel protein in apple, is associated with the major allergen Mal d 1; Puehringer HM et al.; Mal d 1, an 18-kDa intracellular pathogenesis-related protein (PR-10), has been known since long as the major apple allergen in Middle and Northern Europe . However, its biological function, as that of many other PR-10 proteins, is still unknown . In order to identify proteins putatively interacting with Mal d 1, an expression library of Malus domestica was screened using the yeast-two-hybrid (Y2H) system . A novel protein binding to two isoforms of Mal d 1 being used as 'bait' was isolated . The deduced amino acid sequence from the corresponding full-length cDNA of the predicted Mal d 1-Associated-Protein (MdAP) do not display any homology to known proteins, but shares 45% identity with a 'hypothetical protein' in Arabidopsis thaliana . Southern analysis of the apple genome indicated that MdAP, comprising 190 amino acids, is encoded by a single gene . The expression pattern of the 1-kb MdAP transcript resembled the expression profile of the different Mal d 1 isoforms in various apple organs, however at a much lower level . Furthermore, a huge variation in transcription levels of Mal d 1 isoforms was observed in apple tissue . For both, Mal d 1 and MdAP highest amounts of mRNAs were measured in ripe fruits and significantly lower amounts in vegetative tissue by quantitative reverse transcription-polymerase chain reaction (RT-PCR).

Mol Cell, 2003 Nov, 12(5), 1333 - 40
Chromatin remodeling in vivo: evidence for a nucleosome sliding mechanism; Fazzio TG et al.; Members of the ISWI family of chromatin remodeling factors exhibit ATP-dependent nucleosome sliding, loading, and spacing activities in vitro . However, it is unclear which of these activities are utilized by ISWI complexes to remodel chromatin in vivo . We therefore sought to identify the mechanisms of chromatin remodeling by Saccharomyces cerevisiae Isw2 complex at its known sites of action in vivo . To address this question, we developed a method of identifying intermediates of the Isw2-dependent chromatin remodeling reaction as it proceeded . We show that Isw2 complex catalyzes nucleosome sliding at two different classes of target genes in vivo, in each case sliding nucleosomes closer to the promoter regions . In contrast to its biochemical activities in vitro, nucleosome sliding by Isw2 complex in vivo is unidirectional and localized to a few nucleosomes at each site, suggesting that Isw2 activity is constrained by cellular factors.

Mol Cell, 2003 Nov, 12(5), 1325 - 32
Methylation of histone H3 K4 mediates association of the Isw1p ATPase with chromatin; Santos-Rosa H et al.; Set1p methylates lysine 4 (K4) of histone H3 and regulates the expression of many genes in yeast . Here we use a biochemical approach to identify a protein, Isw1p, which recognizes chromatin preferentially when it is di- and trimethylated at K4 H3 . We show that on certain actively transcribed genes, the Isw1p chromatin remodeling ATPase requires K4 H3 methylation to associate with chromatin in vivo . Analysis of one such gene, MET16, shows that the enzymatic activities of Set1p and Isw1p are functionally connected: Set1p methylation and Isw1p ATPase generate specific chromatin changes at the 5' end of the gene, are necessary for the correct distribution of RNA polymerase II over the coding region, and are required for the recruitment of the cleavage and polyadenylation factor Rna15p . These results indicate that K4 H3 methylation and Isw1p ATPase activity are intimately linked in regulating transcription of certain genes in yeast.

Mol Cell, 2003 Nov, 12(5), 1239 - 50
Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer; Seubert N et al.; Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation . By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations . We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor . The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures . We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.

Cell, 2003 Oct 31, 115(3), 355 - 67
The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis; Cao K et al.; Spindle disassembly at the end of mitosis is a complex and poorly understood process . Here, we report that the AAA-ATPase Cdc48/p97 and its adapters Ufd1-Npl4, which have a well-established role in membrane functions, also regulate spindle disassembly by modulating microtubule dynamics and bundling at the end of mitosis . In the absence of p97-Ufd1-Npl4 function, microtubules in Xenopus egg extracts remain as monopolar spindles attached to condensed chromosomes after Cdc2 kinase activity has returned to the interphase level . Consequently, interphase microtubule arrays and nuclei are not established . Genetic analyses of Cdc48, the yeast homolog of p97, reveal that Cdc48 is also required for disassembly of mitotic spindles after execution of the mitotic exit pathway . Furthermore, Cdc48/p97-Ufd1-Npl4 directly binds to spindle assembly factors and regulates their interaction with microtubules at the end of mitosis . Therefore, Cdc48/p97-Ufd1-Npl4 is an essential chaperone that regulates transformation of the microtubule structure as cells reenter interphase.

Biochemistry, 2003 Dec 2, 42(47), 13869 - 78
Near native structure in an RNA collapsed state; Buchmueller KL et al.; Many large RNAs form conformationally collapsed, but non-native, states prior to folding to the native state or assembling with protein cofactors . Although RNA collapsed states play fundamental roles in RNA folding and ribonucleoprotein assembly processes, their structures have been poorly understood . We obtained 12 high-quality structural constraints for the collapsed state formed by the catalytic core of the bI5 intron RNA using site-specific cross-linking mediated by a short-lived reactant . RNA tertiary structures in the collapsed and native states are indistinguishable, even though only the native state forms a solvent-inaccessible core . Thus, structural neighbors in the collapsed state, including several long-range tertiary interactions, are approximately as close in space as in the native state, but RNA packing is sufficiently loose or dynamic to allow access by solvent . Binding by the obligate CBP2 protein cofactor has almost no effect on structural neighbors reported by cross-linking, even though protein binding chases the RNA from the collapsed state to the native state . Protein binding thus appears to promote only the final few angstroms of RNA folding rather than mediate global conformational rearrangements in the catalytic core . The bI5 RNA collapsed state functions to self-chaperone ribonucleoprotein assembly because this conformationally restrained structure lies very near that of the native state and excludes structures that otherwise misassemble efficiently.

Nat Cell Biol, 2003 Dec, 5(12), 1090 - 4 Epub 2003 Nov 23.
Securin and B-cyclin/CDK are the only essential targets of the APC; Thornton BR et al.; The anaphase-promoting complex/cyclosome (APC) is a highly conserved ubiquitin ligase that controls passage through the cell cycle by targeting many proteins for proteolysis . The complex is composed of at least thirteen core subunits, eight of which are essential, and two activating subunits, Cdc20 (essential) and Cdh1/Hct1 (non-essential) . Previously, it was not known which APC targets are sufficient to explain the essential nature of the complex . Here, we show that each of the eight normally essential APC subunits is rendered non-essential ('bypass-suppressed') by the simultaneous removal/inhibition of the APC substrates securin (Pds1) and B-type cyclin/CDK (Clb/CDK) . In strains lacking the APC, levels of Clb2 and Clb3 remain constant, but Clb/CDK activity oscillates as cells cycle . This suggests that in the absence of B-type cyclin destruction, oscillation of the Clb/CDK-inhibitor Sic1 is sufficient to trigger the feedback loops necessary for the bi-stable nature of Clb/CDK activity . These results strongly suggest that securin and B-type cyclin/CDK activity are the only obligatory targets of the APC in Saccharomyces cerevisiae.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14695 - 700 Epub 2003 Nov 21.
The RNA polymerase III transcriptome revealed by genome-wide localization and activity-occupancy relationships; Roberts DN et al.; RNA polymerase III (Pol III) transcribes small untranslated RNAs, such as tRNAs . To define the Pol III transcriptome in Saccharomyces cerevisiae, we performed genome-wide chromatin immunoprecipitation using subunits of Pol III, TFIIIB and TFIIIC . Virtually all of the predicted targets of Pol III, as well as several novel candidates, were occupied by Pol III machinery . Interestingly, TATA box-binding protein occupancy was greater at Pol III targets than virtually all Pol II targets, and the highly occupied Pol II targets are generally strongly transcribed . The temporal relationships between factor occupancy and gene activity were then investigated at selected targets . Nutrient deprivation rapidly reduced both Pol III transcription and Pol III occupancy of both a tRNA gene and RPR1 . In contrast, TFIIIB remained bound, suggesting that TFIIIB release is not a critical aspect of the onset of repression . Remarkably, TFIIIC occupancy increased dramatically during repression . Nutrient addition generally reestablished transcription and initial occupancy levels . Our results are consistent with active Pol III displacing TFIIIC, and with inactivation/release of Pol III enabling TFIIIC to bind, marking targets for later activation . These studies reveal new aspects of the kinetics, dynamics, and targets of the Pol III system.

J Biol Chem, 2004 Feb 13, 279(7), 6163 - 70 Epub 2003 Nov 21.
The meiosis-specific protein kinase Ime2 directs phosphorylation of replication protein A; Clifford DM et al.; In Saccharomyces cerevisiae, the cellular single-stranded DNA-binding protein replication protein A (RPA) becomes phosphorylated during meiosis in two discrete reactions . The primary reaction is first observed shortly after cells enter the meiotic program and leads to phosphorylation of nearly all the detectable RPA . The secondary reaction, which requires the ATM/ATR homologue Mec1, is induced upon initiation of recombination and only modifies a fraction of the total RPA . We now report that correct timing of both RPA phosphorylation reactions requires Ime2, a meiosis-specific protein kinase that is critical for proper initiation of meiotic progression . Expression of Ime2 in vegetative cells leads to an unscheduled RPA phosphorylation reaction that does not require other tested meiosis-specific kinases and is distinct from the RPA phosphorylation reaction that normally occurs during mitotic growth . In addition, immunoprecipitated Ime2 catalyzes phosphorylation of purified RPA . Our data strongly suggest that Ime2 is an RPA kinase in vivo . We propose that Ime2 directly catalyzes RPA phosphorylation in the primary reaction and indirectly promotes the Mec1-dependent secondary reaction by advancing cells through meiotic progression . Our studies have identified a novel meiosis-specific reaction that targets a key protein required for DNA replication, repair, and recombination . This pathway could be important in differentiating mitotic and meiotic DNA metabolism.

J Biol Chem, 2004 Feb 20, 279(8), 6794 - 804 Epub 2003 Nov 21.
DNA recognition by the homing endonuclease PI-SceI involves a divalent metal ion cofactor-induced conformational change; Noel AJ et al.; PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively . Both domains cooperate in binding the recognition sequence . Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA . Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA . We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques . Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein . The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments . A kinetic scheme was established describing the interaction between PI-SceI and DNA . It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor . Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70 . Deletion of the loop inactivates the enzyme . We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers.

J Comput Biol, 2003, 10(5), 791 - 802
PaTre: a method for paralogy trees construction; Pisanti N et al.; Genomes can be described as a collection of clusters, the gene families, whose members are called paralogs . Paralogs are genes that most probably share duplication history and show a significant similarity in their sequences, even if they perform slightly different biological function . Among the different mechanisms that have led to an increase of the genomic information during biological evolution, gene duplication is probably the most important . To better understand duplication events, the first step is to investigate the history of the gene families in order to detect which duplication events have taken place, and in which relative (partial) order . Here we present a method, called PaTre, that, given a gene family, attempts to construct the paralogy tree of the family . We will work under the hypothesis that every family member derives from a duplication process of another member . By the term paralogy tree, we mean a directed tree in which the root represents the most ancient paralog of the family and each oriented arc (a, b) represents the existence of a duplication event from the template gene a to its copy b . Notice that gene a survives the event and can serve as a template of more than one duplication event; in fact, there can be more than one arc leaving a . PaTre uses new algorithmic techniques motivated by the specific application at hand . The reliability of the inferential process has been tested by means of a simulator that implements different hypotheses on the duplication-with-modification paradigm and on three examples of different biological gene families, belonging either to lower and higher organisms.

Anal Chem, 2003 Aug 15, 75(16), 4081 - 6
Web and database software for identification of intact proteins using "top down" mass spectrometry; Taylor GK et al.; For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available . We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions . Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker . Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results . The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search . Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer . A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation . ProSight PTM is the first search engine and Web environment for identification of intact proteins .

Science, 2003 Nov 21, 302(5649), 1399 - 401
Nucleolar clustering of dispersed tRNA genes; Thompson M et al.; Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis . To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families . The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus . Nucleolar localization requires tRNA gene transcription-complex formation, because inactivation of the promoter at a single locus removes its nucleolar association . This organization of tRNA genes must profoundly affect the spatial packaging of the genome and raises the question of whether gene types might be coordinated in three dimensions to regulate transcription.

Plant Physiol, 2004 Jan, 134(1), 147 - 60 Epub 2003 Nov 20.
Differential expression of sucrose transporter and polyol transporter genes during maturation of common plantain companion cells; Ramsperger-Gleixner M et al.; The cDNAs of two sorbitol transporters, common plantain (Plantago major) polyol transporter (PLT) 1 and 2 (PmPLT1 and PmPLT2), were isolated from a vascular bundle-specific cDNA library from common plantain, a dicot plant transporting Suc plus sorbitol in its phloem . Here, we describe the kinetic characterization of these sorbitol transporters by functional expression in Brewer's yeast (Saccharomyces cerevisiae) and in Xenopus sp . oocytes and for the first time the localization of plant PLTs in specific cell types of the vascular tissue . In the yeast system, both proteins were shown to be uncoupler sensitive and could be characterized as low-affinity and low-specificity polyol symporters . The Km value for the physiological substrate sorbitol is 12 mm for PmPLT1 and even higher for PmPLT2, which showed an almost linear increase in sorbitol transport rates up to 20 mm . These data were confirmed in the Xenopus sp . system, where PmPLT1 was analyzed in detail and characterized as a H+ symporter . Using peptide-specific polyclonal antisera against PmPLT1 or PmPLT2 and simultaneous labeling with the monoclonal antiserum 1A2 raised against the companion cell-specific PmSUC2 Suc transporter, both PLTs were localized to companion cells of the phloem in common plantain source leaves . These analyses revealed two different types of companion cells in the common plantain phloem: younger cells expressing PmSUC2 at higher levels and older cells expressing lower levels of PmSUC2 plus both PLT genes . The putative role of these low-affinity transporters in phloem loading is discussed.

Genes Dev, 2003 Nov 15, 17(22), 2798 - 811
Lipoprotein receptors and a disabled family cytoplasmic adaptor protein regulate EGL-17/FGF export in C . elegans; Kamikura DM et al.; Growth factors and morphogens need to be secreted to act on distant cells during development and in response to injury . Here, we report evidence that efficient export of a fibroblast growth factor (FGF), EGL-17, from the Caenorhabditis elegans developing vulva requires the lipoprotein receptor-related proteins Ce-LRP-1 and Ce-LRP-2 and a cytoplasmic adaptor protein, Ce-DAB-1 (Disabled) . Lipoprotein receptors are transmembrane proteins best known for their roles in endocytosis . Ce-LRP-1 and Ce-LRP-2 possess a conserved intraluminal domain that can bind to EGL-17, as well as a cytosolic FXNPXY motif that can bind to Ce-DAB-1 . Ce-DAB-1 contains signals that confer subcellular localization to Golgi-proximal vesicles . These results suggest a model in which Ce-DAB-1 coordinates selection of receptors and cargo, including EGL-17, for transport through the secretory pathway.

Genes Dev, 2003 Nov 15, 17(22), 2747 - 52
Template boundary definition in mammalian telomerase; Chen JL et al.; Telomerase uses a short template sequence in its intrinsic RNA component to synthesize telomere repeats . Disruption of the helix P1b in human telomerase RNA or alteration of its distance from the template resulted in telomerase copying residues past the normal template boundary both in vivo and in vitro . Therefore, helix P1b is important for template boundary definition in human telomerase . Mouse telomerase RNA lacks helix P1b, and the boundary is established at 2 nt downstream of the 5'-end . The divergent structure of boundary definition elements in mammals, yeast, and ciliates suggests diverse mechanisms for template boundary definition in telomerase.

Biochem Biophys Res Commun, 2003 Dec 5, 312(1), 121 - 30
Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy; Davydov DR et al.; We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate . The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR) . Important barotropic heterogeneity of CYP3A4 was detected in both cases . Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition . The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties . No interconversion between these conformers was observed within the time frame of the experiment . Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes . This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.

J Microsc, 2003 Dec, 212(Pt 3), 254 - 63
Monitoring enzymatic reactions in nanolitre wells; Young IT et al.; We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon . We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions . Such reactions can be found in the glycolytic pathway of yeast . We describe the design, construction and testing of our laboratory-on-a-chip . We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays) . We show that our detection limit for NADH fluorescence is 5 micro m with a microscope-based system and 100 micro m for an embedded photodiode system . The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence.

Neoplasma, 2003, 50(5), 311 - 8
Artemis, a novel guardian of the genome; Dudasova Z et al.; B and T lymphocytes recognize foreign antigen through specialized receptors: the immunoglobulins and the T cell receptors, respectively . The highly polymorphic antigen-recognition regions of these receptors are composed of variable (V), diversity (D), and joining (J) gene segments that undergo somatic rearrangement prior to their expression by the V(D)J recombination process . Proper joining of the V, D, and J segments requires the participation of the Rag proteins as well as the non-homologous end-joining (NHEJ) factors . Recently, a novel V(D)J recombination/NHEJ factor, Artemis, has been identified . Mutations in the ARTEMIS gene cause human severe combined immunodeficiency with increased radiosensitivity (RS-SCID), an autosomal recessive disease characterized by the absence of the T and B lymphocytes and by a defect in the V(D)J recombination . This minireview compiles all mutations in the ARTEMIS gene identified so far . Furthermore, phenotypes of RS-SCID patients and links to the particular mutations are described . Biochemical and structural properties of the Artemis proteins are reviewed and integrated into the processes of V(D)J recombination and NHEJ . A genomic caretaker function is assigned to Artemis.

Nucleic Acids Res . 2003 Dec 1;31(23):e151.
Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling; Baum M et al.; Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop . Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier . Arrays can be designed and produced within hours according to the user's requirements . They are processed in a fully automatic workflow . We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results . The instrument detects sample RNAs present at a frequency of 1:100 000 . Detection is quantitative over more than two orders of magnitude . Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility . In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found . Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and--albeit somewhat less clearly--to data from spotted cDNA arrays obtained from the literature.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6953 - 62
Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p; Ikeuchi A et al.; The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule . The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p . In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology . The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments . Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively . Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures . The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides . The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6741 - 7
Dynamic methylation of histone H3 at lysine 4 in transcriptional regulation by the androgen receptor; Kim J et al.; The methylation of histone H3 correlates with either gene expression or silencing depending on the residues modified . Methylated lysine 4 (H3-K4) is associated with transcription at active gene loci . Furthermore, it was reported that trimethylated but not dimethylated H3-K4 is exclusively associated with active chromatin in Saccharomyces cerevisiae . In the present study, we investigated the H3-K4 methylation at the human prostate specific antigen (PSA) locus following gene activation and repression via androgen receptor (AR) . We show that ligand-induced, AR-mediated transcription was accompanied by rapid decreases in di- and trimethylated H3-K4 at the PSA enhancer and promoter . Moreover, the observed decreases in H3-K4 methylation were reversed when AR was inhibited by a specific AR antagonist, bicalutamide . In contrast to the decreases in methylation at the 5' transcriptional control regions of the PSA gene, H3-K4 methylation in the coding region steadily increased after a lag period of approximately 4 h . The results suggest a novel role of methylated H3-K4 in transcriptional regulation.

Cell Stress Chaperones, 2003 Summer, 8(2), 108 - 13
p23, a simple protein with complex activities; Felts SJ et al.; p23 is a small but important cochaperone for the Hsp90 chaperoning pathway . It appears to facilitate the adenosine triphosphate-driven cycle of Hsp90 binding to client proteins . It enters at a late stage of the cycle and enhances the maturation of client proteins . Although this role of p23 is fairly well established, recent studies suggest that it may have additional functions in the cell that merit further exploration.

Cell Mol Life Sci, 2003 Nov, 60(11), 2303 - 18
Long-range silencing and position effects at telomeres and centromeres: parallels and differences; Perrod S et al.; Most of the human genome is compacted into heterochromatin, a form that encompasses multiple forms of inactive chromatin structure . Transcriptional silencing mechanisms in budding and fission yeasts have provided genetically tractable models for understanding heritably repressed chromatin . These silent domains are typically found in regions of repetitive DNA, that is, either adjacent to centromeres or telomeres or within the tandemly repeated ribosomal DNA array . Here we address the mechanisms of centromeric, telomeric and locus-specific gene silencing, comparing simple and complex animals with yeast . Some aspects are universally shared, such as histone-tail modifications, while others are unique to either centromeres or telomeres . These may reflect roles for heterochromatin in other chromosomal functions, like kinetochore attachment and DNA ends protection.

Cell Mol Life Sci, 2003 Nov, 60(11), 2295 - 302
Composition and conservation of the telomeric complex; Kanoh J et al.; The telomere is composed of telomeric DNA and telomere-associated proteins . Recently, many telomere-associated proteins have been identified, and various telomere functions have been uncovered . In budding yeast, scRap1 binds directly to telomeric DNA, and other telomere regulators (Sir proteins and Rif proteins) are recruited to the telomeres by interacting with scRap1 . Cdc13 binds to the most distal end of the chromosome and recruits telomerase to the telomeres . In fission yeast and humans, TTAGGG repeat binding factor (TRF) family proteins bind directly to telomeric DNA, and Rap1 proteins and other telomere regulators are recruited to the telomeres by interacting with the TRF family proteins . Both organisms have Pot1 proteins at the most distal end of the telomere instead of a budding-yeast Cdc13-like protein . Therefore, fission yeast and humans have in part common telomeric compositions that differ from that of budding yeast, a result that suggests budding yeast has lost some telomere components during the course of evolution.

Nat Cell Biol, 2003 Dec, 5(12), 1062 - 70 Epub 2003 Nov 16.
Scaffold-mediated symmetry breaking by Cdc42p; Irazoqui JE et al.; Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking . In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally . The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization . We found that yeast Cdc42p was effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments . Among a panel of Cdc42p effectors and interacting proteins, we found that the scaffold protein Bem1p was uniquely required for this symmetry-breaking behaviour . Moreover, polarization was dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch.

J Biol Chem, 2004 Feb 27, 279(9), 8469 - 77 Epub 2003 Nov 18.
Analysis on origin recognition complex containing Orc5p with defective Walker A motif; Takahashi N et al.; Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes . To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E) . The strain showed temperature-sensitive growth . Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content . Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not . Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif . These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication . Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures . We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.

J Cell Biochem, 2003 Dec 1, 90(5), 884 - 91
Identification of a novel protein from glial cells based on its ability to interact with NF-kappaB subunits; Sweet T et al.; Nuclear factor kappaB (NF-kappaB) represents a family of inducible DNA-binding transcription factors whose activity is critical for expression of the HIV-1 genome in a broad range of cells . In addition to its interaction with the kappaB DNA sequence, the association of NF-kappaB subunits with other cellular proteins plays an important role in stimulation of HIV-1 gene transcription in astrocytic cells . Here, we utilized a yeast two-hybrid system to screen a cDNA library from a human astrocytic cell line and were able to isolate a partial cDNA belonging to a gene with an open reading frame of 1,871 amino acid residues which binds to both the p50 and p65 subunits of NF-kappaB . This gene, named NF-kappaB-binding protein (NFBP) is located on chromosome 10q24.2-25.1 and hybridized to a single transcript of nearly 6 kb in size . It is localized to the nucleus, specifically the nucleolus of cells . Extensive computer analysis was performed with the sequence of the full length NFBP and significant homology was found between NFBP, and yeast and mouse proteins . A discussion of the potential roles of NFBP in normal and viral infected cells is included .

RNA, 2003 Dec, 9(12), 1476 - 90
Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export; Chekanova JA et al.; Eukaryotic poly(A) binding protein (PABP) is a ubiquitous, essential cellular factor with well-characterized roles in translational initiation and mRNA turnover . In addition, there exists genetic and biochemical evidence that PABP has an important nuclear function . Expression of PABP from Arabidopsis thaliana, PAB3, rescues an otherwise lethal phenotype of the yeast pab1Delta mutant, but it neither restores the poly(A) dependent stimulation of translation, nor protects the mRNA 5' cap from premature removal . In contrast, the plant PABP partially corrects the temporal lag that occurs prior to the entry of mRNA into the decay pathway in the yeast strains lacking Pab1p . Here, we examine the nature of this lag-correction function . We show that PABP (both PAB3 and the endogenous yeast Pab1p) act on the target mRNA via physically binding to it, to effect the lag correction . Furthermore, substituting PAB3 for the yeast Pab1p caused synthetic lethality with rna15-2 and gle2-1, alleles of the genes that encode a component of the nuclear pre-mRNA cleavage factor I, and a factor associated with the nuclear pore complex, respectively . PAB3 was present physically in the nucleus in the complemented yeast strain and was able to partially restore the poly(A) tail length control during polyadenylation in vitro, in a poly(A) nuclease (PAN)-dependent manner . Importantly, PAB3 in yeast also promoted the rate of entry of mRNA into the translated pool, rescued the conditional lethality, and alleviated the mRNA export defect of the nab2-1 mutant when overexpressed . We propose that eukaryotic PABPs have an evolutionarily conserved function in facilitating mRNA biogenesis and export.

RNA, 2003 Dec, 9(12), 1431 - 6
Cic1p/Nsa3p is required for synthesis and nuclear export of 60S ribosomal subunits; Fatica A et al.; Cic1p/Nsa3p was previously reported to be associated with the 26S proteasome and required for the degradation of specific substrates, but was also shown to be associated with early pre-60S particles and to be localized to the nucleolus . Here we report that Cic1p/Nsa3p is required for the synthesis of 60S ribosome subunits . A temperature-sensitive lethal cic1-2 point mutation inhibits synthesis of the mature 5.8S and 25S rRNAs . Release of the pre-60S particles from the nucleolus to the nucleoplasm was also inhibited as judged by the nuclear accumulation of an Rpl11b-GFP reporter construct . We suggest that Cic1p/Nsa3p associates early with nascent preribosomal particles and is required for correct processing and nuclear release of large ribosomal subunit precursors.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13887 - 91 Epub 2003 Nov 17.
Association of the Mediator complex with enhancers of active genes; Kuras L et al.; The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro . Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo . We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes . This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences . These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

J Biol Chem, 2004 Feb 20, 279(8), 6414 - 25 Epub 2003 Nov 17.
Transcriptional profiling of ubp10 null mutant reveals altered subtelomeric gene expression and insurgence of oxidative stress response; Orlandi I et al.; UBP10 codes for a deubiquitinating enzyme of Saccharomyces cerevisiae whose loss of function determines slow growth rate and partial impairment of silencing at telomeres and HM loci . A genome-wide analysis performed on a ubp10 disruptant revealed alterations in expression of subtelomeric genes together with a broad change in the whole transcriptional profile, closely parallel to that induced by oxidative stress . This response was accompanied by intracellular accumulation of reactive oxygen species as well as by DNA fragmentation and phosphatidylserine externalization, two markers of apoptosis . SIR4 inactivation mitigated the wide transcriptome remodeling of the ubp10 null mutant affecting particularly the stress transcriptional profile . Moreover, the ubp10sir4 disruptant did not display apoptotic markers . These results argue in favor of an involvement of deubiquitination in transcriptional control and suggest a linkage between oxidative stress and apoptotic pathway in budding yeast.

J Cell Biol, 2003 Nov 24, 163(4), 729 - 41 Epub 2003 Nov 17.
Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion; Stead K et al.; Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window . However, Pds5p and the cohesin complex likely have distinct roles in cohesion . We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation . Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance . We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants . In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants . Pds5p is SUMO conjugated, with sumoylation peaking during mitosis . SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation . smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis . These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion.

J Cell Biol, 2003 Nov 24, 163(4), 777 - 87 Epub 2003 Nov 17.
Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia; Atorino L et al.; Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP) . Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein . We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane . These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts . The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin . Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin-AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure . These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.

Biochem Biophys Res Commun, 2003 Nov 21, 311(3), 577 - 82
Identification of mouse Vps16 and biochemical characterization of mammalian class C Vps complex; Kim BY et al.; Many multiprotein complexes mediate the fusion of the intracellular membranes . The question how the specificity of the membrane fusion is controlled has not been fully elucidated . Here we report the identification of a mouse homologue Vps16p (mVps16), which exhibits a high homology to the yeast Vps16p, a component of Class C vacuolar protein sorting (Vps) complex implicated in the yeast vacuole membrane fusion . Northern and Western blot analyses reveal that mVps16 is ubiquitously expressed in the mouse peripheral tissues . Biochemical analyses show that mammalian Class C Vps proteins interact with multiple syntaxins and Vps45p, which localizes in the endosomal compartments . The internalization of transferrin (Tf) is not affected by the overexpression of mammalian class C Vps proteins, but the recycling was inhibited . Taken together, this study provides biochemical characteristics of mVps16p in mammalian cells and the potential roles of mammalian Class C Vps proteins in membrane trafficking.

Dev Biol, 2003 Dec 1, 264(1), 50 - 63
Golgi dynamics during meiosis are distinct from mitosis and are coupled to endoplasmic reticulum dynamics until fertilization; Payne C et al.; One current theory of the Golgi apparatus views its organization as containing both a matrix fraction of structural proteins and a reservoir of cycling enzymes . During mitosis, the putative matrix protein GM130 is phosphorylated and relocalized to spindle poles . When the secretory pathway is inhibited during interphase, GM130 redistributes to regions adjacent to vesicle export sites on the endoplasmic reticulum (ER) . Strikingly, meiotic maturation and fertilization in nonrodent mammalian eggs presents a unique experimental environment for the Golgi apparatus, because secretion is inhibited until after fertilization, and because the centrosome is absent until introduced by the sperm . Here, we test the hypothesis that phosphorylated GM130 associates not with meiotic spindle poles, but with ER clusters in the mature bovine oocyte . At the germinal vesicle stage, phosphorylated GM130 is observed as fragments dispersed throughout the cytoplasm . During meiotic maturation, GM130 reorganizes into punctate foci that associate near the ER-resident protein calreticulin and is notably absent from the meiotic spindle . GM130 colocalizes with Sec23, a marker for ER vesicle export sites, but not with Lens culinaris agglutinin, a marker for cortical granules . Because disruption of vesicle transport has been shown to block meiotic maturation and embryonic cleavage in some species, we also test the hypothesis that fertilization and cytokinesis are inhibited with membrane trafficking disruptor brefeldin A (BFA) . Despite Golgi fragmentation after BFA treatment, pronuclei form and unite, and embryos cleave and develop through the eight-cell stage . We conclude that, while the meiotic phosphorylation cycle of GM130 mirrors that of mitosis, absence of a maternal centrosome precludes Golgi association with the meiotic spindle . Fertilization introduces the sperm centrosome that can reorganize Golgi proteins, but neither fertilization nor cytokinesis prior to compaction requires a functional Golgi apparatus.

Cell, 2003 Nov 14, 115(4), 475 - 87
A pathway for association of receptors, adaptors, and actin during endocytic internalization; Kaksonen M et al.; In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition . Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility . Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions . Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly . In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes . Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization . Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.

Cell, 2003 Nov 14, 115(4), 425 - 35
Isw1 chromatin remodeling ATPase coordinates transcription elongation and termination by RNA polymerase II; Morillon A et al.; We demonstrate that distinct forms of the yeast chromatin-remodeling enzyme Isw1p sequentially regulate each stage of the transcription cycle . The Isw1a complex (Iswlp/Ioc3p) represses gene expression at initiation through specific positioning of a promoter proximal dinucleosome, whereas the Isw1b complex (Iswlp/Ioc2p/Ioc4p) acts within coding regions to control the amount of RNA polymerase (RNAPII) released into productive elongation and to coordinate elongation with termination and pre-mRNA processing . These effects of Isw1b are controlled via phosphorylation of the heptad repeat carboxy-terminal domain (CTD) of RNAPII and methylation of the chromatin template . The transcription elongation factor Spt4p antagonizes Isw1p and overcomes the Isw1p dependent pausing of RNAPII at the onset of the elongation cycle . Overall these studies establish the central role played by Isw1p in the coordination of transcription.

Mol Microbiol, 2003 Nov, 50(4), 1309 - 18
Candida glabrata STE12 is required for wild-type levels of virulence and nitrogen starvation induced filamentation; Calcagno AM et al.; The highly conserved fungal Ste12 transcription factor family of proteins play critical roles in the regulation of many cellular processes including mating, cell wall biosynthesis, filamentation and invasive growth . They are also important mediators of fungal virulence . The Candida glabrata STE12 homologue was cloned . The encoded protein has a single DNA binding homeodomain but lacks both a C2H2 zinc finger DNA binding domain and an apparent Dig1/Dig2 regulatory motif . Candida glabrata STE12 can functionally complement the nitrogen starvation induced filamentation and mating defects of Saccharomyces cerevisiae ste12 mutants . We also show that C . glabrata STE12 is required for nitrogen starvation-induced filamentation as ste12 mutants rarely produce pseudohyphae on nitrogen depleted media . Finally we describe a novel murine model of C . glabrata systemic disease and use this to demonstrate that C . glabrata ste12 mutants, although still able to cause disease, are attenuated for virulence compared with STE12 reconstituted strains . Candida glabrata STE12 is therefore the first virulence factor encoding gene to be described in this increasingly important fungal pathogen.

Eur J Biochem, 2003 Nov, 270(21), 4254 - 63
Identification and characterization of eukaryotic initiation factor 5A-2; Clement PM et al.; The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post-translationally derived amino acid hypusine {Nepsilon-(4-amino-2-hydroxybutyl)lysine} . Both eIF5A and its hypusine modification are essential for sustained cell proliferation . Normally only one eIF5A protein is expressed in human cells . Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A-2) of 84% sequence identity . Overexpression of eIF5A-2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene . However, eIF5A-2 protein has not been described in human or mammalian cells heretofore . Here, we describe the identification of eIF5A-2 protein in human colorectal and ovarian cancer lines, SW-480 and UACC-1598, that overexpress eIF5A-2 mRNAs . Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted . This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A-2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A-1 . Detectable structural differences were also noted, including lack of immunological cross-reactivity, formation of different complexes with deoxyhypusine synthase, and Km values (1.5 +/- 0.2 vs . 8.3 +/- 1.4 microm for eIF5A-1 and -2, respectively) as substrates for deoxyhypusine synthase in vitro . These physical characteristics and distinct amino acid sequences in the C-terminal domain together with differences in gene expression patterns imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer.

Eur J Biochem, 2003 Nov, 270(22), 4587 - 93
The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure; Kato M et al.; The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa . Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety . In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition . These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding . The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.

Genes Cells, 2003 Nov, 8(11), 857 - 72
Caenorhabditis elegans RBX1 is essential for meiosis, mitotic chromosomal condensation and segregation, and cytokinesis; Sasagawa Y et al.; BACKGROUND: The RING-H2 finger protein RBX1 (ROC1/HRT1) is a common subunit of SKP1-CDC53/CUL1-F-box (SCF), other cullins and von Hippel-Lindau (VHL) tumour suppressor E3 ubiquitin ligase complexes . RBX1 protein sequences are highly conserved in various species, including yeasts, Drosophila melanogaster, mice and humans . In Saccharomyces cerevisiae, RBX1 is essential for the G1/S transition . RESULTS: Caenorhabditis elegans RBX1 is strongly expressed in early embryos and in the gonad, including meiotic cells . Depletion of RBX1 by RNA-mediated interference (RNAi) caused pronounced defects in the first meiotic division . Several irregular phenotypes were identified in embryos that escaped from meiotic arrest: defects in mitotic chromosomal condensation and segregation, abnormal chromosome bridges, giant nuclei, abnormal cortical protrusion, multinucleate cells and defects in germ cell proliferation . Moreover, histone H3 phosphorylation at Ser10 and Ser28 was significantly reduced in these embryos . The histone H3 phosphorylation defect of embryos was rescued by the additional depletion of protein phosphatase 1 (GLC7alpha/beta) by RNAi . CONCLUSION: These results indicate that the RBX1 protein participates in diverse functions relevant to chromosome metabolism and cell cycle control.

Biochemistry, 2003 Nov 25, 42(46), 13637 - 45
Elimination of the disulfide bridge in the Rieske iron-sulfur protein allows assembly of the {2Fe-2S} cluster into the Rieske protein but damages the ubiquinol oxidation site in the cytochrome bc1 complex; Merbitz-Zahradnik T et al.; The {2Fe-2S} cluster of the Rieske iron-sulfur protein is held between two loops of the protein that are connected by a disulfide bridge . We have replaced the two cysteines that form the disulfide bridge in the Rieske protein of Saccharomyces cerevisiae with tyrosine and leucine, and tyrosine and valine, to evaluate the effects of the disulfide bridge on assembly, stability, and thermodynamic properties of the Rieske iron-sulfur cluster . EPR spectra of the Rieske proteins lacking the disulfide bridge indicate the iron-sulfur cluster is assembled in the absence of the disulfide bridge, but there are significant shifts in all g values, indicating a change in the electronic structure of the {2Fe-2S} iron-sulfur center . In addition, the midpoint potential of the iron-sulfur cluster is lowered from 265 mV in the Rieske protein from wild-type yeast to 150 mV in the protein from the C164Y/C180L mutant and to 160 mV in the protein from the C164Y/C180V mutant . Ubiquinol-cytochrome c reductase activities of the bc(1) complexes with Rieske proteins lacking the disulfide bridge are less than 1% of the activity of the bc(1) complex from wild-type yeast, even though normal amounts of the iron-sulfur protein are present as judged by Western blot analysis . These activities are lower than the 105-115 mV decrease in the midpoint potential of the Rieske iron-sulfur cluster can account for . Pre-steady-state reduction of the bc(1) complexes with menadiol indicates that quinol is not oxidized through center P but is oxidized through center N . In addition, the levels of stigmatellin and UHDBT binding are markedly diminished, while antimycin binding is unaffected, in the bc(1) complexes with Rieske proteins lacking the disulfide bridge . Taken together, these results indicate that the ubiquinol oxidation site at center P is damaged in the bc(1) complexes with Rieske proteins lacking the disulfide bridge even though the iron-sulfur cluster is assembled into the Rieske protein.

Mol Cell Biochem, 2003 Nov, 253(1-2), 217 - 22
The cell cycle of Entamoeba histolytica; Lohia A; Entamoeba histolytica, is a microaerophilic protist, which causes amoebic dysentery in humans . This unicellular organism proliferates in the human intestine as the motile trophozoite and survives the hostile environment outside the human host as the dormant quadri-nucleate cyst . Lack of organelles--such as mitochondria and Golgi bodies--and an unequal mode of cell division, led to the popular belief, that this organism preceded other eukaryotes during evolution . However, data from several laboratories have shown that, contrary to this belief, E . histolytica is remarkable in its divergence from other eukaryotes . This uniqueness is witnessed in many aspects of its biochemical pathways, cellular biology and genetic diversity . In this context, I have analysed the cell division cycle of this organism and compared it to that of other eukaryotes . Studies on E . histolytica, suggest that in its proliferative phase, this organism may accumulate polyploid cells . Thus 'checkpoints' regulating alternation of genome duplication and cell division appear to be absent in this unicellular protist . Sequence homologs of several cell cycle regulating proteins have been identified in amoeba, but their structural divergence suggests that they may not have equivalent function in this organism . The regulation of cell proliferation in E . histolytica, may be ideally suited to survival of a parasite in a complex host . Analysis of these molecular details may offer solutions for eradicating the pathogen by hitherto unknown methods.

Planta, 2004 Feb, 218(4), 640 - 6 Epub 2003 Nov 14.
Degradation of proliferating cell nuclear antigen by 26S proteasome in rice (Oryza sativa L.); Yamamoto T et al.; To study whether metabolic control of proliferating cell nuclear antigen (PCNA) during the cell cycle is similar to that of associated protein factors, two-hybrid analysis with PCNA from rice (Oryza sativa L . cv . Nipponbare) was performed . PCNA interacted with rice Rpt6, which is the ATPase subunit of 26S proteasome, both in vitro and in vivo, and the degradation of PCNA was disrupted by the proteasome in vivo . The tissue-specific expression pattern of the transcripts of Rpt6 and PCNA suggested that the rice proteasome played important roles in DNA replication involving PCNA . These findings indicate a proteasome-dependent degradation of PCNA.

Mol Biol Cell, 2004 Jan, 15(1), 1 - 10 Epub 2003 Nov 14.
Interactions between Sec complex and prepro-alpha-factor during posttranslational protein transport into the endoplasmic reticulum; Plath K et al.; Posttranslational translocation of prepro-alpha-factor (ppalphaF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex . We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step . We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules . To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppalphaF was generated with photoreactive probes incorporated into the mature portion of the polypeptide . Cross-linking to multiple regions of Sec61p was observed . In contrast to the signal sequence, neighboring positions of the mature portion of ppalphaF had similar interactions with Sec61p . These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.

J Biol Chem, 2004 Feb 13, 279(7), 5894 - 903 Epub 2003 Nov 13.
Topography of the euryarchaeal transcription initiation complex; Bartlett MS et al.; Transcription in the Archaea is carried out by RNA polymerases and transcription factors that are highly homologous to their eukaryotic counterparts, but little is known about the structural organization of the archaeal transcription complex . To address this, transcription initiation complexes have been formed with Pyrococcus furiosus transcription factors (TBP and TFB1), RNA polymerase, and a linear DNA fragment containing a strong promoter . The arrangement of proteins from base pair -35 to +20 (relative to the transcriptional start site) has been analyzed by photochemical protein-DNA cross-linking . TBP cross-links to the TATA box and TFB1 cross-links both upstream and downstream of the TATA box, as expected, but the sites of most prominent TFB1 cross-linking are located well downstream of the TATA box, reaching as far as the start site of transcription, suggesting a role for TFB1 in initiation of transcription that extends beyond polymerase recruitment . These cross-links indicate the transcription factor orientation in the initiation complex . The pattern of cross-linking of four RNA polymerase subunits (B, A', A", and H) to the promoter suggests a path for promoter DNA relative to the RNA polymerase surface in this archaeal transcription initiation complex . In addition, an unidentified protein approximately the size of TBP cross-links to the non-transcribed DNA strand near the upstream edge of the transcription bubble . Cross-linking is specific to the polymerase-containing initiation complex and requires the gdh promoter TATA box . The location of this protein suggests that it, like TFB1, could also have a role in transcription initiation following RNA polymerase recruitment.

Traffic, 2003 Dec, 4(12), 857 - 68
The ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells; Khor R et al.; Influenza virus enters cells by endocytosis, and requires the low pH of the late endosome for successful infection . Here, we investigated the requirements for sorting into the multivesicular body pathway of endocytosis . We show that treatment of host cells with the proteasome inhibitors MG132 and lactacystin directly affects the early stages of virus replication . Unlike other viruses, such as retroviruses, influenza virus budding was not affected . The requirement for proteasome function was not shared by two other pH-dependent viruses: Semliki Forest virus and vesicular stomatitis virus . With MG132 treatment, incoming influenza viruses were retained in endosomes that partially colocalized with mannose 6-phosphate receptor, but not with classical markers of early or late endosomes . Colocalization was also observed with Rme-1, which is part of the recycling pathway of endocytosis . In addition, influenza virus entry was dependent on the vacuolar protein sorting pathway, as over-expression of dominant-negative hVPS4 caused arrest of viruses in endosome-like populations that partially colocalized with the hVPS4 protein . Overall, we conclude that influenza virus selectively requires the ubiquitin/vacuolar protein sorting pathway for entry into host cells, and that it must communicate with a specific cellular machinery for intracellular sorting during the initial phase of virus infection.

Plant J, 2003 Nov, 36(4), 471 - 84
Regiospecific hydroxylation of isoflavones by cytochrome p450 81E enzymes from Medicago truncatula; Liu CJ et al.; Mining of Medicago truncatula EST databases and screening of a root cDNA library led to the identification of three cytochrome p450 81E subfamily members . Two were functionally characterized by expression in yeast . The recombinant enzymes in yeast microsomes utilized the same isoflavone substrates, but produced different products hydroxylated at the 2' and/or 3' positions of the B-ring . When transiently expressed in alfalfa leaves, green fluorescent protein (GFP) fusions of the isoflavone 2'- and 3'-hydroxylases localized to the endoplasmic reticulum . The isoflavone 2'-hydroxylase was functional when expressed in Arabidopsis . Differential tissue-specific and biotic/abiotic stress-dependent expression patterns were observed for the isoflavone 2'-hydroxylase and 3'-hydroxylase genes, suggesting differential involvement of 2'- and 3'-hydroxylated isoflavonoids in pathogen defense and insect-induced responses, respectively, in Medicago.

Annu Rev Genet, 2003, 37, 251 - 82
The spindle assembly and spindle position checkpoints; Lew DJ et al.; The mitotic spindle segregates chromosomes to opposite ends of the cell in preparation for cell division . Chromosome attachment to the spindle is monitored by the spindle assembly checkpoint, and at least in yeast cells, penetration of one spindle pole into the bud is monitored by the spindle position checkpoint . We review the historical origins of these checkpoints and recent progress in understanding their surveillance pathways . We also highlight fascinating but as yet unresolved questions, and examine crosstalk between the checkpoints.

Anal Bioanal Chem, 2004 Jan, 378(1), 76 - 83 Epub 2003 Nov 13.
Use of a gas-sensor array for detecting volatile organic compounds (VOC) in chemically induced cells; Pasini P et al.; An application of gas sensors for rapid bioanalysis is presented . An array of temperature-modulated semiconductor sensors was used to characterize the headspace above a cell culture . Recombinant Saccharomyces cerevisiae yeast cells, able to respond to 17 beta-estradiol by producing a reporter protein, were used as a model system . Yeast cells had the DNA sequence of the human estrogen receptor stably integrated into the genome, and contained expression plasmids carrying estrogen-responsive sequences and the reporter gene lac-Z, encoding the enzyme beta-galactosidase . The sensor-response profiles showed small but noticeable discrimination between cell samples induced with 17 beta-estradiol and non-induced cell samples . The sensor array was capable of detecting changes in the volatile organic compound composition of the headspace above the cultured cells, which can be associated with metabolic changes induced by a chemical compound . This finding suggests the possibility of using cross-selective gas-sensor arrays for analysis of drugs or bioactive molecules through their interaction with cell systems, with the advantage of providing information on their bioavailability.

Plant Cell, 2003 Dec, 15(12), 2885 - 99 Epub 2003 Nov 13.
The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways; Surpin M et al.; The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13 . Only VTI11 and VTI12 are expressed at appreciable levels . Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant . VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig . Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport . An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions . However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway . VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal . These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

Science, 2003 Nov 14, 302(5648), 1208 - 12
Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI; Gstaiger M et al.; Prefoldins (PFDs) are members of a recently identified, small-molecular weight protein family able to assemble into molecular chaperone complexes . Here we describe an unusually large member of this family, termed URI, that forms complexes with other small-molecular weight PFDs and with RPB5, a shared subunit of all three RNA polymerases . Functional analysis of the yeast and human orthologs of URI revealed that both are targets of nutrient signaling and participate in gene expression controlled by the TOR kinase . Thus, URI is a component of a signaling pathway that coordinates nutrient availability with gene expression.

Curr Genet, 2004 Feb, 45(1), 28 - 36 Epub 2003 Nov 12.
Structure and expression of a phosphate deficiency-inducible ribonuclease gene in Pholiota nameko; Tasaki Y et al.; Thirty-one cDNAs corresponding to pdi genes {inorganic phosphate (Pi) deficiency-inducible genes} were previously isolated through the differential screening of a cDNA library constructed from the mycelium of Pholiota nameko . Among the cDNAs, pdi370 was analyzed here . The deduced amino acid sequence showed high similarity to fungal ribonucleases (RNases) and contained two signature sequences conserved in T2 family RNases: CAS1 and CAS2 . Genomic DNA harboring the pdi370 gene was isolated from a genomic library of P . nameko . Sequence analysis showed that the pdi370 gene is interrupted by 16 introns and that the promoter region contains two cis-acting sequences found in Pi deficiency-induced genes from Saccharomyces cerevisiae, together with several known functional elements, such as a TATA box . RNase activity in the mycelium and culture filtrate increased 5.6-fold and 5.2-fold, respectively, under Pi-deficient conditions . Staining for RNase activity showed that at least four RNases are induced and secreted under the conditions . The N-terminal sequence of one of them agreed with that of the pdi370 gene product.

FEMS Yeast Res, 2003 Nov, 4(2), 207 - 15
The Hansenula polymorpha (strain CBS4732) genome sequencing and analysis; Ramezani-Rad M et al.; The methylotrophic yeast Hansenula polymorpha is a recognised model system for investigation of peroxisomal function, special metabolic pathways like methanol metabolism, of nitrate assimilation or thermostability . Strain RB11, an odc1 derivative of the particular H . polymorpha isolate CBS4732 (synonymous to ATCC34438, NRRL-Y-5445, CCY38-22-2) has been developed as a platform for heterologous gene expression . The scientific and industrial significance of this organism is now being met by the characterisation of its entire genome . The H . polymorpha RB11 genome consists of approximately 9.5 Mb and is organised as six chromosomes ranging in size from 0.9 to 2.2 Mb . Over 90% of the genome was sequenced with concomitant high accuracy and assembled into 48 contigs organised on eight scaffolds (supercontigs) . After manual annotation 4767 out of 5933 open reading frames (ORFs) with significant homologies to a non-redundant protein database were predicted . The remaining 1166 ORFs showed no significant similarity to known proteins . The number of ORFs is comparable to that of other sequenced budding yeasts of similar genome size.

Int J Oncol, 2003 Dec, 23(6), 1515 - 9
Functional and expression analyses of mgl-1, a mouse orthologue of lethal giant larvae recessive oncogene; Kim YS et al.; We recently isolated a few mammalian homologue of Drosophila lethal giant larvae (lgl) recessive oncogene, suggesting that there is functional conservation among proteins of this family . The comparison of amino acid sequence for mgl-1 with other lgl family members using the clustal method showed that they are highly homologous . Therefore, we investigated the biological function of mgl-1, a mouse orthologue of lgl, in the absence of Saccharomyces cerevisiae Sop1 and Sop2, the yeast homologues of the lgl recessive oncogene . Functional analysis showed that the expression of mgl-1 cDNA partially restored salt tolerance in yeast, indicating the evolutionary conservation of lgl family members . Since the developmental expression profile of mgl-1 has not been elucidated, the temporal and spatial expression patterns of mouse mgl-1 during early embryonic development were analyzed . The temporal expression analysis revealed that mgl-1 is expressed throughout embryonic development from days E4.5 to E18.5 with the strong expression at E10.5 . The analysis of spatial expression showed that mgl-1 mRNA is detected in CNS, craniofacial region, eyes, limbs, and the gut.

J Biol Chem, 2004 Feb 6, 279(6), 4017 - 26 Epub 2003 Nov 11.
Intracellular interleukin-1alpha functionally interacts with histone acetyltransferase complexes; Buryskova M et al.; Interleukin-1alpha (IL-1alpha) is an inflammatory cytokine acting extracellularly via membrane receptors . Interestingly, a significant portion of synthesized IL-1alpha is not secreted; instead, it is actively translocated into the cell nucleus . IL-1alpha was indeed shown to be involved in certain intracellular processes, such as control of proliferation, apoptosis, or migration, however, the mechanisms of such actions are not known . Here we show that intracellular IL-1alpha fused to the Gal4p DNA-binding domain (Gal4BD) possesses strong transactivation potential that can be boosted by overexpression of the transcriptional coactivator p300 . We demonstrate that the IL-1alpha precursor interacts via its N-terminal peptide (IL-1NTP) with histone acetyltransferases p300, PCAF, Gcn5 and with the adaptor component Ada3, and that it integrates into the PCAF.p300 complex in a non-destructive manner . In analogy with known acidic coactivators, yeast strains expressing Gal4BD/IL-1NTP display a toxic phenotype that can be relieved by depletion of various components of the SAGA complex . Our data provide the first solid evidence for the nuclear target of the IL-1alpha precursor and suggest its novel function in transcriptional control.

J Biol Chem, 2004 Feb 13, 279(7), 5934 - 46 Epub 2003 Nov 10.
CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization; Suzuki N et al.; Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA . To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis . An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells . Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase . These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA . In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.

Mol Cell Biol, 2003 Dec, 23(23), 8829 - 45
Recruitment of SWI/SNF by Gcn4p does not require Snf2p or Gcn5p but depends strongly on SWI/SNF integrity, SRB mediator, and SAGA; Yoon S et al.; The nucleosome remodeling complex SWI/SNF is a coactivator for yeast transcriptional activator Gcn4p . We provide strong evidence that Gcn4p recruits the entire SWI/SNF complex to its target genes ARG1 and SNZ1 but that SWI/SNF is dispensable for Gcn4p binding to these promoters . It was shown previously that Snf2p/Swi2p, Snf5p, and Swi1p interact directly with Gcn4p in vitro . However, we found that Snf2p is not required for recruitment of SWI/SNF by Gcn4p nor can Snf2p be recruited independently of other SWI/SNF subunits in vivo . Snf5p was not recruited as an isolated subunit but was required with Snf6p and Swi3p for optimal recruitment of other SWI/SNF subunits . The results suggest that Snf2p, Snf5p, and Swi1p are recruited only as subunits of intact SWI/SNF, a model consistent with the idea that Gcn4p makes multiple contacts with SWI/SNF in vivo . Interestingly, Swp73p is necessary for efficient SWI/SNF recruitment at SNZ1 but not at ARG1, indicating distinct subunit requirements for SWI/SNF recruitment at different genes . Optimal recruitment of SWI/SNF by Gcn4p also requires specific subunits of SRB mediator (Gal11p, Med2p, and Rox3p) and SAGA (Ada1p and Ada5p) but is independent of the histone acetyltransferase in SAGA, Gcn5p . We suggest that SWI/SNF recruitment is enhanced by cooperative interactions with subunits of SRB mediator and SAGA recruited by Gcn4p to the same promoter but is insensitive to histone H3 acetylation by Gcn5p.

Mol Cell Biol, 2003 Dec, 23(23), 8718 - 28
The Cdk-activating kinase Cak1p promotes meiotic S phase through Ime2p; Schindler K et al.; CAK1 encodes an essential protein kinase in Saccharomyces cerevisiae that is required for activation of the Cdc28p Cdk . CAK1 also has several CDC28-independent functions that are unique to meiosis . The earliest of these functions is to induce S phase, which is regulated differently in meiosis than in mitosis . In mitosis, Cdc28p controls its own S-phase-promoting activity by signaling the destruction of its inhibitor, Sic1p . In meiosis, Sic1p destruction is signaled by the meiosis-specific Ime2p protein kinase . Our data show that Cak1p is required to activate Ime2p through a mechanism that requires threonine 242 and tyrosine 244 in Ime2p's activation loop . This activation promotes autophosphorylation and accumulation of multiply phosphorylated forms of Ime2p during meiotic development . Consistent with Cak1p's role in activating Ime2p, cells lacking Cak1p are deficient in degrading Sic1p . Deletion of SIC1 or overexpression of IME2 can partially suppress the S-phase defect in cak1 mutant cells, suggesting that Ime2p is a key target of Cak1p regulation . These data show that Cak1p is required for the destruction of Sic1p in meiosis, as in mitosis, but in meiosis, it functions through a sporulation-specific kinase.

Mol Cell Biol, 2003 Dec, 23(23), 8471 - 85
Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein; Zhang B et al.; Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level . Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress . Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements . In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide . Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers . We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein . This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1 . Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1 . The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.

Mol Cell Biol, 2003 Dec, 23(23), 8450 - 61
End resection initiates genomic instability in the absence of telomerase; Hackett JA et al.; Telomere dysfunction causes genomic instability . However, the mechanism that initiates this instability when telomeres become short is unclear . We measured the mutation rate and loss of heterozygosity along a chromosome arm in diploid yeast that lacked telomerase to distinguish between mechanisms for the initiation of instability . Sequence loss was localized near chromosome ends in the absence of telomerase but not after breakage of a dicentric chromosome . In the absence of telomerase, the increase in mutation rate is dependent on the exonuclease Exo1p . Thus, exonucleolytic end resection, rather than chromosome fusion and breakage, is the primary mechanism that initiates genomic instability when telomeres become short.

Mol Cell Biol, 2003 Dec, 23(23), 8440 - 9
A conserved telomerase motif within the catalytic domain of telomerase reverse transcriptase is specifically required for repeat addition processivity; Lue NF et al.; Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats . The catalytic protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT) domain that mediates nucleotide addition . The RT domain of TERT is distinguishable from retroviral and retrotransposon RTs in having a sizable insertion between conserved motifs A and B', within the so-called fingers domain . Sequence analysis revealed the existence of conserved residues in this region, named IFD (insertion in fingers domain) . Mutations of some of the conserved residues in Saccharomyces cerevisiae TERT (Est2p) abolished telomerase function in vivo, testifying to their importance . Significant effects of the mutations on telomerase activity in vitro were observed, with most of the mutants exhibiting a uniform reduction in activity regardless of primer sequence . Remarkably, one mutant manifested a primer-specific defect, being selectively impaired in extending primers that form short hybrids with telomerase RNA . This mutant also accumulated products that correspond to one complete round of repeat synthesis, implying an inability to effect the repositioning of the DNA product relative to the RNA template that is necessary for multiple repeat addition . Our results suggest that the ability to stabilize short RNA-DNA hybrids is crucial for telomerase function in vivo and that this ability is mediated in part by a more elaborate fingers domain structure.

Genome Biol . 2003;4(11):R76 . Epub 2003 Oct 24.
Application of independent component analysis to microarrays; Lee SI et al.; We apply linear and nonlinear independent component analysis (ICA) to project microarray data into statistically independent components that correspond to putative biological processes, and to cluster genes according to over- or under-expression in each component . We test the statistical significance of enrichment of gene annotations within clusters . ICA outperforms other leading methods, such as principal component analysis, k-means clustering and the Plaid model, in constructing functionally coherent clusters on microarray datasets from Saccharomyces cerevisiae, Caenorhabditis elegans and human.

Genome Biol . 2003;4(11):236 . Epub 2003 Oct 27.
The septins; Kinoshita M; The septins are guanine-nucleotide binding proteins that mostly form filaments . They are important in cytokinesis and also have roles in sporulation in yeasts and embryonic development and in the nervous system in animals.

Zhejiang Da Xue Xue Bao Yi Xue Ban, 2003 Oct, 32(5), 393 - 7
{Cloning and bioinformatics of human REV3 gene promoter region and its response to carcinogen N-methyl-N'-nitro-N-nitrosoguanidine}; Zhu F et al.; OBJECTIVE: To understand the up regulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . METHODS: Bioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites . Cloning of human REV3 gene promoter region was performed by nested PCR . Response of human REV3 gene promoter to the chemical carcinogen MNNG was measured by transient transfection assay based on the dual luciferase reporter assay system . RESULT: Bioinformatic analysis showed that human REV3 gene promoter region was located on chromosome 6 PAC clone RP3-415N12, and that the hypothetical promoter region contained promoter sequences, rich CpG islands, and putative recognition sites for several transcriptional factors, including AP-1/c-Jun/c-Fos, AP-2, STAT, CREBP, and NF-kappaB . Reconstructed reporter plasmid pGL3- 2582 was established by inserting 2582 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic . Transient transfection assay showed the hypothetical REV3 promoter region had promoter function, and it responded to MNNG treatment (P<0.01) . CONCLUSION: Human mutator REV3 gene promoter region has been successfully cloned . The response of REV3 promoter region to MNNG suggests that REV3 gene can be regulated at transcriptional level under conditions of genotoxic stress.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13857 - 62 Epub 2003 Nov 10.
ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA; Perriman R et al.; Stable addition of U2 small nuclear ribonucleoprotein (snRNP) to form the prespliceosome is the first ATP-dependent step in splicing, and it requires the DEXD/H box ATPase Prp5p . However, prespliceosome formation occurs without ATP in extracts lacking the U2 snRNP protein Cus2p . Here we show that Prp5p is required for the ATP-independent prespliceosome assembly that occurs in the absence of Cus2p . Addition of recombinant Cus2p can restore the ATP dependence of prespliceosome assembly, but only if it is added before Prp5p . Prp5p with an altered ATP-binding domain (Prp5-GNTp) can support growth in vivo, but only in a cus2 deletion strain, mirroring the in vitro results . Other Prp5 ATP-binding domain substitutions are lethal, even in the cus2 deletion strain, but can be suppressed by U2 small nuclear RNA mutations that hyperstabilize U2 stem IIa . We infer that the presence of Cus2p and stem IIa-destabilized forms of U2 small nuclear RNA places high demands on the ATP-driven function of Prp5p . Because Prp5p is not dispensable in vitro even in the absence of ATP, we propose that the core Prp5p function in bringing U2 to the branchpoint is not directly ATP-dependent . The positive role of Cus2p in rescuing mutant U2 can be reconciled with its antagonistic effect on Prp5 function in a model whereby Cus2p first helps Prp5p to activate the U2 snRNP for prespliceosome formation but then is displaced by Prp5p before or during the stabilization of U2 at the branchpoint.

J Virol, 2003 Dec, 77(23), 12795 - 806
Relationship between RNA lariat debranching and Ty1 element retrotransposition; Salem LA et al.; The Saccharomyces cerevisiae DBR1 gene encodes a 2'-5' phosphodiesterase that debranches intron RNA lariats following splicing . Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3 . We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching . Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p . Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition . Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine . We have developed an RNase protection assay to quantitate intron RNA accumulation in cells . The assay uses RNA probes that hybridize to ACT1 intron RNA . Protection patterns confirm that sequences from the 5' end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation . RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2'-5' phosphodiesterase activity in Ty1 transposition . A Ty1 element lacking most of its internal sequences transposes independently of DBR1 . The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.

J Biol Chem, 2004 Jan 23, 279(4), 2616 - 22 Epub 2003 Nov 10.
Stress-specific activation mechanisms for the "cell integrity" MAPK pathway; Harrison JC et al.; Many environmental stresses trigger cellular responses by activating mitogen-activated protein kinase (MAPK) pathways . Once activated, these highly conserved protein kinase cascades can elicit cellular responses such as transcriptional activation of response genes, cytoskeletal rearrangement, and cell cycle arrest . The mechanism of pathway activation by environmental stresses is in most cases unknown . We have analyzed the activation of the budding yeast "cell integrity" MAPK pathway by heat shock, hypoosmotic shock, and actin perturbation, and we report that different stresses regulate this pathway at different steps . In no case can MAPK activation be explained by the prevailing view that stresses simply induce GTP loading of the Rho1p GTPase at the "top" of the pathway . Instead, our findings suggest that the stresses can modulate at least three distinct kinases acting between Rho1p and the MAPK . These findings suggest that stresses provide "lateral" inputs into this regulatory pathway, rather than operating in a linear "top-down" manner.

J Biol Chem, 2004 Feb 13, 279(7), 5655 - 60 Epub 2003 Nov 10.
Aurora-B phosphorylation in vitro identifies a residue of survivin that is essential for its localization and binding to inner centromere protein (INCENP) in vivo; Wheatley SP et al.; The chromosomal passengers, aurora-B kinase, inner centromere protein (INCENP), and survivin, are essential proteins that have been implicated in the regulation of metaphase chromosome alignment, spindle checkpoint function, and cytokinesis . All three share a common pattern of localization, and it was recently demonstrated that aurora-B, INCENP, and survivin are present in a complex in Xenopus eggs and Saccharomyces cerevisiae . The presence of aurora-B kinase in the complex and its ability to bind the other components directly suggest that INCENP and survivin could potentially be aurora-B substrates . This hypothesis was recently proven for INCENP in vitro . Here we report that human survivin is specifically phosphorylated in vitro by aurora-B kinase at threonine 117 in its carboxyl alpha-helical coil . Mutation of threonine 117 to alanine prevents survivin phosphorylation by aurora-B in vitro but does not alter its localization in HeLa cells . By contrast, a phospho-mimic, in which threonine 117 was mutated to glutamic acid, was unable to localize correctly at any stage in mitosis . Mutation at threonine 117 also prevented immunoprecipitation of INCENP with survivin in vivo . These data suggest that phosphorylation of survivin at threonine 117 by aurora-B may regulate targeting of survivin, and possibly the entire passenger complex, in mammals.

J Biol Chem, 2004 Jan 30, 279(5), 3361 - 9 Epub 2003 Nov 10.
Amyloid nucleation and hierarchical assembly of Ure2p fibrils . Role of asparagine/glutamine repeat and nonrepeat regions of the prion domains; Jiang Y et al.; The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro . This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates . The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic . We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions . Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding) . The Ure2 dimer (h = 2.6 +/- 0.5 nm) and granular particles corresponding to higher order oligomers (h = 4-12 nm) could also be detected . The mutants 15Ure2 and Delta 15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2 . In addition, Delta 15-42Ure2 showed reduced binding to ThT . The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT . Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.

EMBO J, 2003 Nov 17, 22(22), 6045 - 56
Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR; Tsang CK et al.; The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation . However, how TOR is involved remains poorly understood . In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells . In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription . We also present evidence that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4 . Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition . Taken together, our results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

J Muscle Res Cell Motil, 2003, 24(2-3), 205 - 9
Cardiac expression of Gal4 causes cardiomyopathy in a dose-dependent manner; Habets PE et al.; Cardiac expression of a transgene is a common approach for determining the role of gene products in the processes underlying cardiomyopathy and heart failure (HF) . We have generated transgenic mice that express the 'harmless' yeast transcription factor Gal4 in the heart under control of the alpha-myosin heavy chain promoter and found that expression of this gene causes cardiomyopathy and HF, the severity of which correlated with the number of copies of the transgene integrated into the genome and with the expression level . A line with a single copy of the transgene targeted to the hprt locus correctly expressed the transgene but did not develop cardiomyopathy . Our results indicate that expression of a transgene in the heart may non-specifically cause HF in a dose-dependent manner.

Nat Struct Biol, 2003 Dec, 10(12), 1039 - 47 Epub 2003 Nov 09.
Homodirectional changes in transcriptome composition and mRNA translation induced by rapamycin and heat shock; Preiss T et al.; Transcription and mRNA turnover determine the quantitative composition of the cellular transcriptome . The transcriptome in turn serves as a template for the proteome via translation . Treatment of Saccharomyces cerevisiae with the TOR kinase inhibitor rapamycin causes increases and decreases in the mRNA levels of hundreds of genes . We used DNA microarray analysis to monitor simultaneously transcriptome and translational changes for all detectable yeast mRNAs . Notably, genes that are induced in the transcriptome correlate tightly with more efficiently translated mRNAs (based on their relative degree of polyribosome association); similarly, genes that show reduced mRNA levels after rapamycin treatment also show lower translational fitness . Microarray analyses on heat-shocked cells also reveal homodirectional co-regulatory responses . Thus, signal-induced changes in the transcriptome are amplified at the translational level . These results unveil a higher level of coordinated gene regulation that we refer to as 'potentiation.'

EMBO Rep, 2003 Dec, 4(12), 1156 - 62 Epub 2003 Nov 07.
Formation and nuclear export of tRNA, rRNA and mRNA is regulated by the ubiquitin ligase Rsp5p; Neumann S et al.; The yeast ubiquitin-protein ligase Rsp5p regulates processes as diverse as polII transcription and endocytosis . Here, we identify Rsp5p in a screen for tRNA export (tex) mutants . The tex23-1/rsp5-3 mutant, which is complemented by RSP5, not only shows a strong nuclear accumulation of tRNAs at the restrictive temperature, but also is severely impaired in the nuclear export of mRNAs and 60S pre-ribosomal subunits . In contrast, nuclear localization sequence (NLS)-mediated nuclear protein import is unaffected in this mutant . Strikingly, the nuclear RNA export defects seen in the rsp5-3 strain are accompanied by a dramatic inhibition of both rRNA and tRNA processing, a combination of phenotypes that has not been reported for any previously characterized mutation in yeast . These data implicate ubiquitination as a mechanism coordinating the major nuclear RNA biogenesis pathways.

Clin Diagn Lab Immunol, 2003 Nov, 10(6), 1096 - 102
Novel microtechnique for assessment of postnatal maturation of the phagocytic function of neutrophils and monocytes; Muniz-Junqueira MI et al.; We describe a simple test for the evaluation of phagocytosis and provide a chart of reference values to evaluate normal phagocytosis by age . We assessed the postnatal maturation of phagocytic function of neutrophils and monocytes . Phagocytosis was evaluated in newborn children delivered vaginally or by cesarean section, infants, preschool children, schoolchildren, and adult subjects . Two drops of blood were placed on a microscope slide and incubated with Saccharomyces cerevisiae yeasts, and phagocytosis was evaluated by microscopy . Our technique showed results comparable to or better than those obtained by other usual techniques . The neutrophils of newborn children delivered by cesarean section showed a phagocytic capacity 45% higher than those of neonates delivered vaginally, whereas neutrophils from children in the latter group showed the lowest phagocytic capacity of all age groups . Phagocytosis by neutrophils reached the levels seen in adults at about the first year of life, while there were no important variations in phagocytosis by monocytes in the different age groups . The technique described is reliable and fast, uses only a few drops of blood, and allows better preservation of cell function due to the minimal manipulation to which the cells are submitted . The delayed maturation of the phagocytic function by neutrophils may account for the high levels of susceptibility of newborn and infant children to bacterial infections . This practical method of assessment of phagocytosis may allow the diagnosis of primary or secondary phagocytic deficiencies to be made more easily and may allow better monitoring and treatment of those with dysfunctions of these cells.

J Biol Chem, 2004 Feb 6, 279(6), 3980 - 9 Epub 2003 Nov 08.
Distinct steps in dislocation of luminal endoplasmic reticulum-associated degradation substrates: roles of endoplamic reticulum-bound p97/Cdc48p and proteasome; Elkabetz Y et al.; Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation . Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps . The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates . In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain . The second dislocation step is the release of accumulated substrates to the cytosol . In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ER-bound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates . An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin . BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD . We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited . Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.

Cancer Lett, 2003 Nov 25, 201(2), 203 - 10
p55CDC/hCDC20 mutant induces mitotic catastrophe by inhibiting the MAD2-dependent spindle checkpoint activity in tumor cells; Sihn CR et al.; Nondisjunction of chromosomes results in aneuploidy in mammalian cells causing genomic instability . The spindle checkpoint, one of the surveillance systems to maintain genomic stability, prevents missegregation of chromosomes until all the kinetochores are properly attached with bipolar spindles . When this condition is not met, MAD2, a component of the spindle checkpoint complex, associates with p55CDC/hCDC20 to inhibit ubiquitination of substrates by the anaphase-promoting complex (APC) . In this study, we have focused on the biological role of the MAD2-binding domain in p55CDC/hCDC20 in the maintenance of genomic stability . Based on previous studies, we constructed a truncated p55CDC/hCDC20 mutant (F2) that harbors only the MAD2-binding domain . Interestingly, we found that in the yeast two-hybrid system, the interaction of F2 and MAD2 was stronger than that of intact p55CDC/hCDC20 . We also found that in the presence of the microtubule-disrupting drug, nocodazole, U2OS cells expressing p55CDC/hCDC20 mutants bypassed the mitotic arrest and showed apoptotic morphologies, whereas cells harboring vector alone arrested at metaphase . In particular, the apoptotic phenomena were dramatically enhanced in the F2-expressing cells . These mitotic catastrophes also occurred in cells treated with other microtubule disrupting agents, such as taxol and vinblastine . In addition, the mutant cells exhibited chromosomal missegregation during mitosis, even in the absence of nocodazole . Taken together, these results suggest that agents blocking the spindle checkpoint response may induce tumor cells to become more sensitive to spindle poison drugs, providing a powerful tool to improve chemotherapy.

Life Sci, 2003 Dec 5, 74(2-3), 199 - 205
ER signaling in unfolded protein response; Kaneko M et al.; Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death . To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER) . This is known as the unfolded protein response (UPR) . ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system . Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system . Hrd3p interacts with and stabilizes Hrd1p . We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p . Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER . HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death . These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.

Plant Physiol, 2003 Dec, 133(4), 1831 - 42 Epub 2003 Nov 06.
Activation of anthocyanin biosynthesis in Gerbera hybrida (Asteraceae) suggests conserved protein-protein and protein-promoter interactions between the anciently diverged monocots and eudicots; Elomaa P et al.; We have identified an R2R3-type MYB factor, GMYB10, from Gerbera hybrida (Asteraceae) that shares high sequence homology to and is phylogenetically grouped together with the previously characterized regulators of anthocyanin pigmentation in petunia (Petunia hybrida) and Arabidopsis . GMYB10 is able to induce anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum), especially in vegetative parts and anthers . In G . hybrida, GMYB10 is involved in activation of anthocyanin biosynthesis in leaves, floral stems, and flowers . In flowers, its expression is restricted to petal epidermal cell layers in correlation with the anthocyanin accumulation pattern . We have shown, using yeast (Saccharomyces cerevisiae) two-hybrid assay, that GMYB10 interacts with the previously isolated bHLH factor GMYC1 . Particle bombardment analysis was used to show that GMYB10 is required for activation of a late anthocyanin biosynthetic gene promoter, PGDFR2 . cis-Analysis of the target PGDFR2 revealed a sequence element with a key role in activation by GMYB10/GMYC1 . This element shares high homology with the anthocyanin regulatory elements characterized in maize (Zea mays) anthocyanin promoters, suggesting that the regulatory mechanisms involved in activation of anthocyanin biosynthesis have been conserved for over 125 million years not only at the level of transcriptional regulators but also at the level of the biosynthetic gene promoters.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13827 - 32 Epub 2003 Nov 06.
Replication protein A-mediated recruitment and activation of Rad17 complexes; Zou L et al.; The human Rad17-Rfc2-5 and Rad9-Rad1-Hus1 complexes play crucial roles in the activation of the ATR-mediated DNA damage and DNA replication stress response pathways . In response to DNA damage, Rad9 is recruited to chromatin in a Rad17-dependent manner in human cells . However, the DNA structures recognized by the Rad17-Rfc2-5 complex during the damage response have not been defined . Here, we show that replication protein A (RPA) stimulates the binding of the Rad17-Rfc2-5 complex to single-stranded DNA (ssDNA), primed ssDNA, and a gapped DNA structure . Furthermore, RPA facilitates the recruitment of the Rad9-Rad1-Hus1 complex by the Rad17-Rfc2-5 complex to primed and gapped DNA structures in vitro . These findings suggest that RPA-coated ssDNA is an important part of the structures recognized by the Rad17-Rfc2-5 complex . Unlike replication factor C (RFC), which uses the 3' primer/template junction to recruit proliferating cell nuclear antigen (PCNA), the Rad17-Rfc2-5 complex can use both the 5' and the 3' primer/template junctions to recruit the Rad9-Rad1-Hus1 complex, and it shows a preference for gapped DNA structures . These results explain how the Rad17-Rfc2-5 complex senses DNA damage and DNA replication stress to initiate checkpoint signaling.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13839 - 44 Epub 2003 Nov 06.
J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix; D'Silva PD et al.; The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix . Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel . Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate . Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function . However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane . Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix . We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import.

Science, 2003 Dec 19, 302(5653), 2120 - 4 Epub 2003 Nov 06.
Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14; Pereira G et al.; The inner centromere-like protein (INCENP) forms a complex with the evolutionarily conserved family of Aurora Bkinases . The INCENP-Aurora complex helps coordinate chromosome segregation, spindle behavior, and cytokinesis during mitosis . INCENP-Aurora associates with kinetochores in metaphase and with spindle microtubules in anaphase, yet the trigger for this abrupt transfer is unknown . Here we show that the conserved phosphatase Cdc14 regulated the yeast INCENP-Aurora complex, Sli15-Ipl1 . Cdc14 dephosphorylated Sli15 and thereby directed the complex to spindles . Activation of Cdc14 by separase was sufficient for Sli15 dephosphorylation and relocalization . Cdc14 not only regulates mitotic exit but also modulates spindle midzone assembly through Sli15-Ipl1.

Structure (Camb), 2003 Nov, 11(11), 1423 - 30
Crystal structure of a bulged RNA tetraplex at 1.1 a resolution: implications for a novel binding site in RNA tetraplex; Pan B et al.; Bulges are an important structural motif in RNA and can be used as recognition and interaction sites in RNA-protein interaction and RNA-RNA interaction . Here we report the first crystal structure of a bulged RNA tetraplex at 1.1 A resolution . The hexamer r(U)(BrdG)r(UGGU) forms a parallel tetraplex with the uridine sandwiched by guanines bulging out . The bulged uridine adopts the syn glycosidic conformation and its O2 and N3 atoms face outwards, serving as an effective recognition and interaction site . The bulge formation both widens the groove width and changes the groove hydrogen-bonding pattern on its 5' side . However, the bulge does not make any bends or kinks in the tetraplex structure . The present study demonstrates the dramatic difference between uridine and guanine in forming tetraplex structure . In addition, both G(syn) tetrad and G(anti) tetrad have been observed . They display the same base-pairing pattern and similar C1'-C1' distance but different hydrogen-bonding patterns in the groove.

Leukemia, 2004 Jan, 18(1), 92 - 102
MLL fusion partners AF4 and AF9 interact at subnuclear foci; Erfurth F et al.; The MLL gene is involved in translocations associated with both acute lymphoblastic and acute myelogenous leukemia . These translocations fuse MLL with one of over 30 partner genes . Collectively, the MLL partner genes do not share a common structural motif or biochemical function . We have identified a protein interaction between the two most common MLL fusion partners AF4 and AF9 . This interaction is restricted to discrete nuclear foci we have named 'AF4 bodies' . The AF4 body is non-nucleolar and is not coincident with any known nuclear structures we have examined . The AF4-AF9 interaction is maintained by the MLL-AF4 fusion protein, and expression of the MLL-AF4 fusion can alter the subnuclear localization of AF9 . In view of other research indicating that other MLL fusion partners also interact with one another, these results suggest that MLL fusion partners may participate in a web of protein interactions with a common functional goal . The disruption of this web of interactions by fusion with MLL may be important to leukemogenesis.

Sci Aging Knowledge Environ . 2002 Sep 18;2002(37):pe14.
Mitigating the tithonus error: genetic analysis of mortality phenotypes; Pletcher SD; Summarizing an organism's age at death in terms of the mean or maximum life-span is the most popular way to describe genetic effects on aging . In this Perspective, the author describes a new study with the fly Drosophila melanogaster, in which another type of measure is also used: the age-dependent risk of death, or age-specific mortality . Changes in age-specific mortality reflect the underlying physiological deterioration of an organism as it ages . Thus, the author argues that these changes provide a phenotype that is ideal for the genetic analysis of aging.

Nucleic Acids Res, 2003 Nov 15, 31(22), 6493 - 501
Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins; Carvin CD et al.; Cytosine-5 DNA methylation is a critical signal defining heritable epigenetic states of transcription . As aberrant methylation patterns often accompany disease states, the ability to target cytosine methylation to preselected regions could prove valuable in re-establishing proper gene regulation . We employ the strategy of targeted gene methylation in yeast, which has a naturally unmethylated genome, selectively directing de novo DNA methylation via the fusion of C5 DNA methyltransferases to heterologous DNA-binding proteins . The zinc-finger proteins Zif268 and Zip53 can target DNA methylation by M.CviPI or M.SssI 5-52 nt from single zinc-factor binding sites . Modification at specific GC (M.CviPI) or CG (M.SssI) sites is enhanced as much as 20-fold compared with strains expressing either the free enzyme or a fusion protein with the zinc-finger protein moiety unable to bind to DNA . Interestingly, methylation is also selectively targeted as far as 353 nt from the zinc-finger protein binding sites, possibly indicative of looping, nucleosomes or higher-order chromatin structure . These data demonstrate that methylation can be targeted in vivo to a potentially broad range of sequences using specifically engineered zinc-finger proteins . Further more, the selective targeting of methylation by zinc-finger proteins demonstrates that binding of distinct classes of factors can be monitored in living cells.

Dev Cell, 2003 Nov, 5(5), 735 - 45
An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase; Pinsky BA et al.; Chromosome segregation depends on kinetochore biorientation so that sister kinetochores attach to microtubules from opposite poles and come under tension . The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint . We found that checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension . We isolated mtw1-1 dosage suppressors and identified Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins . mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together . Although mtw1 mutant cells contained unattached chromosomes, attachment was restored by impairing Ipl1p function . These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes . We therefore propose that an Mtw1 complex is required for kinetochore biorientation that is monitored by the Ipl1p kinase.

Dev Cell, 2003 Nov, 5(5), 709 - 22
The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4; Marchese A et al.; Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood . Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes . CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes . Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs . We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.

Virology, 2003 Nov 10, 316(1), 75 - 83
The E1A proteins of all six human adenovirus subgroups target the p300/CBP acetyltransferases and the SAGA transcriptional regulatory complex; Shuen M et al.; The N-terminal/conserved region 1 (CR1) portion of the human adenovirus (Ad) 5 E1A protein was previously shown to inhibit growth in the simple eukaryote Saccharomyces cerevisiae . We now demonstrate that the corresponding regions of the E1A proteins of Ad3,-4,-9,-12, and -40, which represent the remaining five Ad subgroups, also inhibit yeast growth . These results suggest that the E1A proteins of all six human Ad subgroups share a common cellular target(s) conserved in yeast . Growth inhibition induced by either full-length or the N-terminal/CR1 portion of Ad5 E1A was relieved by coexpression of the E1A binding portions of the mammalian p300, CBP, and pCAF acetyltransferases . Similarly, growth inhibition by the N-terminal/CR1 portions of the other Ad E1A proteins was suppressed by expression of the same regions of CBP or pCAF known to bind Ad5 E1A . The physical interaction of each of the different Ad E1A proteins with CBP, p300, and pCAF was confirmed in vitro . Furthermore, deletion of the gene encoding yGcn5, the yeast homolog of pCAF and a subunit of the SAGA transcriptional regulatory complex, restored growth in yeast expressing each of the different Ad E1A proteins . This indicates that the SAGA complex is a conserved target of all Ad E1A proteins . Our results demonstrate for the first time that the p300, CBP, and pCAF acetyltransferases are common targets for the E1A proteins of all six human Ad subgroups, highlighting the importance of these interactions for E1A function.

Mol Biochem Parasitol, 2003 Dec, 132(2), 75 - 81
Cloning and analysis of Trypanosoma cruzi lanosterol 14alpha-demethylase; Buckner FS et al.; Trypanosoma cruzi infection, transmitted by insect vectors or blood transfusions, is an important cause of morbidity and mortality in many Latin American countries . Treatments are toxic and frequently ineffective in curing patients with chronic manifestations of the infection (Chagas disease) . Potentially exploitable chemotherapeutic targets of T . cruzi are enzymes of the sterol biosynthesis pathway . In particular, the P450 enzyme, lanosterol 14alpha-demethylase, has been implicated as the target of azole antifungal drugs that have potent anti-T . cruzi activity . In the work reported here, the T . cruzi lanosterol 14alpha-demethylase (Tc14DM) gene was cloned by degenerate PCR . The gene was found to be expressed in both insect and mammalian life-cycle stages of the parasite . Tc14DM was able to complement the function of the homologous gene in yeast (erg11) as demonstrated by restored ergosterol production in an erg11-deficient yeast strain . When the yeast strain was co-transfected with the P450 reductase gene from Trypanosoma brucei, the amount of ergosterol production was increased, indicating that the endogenous yeast P450 reductase was an inefficient partner with Tc14DM . Heterologous expression of Tc14DM in the baculovirus/Sf9 system resulted in a 52kDa product . The protein was observed to have the characteristic absorbance spectra of a P450 enzyme . A typical Type II binding spectrum was produced when the imidazole compound, ketoconazole, was mixed with the Tc14DM, demonstrating that ketoconazole binds the enzyme.

Mol Biochem Parasitol, 2003 Dec, 132(2), 67 - 74
Ribosomal RNA processing and the role of SmMAK16 in ribosome biogenesis in Schistosoma mansoni; Capowski EE et al.; Ribosome biogenesis is an essential and complex biological process with links to cell cycle control, DNA replication and aberrant cell growth . As the process becomes better understood at a mechanistic level, new opportunities arise to exploit its effects on maturation and fertility, two important targets in parasite therapeutics . While the physical structure and sequence of ribosomal RNA (rRNA) in the trematode Schistosoma mansoni have been described, the process of cleavage, modification and assembly of the mature ribonucleoprotein particles is not well characterized . We have investigated the cleavage pathway of rRNA in different life cycle stages of the parasite and present evidence supporting a proposed model . In addition, we have investigated the role of the S . mansoni nucleolar protein SmMAK16 in ribosome biogenesis . Co-immunoprecipitation data and polysome analysis support the hypothesis that SmMAK16 associates with pre-ribosomal precursor complexes . This work represents the first analysis of this important and complex process in schistosomes.

J Cell Biol, 2003 Nov 10, 163(3), 503 - 10 Epub 2003 Nov 03.
Evidence for a two membrane-spanning autonomous mitochondrial DNA replisome; Meeusen S et al.; The unit of inheritance for mitochondrial DNA (mtDNA) is a complex nucleoprotein structure termed the nucleoid . The organization of the nucleoid as well as its role in mtDNA replication remain largely unknown . Here, we show in Saccharomyces cerevisiae that at least two populations of nucleoids exist within the same mitochondrion and can be distinguished by their association with a discrete proteinaceous structure that spans the outer and inner mitochondrial membranes . Surprisingly, this two membrane-spanning structure (TMS) persists and self-replicates in the absence of mtDNA . We tested whether TMS functions to direct the replication of mtDNA . By monitoring BrdU incorporation, we observed that actively replicating nucleoids are associated exclusively with TMS . Consistent with TMS's role in mtDNA replication, we found that Mip1, the mtDNA polymerase, is also a stable component of TMS . Taken together, our observations reveal the existence of an autonomous two membrane-spanning mitochondrial replisome as well as provide a mechanism for how mtDNA replication and inheritance may be physically linked.

J Biol Chem, 2004 Feb 6, 279(6), 4507 - 14 Epub 2003 Nov 03.
p42, a novel cyclin-dependent kinase-activating kinase in mammalian cells; Liu Y et al.; The cyclin-dependent kinase (CDK)-activating kinase (CAK) phosphorylates a conserved threonine residue on CDKs and activates them . Two known classes of CAKs are represented by monomeric Cak1p in budding yeast Saccharomyces cerevisiae and by heterotrimeric CDK7-cyclin H-Mat1 in human and other metazoa . We report here the identification of p42, a novel CAK activity in human cells . p42 has sequence homology to both Cak1p and CDK7 groups of CAKs . p42 is essential for the phosphorylation of Thr-160 and activation of CDK2 . A dominant-negative p42 mutant, T161A, and posttranscriptional gene silencing of p42 with RNA(i)-impaired Thr-160 phosphorylation and activity of CDK2 . Purified p42 phosphorylated glutathione S-transferase-CDK2 at Thr-160 within the T-loop and activated its histone H1 kinase activity . Finally, p42 is indispensable for cell growth . Cells lacking p42 were incapable of growing and forming colonies whereas cells with a reduced level of p42 grew at significantly slower rates than control cells . Our findings suggest that p42 represents a novel CAK activity in mammalian cells.

Development, 2003 Dec, 130(24), 6065 - 73
Regulation of storage protein gene expression in Arabidopsis; Kroj T et al.; The expression of seed storage proteins is under tight developmental regulation and represents a powerful model system to study the regulation of gene expression during plant development . In this study, we show that three homologous B3 type transcription factors regulate the model storage protein gene, At2S3, via two distinct mechanisms: FUSCA3 (FUS3) and LEAFY COTYLEDON2 (LEC2) activate the At2S3 promoter in yeast suggesting that they regulate At2S3 by directly binding its promoter; ABSCISIC ACID INSENSITIVE3 (ABI3), however, appears to act more indirectly on At2S3, possibly as a cofactor in an activation complex . In accordance with this, FUS3 and LEC2 were found to act in a partially redundant manner and differently from ABI3 in planta: At2S3 expression is reduced to variable and sometimes only moderate extent in fus3 and lec2 single mutants but is completely abolished in the lec2 fus3 double mutant . In addition, we found that FUS3 and LEC2 expression patterns, together with an unsuspected regulation of FUS3 by LEC2, enable us to explain the intriguing expression pattern of At2S3 in lec2 or fus3 single mutants . Based on these results, we present a model of At2S3 regulation and discuss its implications for other aspects of seed maturation.

Res Microbiol, 2003 Nov, 154(9), 603 - 10
On the trail of an elusive flux sensor; Bisson LF et al.; The hexokinase PII isozyme has been implicated as an essential component of multiple glucose sensing pathways in the yeast Saccharomyces cerevisiae . Several lines of evidence suggest that the flux through this enzymatic step, but not the levels of substrates, cofactors or products, is the critical process detected by downstream sensing machinery . In spite of intensive research efforts, how the activity of this enzyme is translated into a quantitative signal remains an unresolved question.

Biochemistry, 2003 Nov 11, 42(44), 12941 - 9
Interactions between charged amino acid residues within transmembrane helices in the sulfate transporter SHST1; Shelden MC et al.; The aim of this study was to identify charged amino acid residues important for activity of the sulfate transporter SHST1 . We mutated 10 charged amino acids in or near proposed transmembrane helices and expressed the resulting mutants in a sulfate transport-deficient yeast strain . Mutations affecting four residues resulted in a complete loss of sulfate transport; these residues were D107 and D122 in helix 1 and R354 and E366 in helix 8 . All other mutants showed some reduction in transport activity . The E366Q mutant was unusual in that expression of the mutant protein was toxic to yeast cells . The R354Q mutant showed reduced trafficking to the plasma membrane, indicating that the protein was misfolded . However, transporter function (to a low level) and wild-type trafficking could be recovered by combining the R354Q mutation with either the E175Q or E270Q mutations . This suggested that R354 interacts with both E175 and E270 . The triple mutant E175Q/E270Q/R354Q retained only marginal sulfate transport activity but was trafficked at wild-type levels, suggesting that a charge network between these three residues may be involved in the transport pathway, rather than in folding . D107 was also found to be essential for the ion transport pathway and may form a charge pair with R154, both of which are highly conserved . The information obtained on interactions between charged residues provides the first evidence for the possible spatial arrangement of transmembrane helices within any member of this transporter family . This information is used to develop a model for SHST1 tertiary structure.

Biotechnol Bioeng, 2003 Dec 20, 84(6), 658 - 66
Expression of vitreoscilla hemoglobin improves growth and levels of extracellular enzyme in Yarrowia lipolytica; Bhave SL et al.; Enhancement in oxygen uptake by high-cell-density cultivations has been achieved previously by expression of the bacterial hemoglobin gene from Vitreoscilla . The Vitreoscilla hemoglobin (VHb) gene was expressed in the yeast Yarrowia lipolytica to study the effect of expression in this commercially important yeast . The expression of VHb in this yeast was found to enhance growth, contrary to reported observations in wild-type Saccharomyces cerevisiae in which there was no significant growth enhancement . VHb-expressing Y . lipolytica exhibited higher specific growth rate, enhanced oxygen uptake rate, and higher respiratory activity . We report the beneficial effects of VHb expression on growth under microaerobic as well as under nonlimiting dissolved oxygen conditions . Earlier studies in Y . lipolytica have demonstrated inhibition of mycelia formation by respiratory inhibitors and poor nitrogen source, conditions poor for growth . VHb(+) Y . lipolytica cells were more efficient at forming mycelia, indicating better utilization of available oxygen as compared with the VHb(-) cells . Expression of VHb was also found to increase the levels of enzyme ribonuclease secreted into the medium, a property that may be beneficial for producing heterologous proteins in Y . lipolytica .

Nat Struct Biol, 2003 Dec, 10(12), 988 - 94 Epub 2003 Nov 02.
Tom40 protein import channel binds to non-native proteins and prevents their aggregation; Esaki M et al.; Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel . Here we report the role of Tom40 in protein translocation across the membrane . The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40 . Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate . A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria . These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation.

Mol Biol Cell, 2004 Feb, 15(2), 532 - 42 Epub 2003 Oct 31.
Unique and redundant roles for HOG MAPK pathway components as revealed by whole-genome expression analysis; O'Rourke SM et al.; The Saccharomyces cerevisiae high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway is required for osmoadaptation and contains two branches that activate a mitogen-activated protein kinase (Hog1) via a mitogen-activated protein kinase kinase (Pbs2) . We have characterized the roles of common pathway components (Hog1 and Pbs2) and components in the two upstream branches (Ste11, Sho1, and Ssk1) in response to elevated osmolarity by using whole-genome expression profiling . Several new features of the HOG pathway were revealed . First, Hog1 functions during gene induction and repression, cross talk inhibition, and in governing the regulatory period . Second, the phenotypes of pbs2 and hog1 mutants are identical, indicating that the sole role of Pbs2 is to activate Hog1 . Third, the existence of genes whose induction is dependent on Hog1 and Pbs2 but not on Ste11 and Ssk1 suggests that there are additional inputs into Pbs2 under our inducing conditions . Fourth, the two upstream pathway branches are not redundant: the Sln1-Ssk1 branch has a much more prominent role than the Sho1-Ste11 branch for activation of Pbs2 by modest osmolarity . Finally, the general stress response pathway and both branches of the HOG pathway all function at high osmolarity . These studies demonstrate that cells respond to increased osmolarity by using different signal transduction machinery under different conditions.

J Biol Chem, 2004 Jan 23, 279(4), 2984 - 92 Epub 2003 Oct 31.
The role of lysine 532 in the catalytic mechanism of human topoisomerase I; Interthal H et al.; Based on co-crystal structures of human topoisomerase I with bound DNA, Lys(532) makes a minor groove contact with the strongly preferred thymidine residue at the site of covalent attachment (-1 position) . Replacement of Lys(532) with either arginine or alanine has essentially no effect on the sequence preference of the enzyme, indicating that this interaction is not required for the preference for a T at the -1 position . Although both the cleavage and religation activities of the K532R mutant enzyme are reduced, cleavage is reduced to a greater extent than religation . The reverse is true for the K532A mutant enzyme with religation so impaired that the nicked intermediate accumulates during plasmid relaxation assays . Consistent with the shift in the cleavage religation equilibrium toward cleavage for the K532A mutant enzyme, expression of the mutant enzyme in Saccharomyces cerevisiae is cytotoxic, and thus this mutant enzyme mimics the effects of the anticancer drug camptothecin . Cleavage assays with the mutant enzymes using an oligonucleotide containing a 5'-bridging phosphorothiolate indicate that Lys(532) functions as a general acid during cleavage to protonate the leaving 5'-oxygen . It is possible that the contact with the -1 base is important during catalysis to provide positional rigidity to the active site . The corresponding residues in the vaccinia virus topoisomerase and the tyrosine recombinases may have similar critical roles in catalysis.

J Biol Chem, 2004 Jan 16, 279(3), 1907 - 15 Epub 2003 Oct 31.
The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding; Johansson E et al.; We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase delta (Pol delta) . Interactions with POL31, the second subunit of Pol delta, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region . Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase alpha, POL1, requires the carboxyl-proximal region of Pol32 . The amino-terminal region of Pol32 is essential for damage-induced mutagenesis . However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage . In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol delta interactions . However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol delta complex . Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits . Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol delta localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.

J Biol Chem, 2004 Jan 30, 279(5), 3525 - 34 Epub 2003 Oct 30.
Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum; Kikkert M et al.; The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated . E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded . The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER . In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1 . We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain . In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation . Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols . Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.

J Biol Chem, 2003 Dec 26, 278(52), 51989 - 92 Epub 2003 Oct 30.
Deubiquitination, a new player in Golgi to endoplasmic reticulum retrograde transport; Cohen M et al.; Modification by ubiquitin plays a major role in a broad array of cellular functions . Although reversal of this process, deubiquitination, likely represents an important regulatory step contributing to cellular homeostasis, functions of deubiquitination enzymes still remain poorly characterized . We have previously shown that the ubiquitin protease Ubp3p requires a co-factor, Bre5p, to specifically deubiquitinate the coat protein complex II (COPII) subunit Sec23p, which is involved in anterograde transport between endoplasmic reticulum and Golgi compartiments . In the present report, we show that disruption of BRE5 gene also led to a defect in the retrograde transport from the Golgi to the endoplasmic reticulum . Further analysis indicate that the COPI subunit beta'-COP represents another substrate of the Ubp3p.Bre5p complex . All together, our results indicate that the Ubp3p.Bre5p deubiquitination complex co-regulates anterograde and retrograde transports between endoplasmic reticulum and Golgi compartments.

J Biol Chem, 2004 Jan 23, 279(4), 2507 - 12 Epub 2003 Oct 30.
Protein export across the inner membrane of mitochondria: the nature of translocated domains determines the dependence on the Oxa1 translocase; Herrmann JM et al.; The biogenesis of mitochondria requires the insertion of both nuclear and mitochondrially encoded proteins into the inner membrane . The inner membrane protein Oxa1 plays an important role in this process . Translocation of the terminal intermembrane space domains of subunit 2 of the cytochrome oxidase complex, Cox2, strictly depends on Oxa1 . In contrast, other Oxa1 substrates can be inserted independently of Oxa1 function, although at reduced efficiency . A Saccharomyces cerevisiae mutant containing a large deletion in its mitochondrial genome allowed us to analyze the insertion process of a fusion protein of cytochrome b and Cox2 . In this mutant, the N-terminal domain of Cox2 is synthesized as a hairpin loop that is flanked by hydrophobic transmembrane segments on both sides . Both genetic and biochemical evidences indicate that translocation of this region across the inner membrane still requires Oxa1 function . Thus, the position of intermembrane space domains within protein sequences does not appear to determine their dependence on the Oxa1 translocase . Our observations rather suggest that the dependence on Oxa1 correlates with the net charge of the domain that has to be translocated across the lipid bilayer.

EMBO J, 2003 Nov 3, 22(21), 5951 - 61
Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p; Perez-Martinez X et al.; The post-transcriptional role of Mss51p in mitochondrial gene expression is of great interest since MSS51 mutations suppress the respiratory defect caused by shy1 mutations . SHY1 is a Saccharomyces cerevisiae homolog of human SURF1, which when mutated causes a cytochrome oxidase assembly defect . We found that MSS51 is required for expression of the mitochondrial reporter gene ARG8(m) when it is inserted at the COX1 locus, but not when it is at COX2 or COX3 . Unlike the COX1 mRNA-specific translational activator PET309, MSS51 has at least two targets in COX1 mRNA . MSS51 acts in the untranslated regions of the COX1 mRNA, since it was required to synthesize Arg8p when ARG8(m) completely replaced the COX1 codons . MSS51 also acts on a target specified by the COX1 coding region, since it was required to translate either COX1 or COX1:: ARG8(m) coding sequences from an ectopic COX2 locus . Mss51p was found to interact physically with newly synthesized Cox1p, suggesting that it could coordinate Cox1p synthesis with insertion into the inner membrane or cytochrome oxidase assembly.

Arch Biochem Biophys, 2003 Nov 15, 419(2), 222 - 33
Multiple cellular consequences of isocitrate dehydrogenase isozyme dysfunction; McCammon MT et al.; To probe the functions of multiple forms of isocitrate dehydrogenase in Saccharomyces cerevisiae, mutants lacking three of the isozymes were constructed and analyzed . Results show that, while the mitochondrial NAD+-dependent enzyme, IDH (composed of Idh1p and Idh2p subunits) is not the major contributor to total isocitrate dehydrogenase activity under any growth condition, loss of IDH produces the most dramatic growth phenotypes . These include reduced growth in the absence of glutamate, as well as an increase in expression of Idp2p (the cytosolic NADP+-dependent enzyme) under some growth conditions . In this study, we have focused on another phenotype associated with loss of IDH, an elevated frequency of petite mutations indicating loss of functional mtDNA . Using mutant forms of IDH with altered active site residues, a correlation was observed between the high frequency of petite mutations and the loss of catalytic activity . Loss of Idp1p (the mitochondrial NADP+-dependent enzyme) and Idp2p contributes to the loss of functional mtDNA, but only in an IDH dysfunctional background . Surprisingly, overexpression of Idp1p, but not of Idp2p, was found to result in an elevated petite frequency independent of the functional state of IDH . This is the first phenotype associated with altered Idp1p . Finally, throughout this study we examined effects of loss of mitochondrial citrate synthase (Cit1p) on isocitrate dehydrogenase mutants, since defects in the CIT1 gene were previously shown to enhance growth of IDH dysfunctional strains on nonfermentable carbon sources . Loss of Cit1p was found to suppress the petite phenotype of strains lacking IDH, suggesting that these phenotypes may be linked.

Curr Biol, 2003 Oct 28, 13(21), R843 - 5
Aging: the sins of the parents; Haber JE; Yeast cells have an asymmetric, stem-cell-like division . As the mother cell ages it becomes 100 times more genetically unstable, but it is only the daughter cells that exhibit loss of heterozygosity; the latter effect is not connected to SIR2-dependent aging, but seems to be accompanied by a loss of the DNA damage checkpoint.

Mol Cell Biol, 2003 Nov, 23(22), 8323 - 33
The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo; Mason PB et al.; The FACT complex facilitates transcription on chromatin templates in vitro, and it has been functionally linked to nucleosomes and putative RNA polymerase II (Pol II) elongation factors . In Saccharomyces cerevisiae cells, FACT specifically associates with active Pol II genes in a TFIIH-dependent manner and travels across the gene with elongating Pol II . Conditional inactivation of the FACT subunit Spt16 results in increased Pol II density, transcription, and TATA-binding protein (TBP) occupancy in the 3' portion of certain coding regions, indicating that FACT suppresses inappropriate initiation from cryptic promoters within coding regions . Conversely, loss of Spt16 activity reduces the association of TBP, TFIIB, and Pol II with normal promoters . Thus, FACT is required for wild-type cells to restrict initiation to normal promoters, thereby ensuring that only appropriate mRNAs are synthesized . We suggest that FACT contributes to the fidelity of Pol II transcription by linking the processes of initiation and elongation.

Mol Cell Biol, 2003 Nov, 23(22), 8202 - 15
The Ku heterodimer performs separable activities at double-strand breaks and chromosome termini; Bertuch AA et al.; The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks . The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends . Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection . Detailed characterization of these mutations, referred to as yku80(tel) alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation . While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini . Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations . Based on homology modeling with the human Ku structure, five of the yku80(tel) alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.

Mol Cell Biol, 2003 Nov, 23(22), 8124 - 36
Biochemical characterization of the Ran-RanBP1-RanGAP system: are RanBP proteins and the acidic tail of RanGAP required for the Ran-RanGAP GTPase reaction?
Seewald MJ, Kraemer A, Farkasovsky M, Korner C, Wittinghofer A, Vetter IR.
RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran . Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions . We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product P(i) . It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 x 10(8) M(-1) s(-1)) . Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction . Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis . We propose that the acidic tail of RanGAP is required for a process during mitosis.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13767 - 72 Epub 2003 Oct 29.
Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality; Woo MH et al.; Eukaryotic DNA topoisomerase I (Top1) is a monomeric protein clamp that functions in DNA replication, transcription, and recombination . Opposable "lip" domains form a salt bridge to complete Top1 protein clamping of duplex DNA . Changes in DNA topology are catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723 and a single DNA strand . Substantial protein domain movements are required for DNA binding, whereas the tight packing of DNA within the covalent Top1-DNA complex necessitates some DNA distortion to allow rotation . To investigate the effects of Top1-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and Ser-534, to crosslink protein loops more proximal to the active-site tyrosine than the protein loops held by the Lys-369-Glu-497 salt bridge . In reducing environments, Top1-Clamp was catalytically active . However, contrary to crosslinking the salt-bridge loops {Carey, J . F., Schultz, S . J., Sission, L., Fazzio, T . G . & Champoux, J . J . (2003) Proc . Natl . Acad . Sci . USA 100, 5640-5645}, crosslinking the active-site proximal loops inhibited DNA rotation . Apparently, subtle alterations in Top1 clamp flexibility impact enzyme catalysis in vitro . Yet, the catalytically active Top1-Clamp was cytotoxic, even in the reducing environment of yeast cells . Remarkably, a shift in redox potential in glr1Delta cells converted the catalytically inactive Top1Y723F mutant clamp into a cellular toxin, which failed to induce an S-phase terminal phenotype . This cytotoxic mechanism is distinct from that of camptothecin chemotherapeutics, which stabilize covalent Top1-DNA complexes, and it suggests that the development of novel therapeutics that promote Top1-clamp closure is possible.

Org Biomol Chem, 2003 Oct 7, 1(19), 3362 - 76
Chemistry and biology of khafrefungin . Large-scale synthesis, design, and structure-activity relationship of khafrefungin, an antifungal agent; Nakamura M et al.; Large-scale synthesis, design, and structure-activity relationships of khafrefungin are reported . Khafrefungin is an antifungal agent that inhibits inositol phosphorylceramide (IPC) synthase, an enzyme involved in fungal sphingolipid biosynthesis . Unlike other inhibitors that inhibit the corresponding enzyme in fungi and mammals to the same extent, khafrefungin does not impair sphingolipid synthesis in mammals . We have developed an efficient method for large-scale synthesis of khafrefungin, and various khafrefungin derivatives were synthesized based on this method . While most of the khafrefungin derivatives lost antifungal activity, a lactone-type derivative had almost the same activity as khafrefungin . We also designed and synthesized derivatives which contain a five- or six-membered ring at the central part of the structure based on NOE experiments of khafrefungin . A macrocyclic khafrefungin derivative was also synthesized, but the antifungal activity was lost . These results suggest that the structure of khafrefungin might be strictly recognized in fungi.

J Biol Chem, 2004 Jan 23, 279(4), 2470 - 9 Epub 2003 Oct 28.
Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated gain-of-function mechanism but not immunosuppression; Baumgrass R et al.; Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling . We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18 . {Methylsarcosine(3)}CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm . {Dimethylaminoethylthiosarcosine(3)}CsA ({Dat-Sar(3)}CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the {Dat-Sar(3)}CsA-cyclophilin 18 binary complex . Despite their different inhibitory properties, both CsA and {Dat-Sar(3)}CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression . Furthermore, to demonstrate that {Dat-Sar(3)}CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted . {Dat-Sar(3)}CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.

J Biol Chem, 2004 Jan 9, 279(2), 1546 - 52 Epub 2003 Oct 28.
Crystal structure of the homolog of the oncoprotein gankyrin, an interactor of Rb and CDK4/6; Padmanabhan B et al.; The oncoprotein gankyrin plays a central role in tumorigenesis and cell proliferation . Gankyrin interacts with the retinoblastoma tumor suppressor (Rb) and cyclin-dependent kinase 4/6 (CDK4/6), increases phosphorylation at specific residues of Rb by CDK4/6 in vivo, and promotes tumorigenesis . The phosphorylation of Rb by CDK4/6 leads to the deregulation of the cell cycle during G1/S transition . Although how phosphorylation occurs on Rb has been studied extensively, the mechanism of site-specific phosphorylation of Rb remains unclear due to a lack of information on the structural arrangement of Rb and CDK4/6 . Here, we have determined and refined to 2.3-A resolution the crystal structure of a gankyrin homolog, the non-ATPase subunit 6 (Nas6p) of the proteasome from yeast . The crystal structure reveals that Nas6p contains seven ankyrin repeats . The number of the repeats is different from that predicted from the primary structure . Nas6p also possesses an unusual curved structure with two acidic regions at the N- and C-terminal regions separated by one basic region, suggesting that it has at least two functional surfaces . The tertiary structure of Nas6p, together with the previous biochemical studies, indicates that the CDK4/6 and Rb binding surfaces of gankyrin are located at the N- and C-terminal regions, respectively, and face the same side of gankyrin . These observations suggest that gankyrin brings Rb and CDK4/6 together through gankyrin-Rb and gankyrin-CDK4/6 interactions and determines the relative positioning of the substrate (Rb) and the enzyme (CDK4/6) . Our findings provide mechanistic insight into site-specific phosphorylation of Rb caused by CDK4/6.

J Biol Chem, 2004 Jan 9, 279(2), 1383 - 91 Epub 2003 Oct 28.
Identification of a human cytoplasmic poly(A) nuclease complex stimulated by poly(A)-binding protein; Uchida N et al.; The poly(A) tail shortening in mRNA, called deadenylation, is the first rate-limiting step in eukaryotic mRNA turnover, and the polyadenylate-binding protein (PABP) appears to be involved in the regulation of this step . However, the precise role of PABP remains largely unknown in higher eukaryotes . Here we identified and characterized a human PABP-dependent poly(A) nuclease (hPAN) complex consisting of catalytic hPan2 and regulatory hPan3 subunits . hPan2 has intrinsically a 3' to 5' exoribonuclease activity and requires Mg2+ for the enzyme activity . On the other hand, hPan3 interacts with PABP to simulate hPan2 nuclease activity . Interestingly, the hPAN nuclease complex has a higher substrate specificity to poly(A) RNA upon its association with PABP . Consistent with the roles of hPan2 and hPan3 in mRNA decay, the two subunits exhibit cytoplasmic co-localization . Thus, the human PAN complex is a poly(A)-specific exoribonuclease that is stimulated by PABP in the cytoplasm.

Brief Bioinform, 2003 Sep, 4(3), 228 - 35
Inferring gene networks from time series microarray data using dynamic Bayesian networks; Kim SY et al.; Dynamic Bayesian networks (DBNs) are considered as a promising model for inferring gene networks from time series microarray data . DBNs have overtaken Bayesian networks (BNs) as DBNs can construct cyclic regulations using time delay information . In this paper, a general framework for DBN modelling is outlined . Both discrete and continuous DBN models are constructed systematically and criteria for learning network structures are introduced from a Bayesian statistical viewpoint . This paper reviews the applications of DBNs over the past years . Real data applications for Saccharomyces cerevisiae time series gene expression data are also shown.

Plant Cell Physiol, 2003 Oct, 44(10), 982 - 9
Arabidopsis NDK1 is a component of ROS signaling by interacting with three catalases; Fukamatsu Y et al.; Plants sense various environmental stimuli and have specific signaling pathways to respond to these cues . We focused on light responsive components and found that NDKs were phosphorylated specifically after red light irradiation in Pisum sativum {Tanaka et al . (1998) J . Photochem . Photobiol . B 45: 113} and after blue light irradiation in Neurospora crassa {Oda and Hasunuma (1997) Mol . Gen . Genet . 256: 593, Ogura et al . (2001) J . Biol . Chem . 276: 21228} . We performed yeast two-hybrid screening using AtNDK1, the counterpart of NDK-P1 (Pisum sativum NDK1) in Arabidopsis, as bait, and isolated catalase3 (AtCat3) . Interactions between AtNDK1-AtCAT1 and AtNDK1-AtCAT2 were also detected with the two-hybrid system . Non-denaturing two-dimensional gel electrophoresis of crude extracts from plants revealed that catalase and NDK activities co-migrated in the same area of the gel . Transgenic plants expressing AtNDK1 under control of the CaMV 35S promoter exhibited tolerance to paraquat and high ability to eliminate exogenous H2O2 . These results indicate that AtNDK1 has a role in ROS response.

J Biol Chem, 2004 Jan 16, 279(3), 1729 - 38 Epub 2003 Oct 27.
Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome; Guterman A et al.; Substrates destined for degradation by the 26 S proteasome are labeled with polyubiquitin chains . These chains can be dismantled by deubiquitinating enzymes (DUBs) . A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome . We measured deubiquitination by both isolated lid and base-core particle subcomplexes, suggesting that at least two different DUBs are intrinsic components of 26 S proteasome holoenzymes . In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the lid and base subcomplexes, respectively . To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null . Interestingly, in both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles . In accord, the double mutant is synthetically lethal . In contrast to WT proteasomes, proteasomes lacking the lid subcomplex or those purified from the rpn11 mutant are less sensitive to metal chelators, supporting the prediction that Rpn11 may be a metalloprotein . Treatment of proteasomes with ubiquitin-aldehyde or with cysteine modifiers also inhibited deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag . Degradation is unique to 26 S proteasome holoenzymes; we could not detect degradation of a ubiquitinated protein by "lidless" proteasomes, although they were competent for deubiquitination . The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation.

J Cell Biol, 2003 Oct 27, 163(2), 351 - 62
The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains; Folsch H et al.; Most epithelial cells contain two AP-1 clathrin adaptor complexes . AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes . AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface . Both AP-1 complexes are heterotetramers and differ only in their 50-kD mu1A or mu1B subunits . Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region . Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport . By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B-containing membranes that were both distinct from AP-1A-positive TGN elements and more closely apposed to transferrin receptor-positive recycling endosomes . Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.

J Cell Biol, 2003 Oct 27, 163(2), 237 - 43
Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome; Bilodeau PS et al.; Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs) . Two yeast endosomal protein complexes have been proposed as Ub-sorting "receptors," the Vps27-Hse1 complex and the ESCRT-I complex . We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23 . Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo . Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus . Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen . However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected . In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation . These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.

Neuroscience, 2003, 121(4), 899 - 906
Identification of human SEP1 as a glial cell line-derived neurotrophic factor-inducible protein and its expression in the nervous system; Shimoyama Y et al.; Glial cell line-derived neurotrophic factor (GDNF) signals through multisubunit receptor complex consisting of RET tyrosine kinase and a glycosylphosphatidylinositol-anchored coreceptor called GDNF family receptor alpha1 (GFRalpha1) . In the current study, we cloned a human SEP1 gene as a GDNF-inducible gene using human neuroblastoma cells that express RET and GFRalpha1 . The induction of the SEP1 gene showed two peaks at 0.5-2 h and 24-48 h after GDNF stimulation by Northern blotting and quantitative real-time reverse transcriptase polymerase chain reaction . The late induction was also confirmed at protein levels by Western blotting with anti-SEP1 antibody . Immunostaining revealed that the expression of the SEP1 protein was detected in cell body, elongated neurites and growth cone-like structure of neuroblastoma cells treated with GDNF . In addition, we found a high level of SEP1 expression in neurons of the dorsal root and superior cervical ganglia and motor neurons of the spinal cord of mice in which RET is also expressed . SEP1 was co-immunoprecipitated with alpha- and beta-tubulins from the lysate of mouse brain . These results thus suggested that SEP1 is a GDNF-inducible and microtubule-associated protein that may play a role in the nervous system.

Mol Cell, 2003 Oct, 12(4), 983 - 90
Targeting activity is required for SWI/SNF function in vivo and is accomplished through two partially redundant activator-interaction domains; Prochasson P et al.; The SWI/SNF complex is required for the expression of many yeast genes . Previous studies have implicated DNA binding transcription activators in targeting SWI/SNF to UASs and promoters . To determine how activators interact with the complex and to examine the importance of these interactions, relative to other potential targeting mechanisms, for SWI/SNF function, we sought to identify and mutate the activator-interaction domains in the complex . Here we show that the N-terminal domain of Snf5 and the second quarter of Swi1 are sites of activation domain contact . Deletion of both of these domains left the SWI/SNF complex intact but impaired its ability to bind activation domains . Importantly, while deletion of either domain alone had minor phenotypic effect, deletion of both resulted in strong SWI/SNF related phenotypes . Thus, two distinct activator-interaction domains play overlapping roles in the targeting activity of SWI/SNF, which is essential for its function in vivo.

Exp Appl Acarol, 2003, 29(1-2), 89 - 107
Trophic determinants of hypopus induction in the stored-product mite Lepidoglyphus destructor (Acari: Astigmata); Corente Ch et al.; A unique (synapomorphic) characteristic of astigmatic mites is the heteromorphic deuteronymph also called hypopus . It is a non-feeding and facultative instar between protonymph and tritonymph . The hypopus is adapted for dispersal and sometimes also for dormancy, as in Lepidoglyphus destructor . The experiments reveal a correlation between the composition of the foodstuff, the duration of development of homomorphic instars, the mortality of protonymphs and the production of hypopodes . As food quality decreases, development lasts longer, mortality increases and hypopodes are produced in greater numbers . Disadvantageous trophic conditions of varied chemical nature favour the induction of hypopodes . The experimental data show that hypopus incidences (as percentage individuals of a population) depend on the relative proportions of constituents of an ingested foodstuff . What matters is the ratio between nourishing foodstuff components and those that are of little or no nutritional value . When a certain ratio does not meet a presumed metabolically required level of nutrients a nutritional deficiency results and hypopus induction is triggered, provided that adequate genetic propensities for hypopus production are present (L . destructor is highly polymorphic for hypopus production) . Specific key substances are apparently not involved, and composite properties of a foodstuff are crucial for hypopus induction . Decrease of food quality (not poor food per se) during the hypopus-inducible period (late larval to early protonymphal phase) promotes hypopus induction . The interpretation matches the ecological scene . When trophic deterioration of a patch habitat sets in, often as a result of overcrowding, conditions will eventually become untenable . As a response to incurring nutritional deficiencies the mites will induce hypopodes, which provide for escape from or survival at the decaying habitat patch . Experiments support the threshold model of quantitative genetics for hypopus expression as previously inferred from other experiments with L . destructor.

Invest Ophthalmol Vis Sci, 2003 Nov, 44(11), 4715 - 23
Pinin/DRS/memA interacts with SRp75, SRm300 and SRrp130 in corneal epithelial cells; Zimowska G et al.; PURPOSE: Pinin (Pnn/DRS/memA) is a cell-adhesion-related and nuclear protein that has been identified as central in the establishment and maintenance of corneal epithelial cell-cell adhesion . To begin the elucidation of the role of Pnn within the nucleus of corneal epithelial cells, this study was undertaken to identify the proteins that bind to Pnn . METHODS: Yeast two-hybrid analyses were performed . A human cDNA library in the pGAD-10 vector and C-terminal region of human Pnn (465-717) in a pAS2-1 vector were cotransformed into the PJ69-4A yeast strain, containing the lacZ, HIS3, and ADE2 reporter genes . To dissect domains of Pnn responsible for mediating the interaction with the identified proteins, PNN fragments were ligated with the DNA-binding domain of the pAS2-1 vector . Human corneal epithelial cells (HCE-T, RCB1384) and HEK-293 cells were cotransfected with mammalian expression vectors containing Pnn with identified interacting partners and subsequently immunostained and immunoblotted to determine expressed and endogenous proteins . RESULTS: Pnn colocalized and copurified with serine-arginine (SR) proteins . Three SR-rich proteins were identified that interact with the C-terminus of Pnn: SRp75 and SRm300, known components of spliceosome machinery, and a novel 130-kDa nuclear protein, SRrp130 . All of these proteins colocalized and coimmunoprecipitated with one another and exhibited speckled nuclear distribution that aligned with components of the pre-mRNA splicing machinery . The cDNA for SRrp130 encoded a protein of 805 amino acid residues and contained multiple arginine-serine (RS) repeats but had no RNA recognition motif . Analysis of the Pnn motifs using two-hybrid system assays demonstrated that the polyserine/RS motif within Pnn plays a central but not exclusive role in mediating molecular interactions with identified SR-rich proteins . CONCLUSIONS: The results suggest that Pnn and SR-rich proteins may be part of a multiprotein complex within the nucleus and may be involved in pre-mRNA processing.

Lipids, 2003 Aug, 38(8), 881 - 4
Lipid synthesis and acyl-CoA synthetase in developing rice seeds; Ichihara K et al.; Developing rice seeds rapidly accumulated storage lipids between 5 and 12 d after flowering . The contents of palmitic, oleic, and linoleic acids increased throughout seed development, while the alpha-linolenic acid content remained low . The activity of acyl-CoA synthetase varied coincidentally during the period of lipid accumulation, and rice seeds had a sufficient capacity to supply acyl-CoA substrates for TAG synthesis . Acyl-CoA synthetase showed a broad specificity for native FA of rice seeds except for stearic acid, and pi electrons of a delta9-delta11 double bond in the C16-C18 acyl chains were required for its maximal activity.

Science, 2003 Oct 24, 302(5645), 646 - 50
Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase; Rak A et al.; Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells whose function is governed by the guanosine diphosphate (GDP) dissociation inhibitor (RabGDI) . Using a combination of chemical synthesis and protein engineering, we generated and crystallized the monoprenylated Ypt1:RabGDI complex . The structure of the complex was solved to 1.5 angstrom resolution and provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins . Isoprenoid binding requires a conformational change that opens a cavity in the hydrophobic core of its domain II . Analysis of the structure provides a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.

J Cell Sci, 2003 Nov 15, 116(Pt 22), 4483 - 91
Nongenic transcription, gene regulation and action at a distance; Cook PR; In eukaryotes, motifs such as silencers, enhancers and locus control regions act over thousands of base pairs to regulate adjacent genes; insulators limit such effects, and barriers confine repressive heterochromatin to particular chromosomal segments . Recent results show that many of these motifs are nongenic transcription units, and two of them directly contact their targets lying further down the chromosome to loop the intervening DNA: the barriers (scs and scs') flanking the 87A7 heat-shock locus in the fly contact each other, and a locus control region touches the beta-globin gene in the mouse . I hypothesize that the act of transcription underlies the function of these regulators; active polymerizing complexes tend to cluster into 'factories' and this facilitates molecular contact between the transcribed regulator and its distant (and transcribed) target.

Nucleic Acids Res, 2003 Nov 1, 31(21), 6334 - 43
Human EFO1p exhibits acetyltransferase activity and is a unique combination of linker histone and Ctf7p/Eco1p chromatid cohesion establishment domains; Bellows AM et al.; Proper segregation of chromosomes during mitosis requires that the products of chromosome replication are paired together-termed sister chromatid cohesion . In budding yeast, Ctf7p/Eco1p is an essential protein that establishes cohesion between sister chromatids during S phase . In fission yeast, Eso1p also functions in cohesion establishment, but is comprised of a Ctf7p/Eco1p domain fused to a Rad30p domain (a DNA polymerase) both of which are independently expressed in budding yeast . In this report, we identify and characterize the first candidate human ortholog of Ctf7p/Eco1p, which we term hEFO1p (human Establishment Factor Ortholog) . As in fission yeast Eso1p, the hEFO1p open reading frame extends well upstream of the C-terminal Ctf7p/Eco1p domain . However, this N-terminal extension in hEFO1p is unlike Rad30p, but instead exhibits significant homology to linker histone proteins . Thus, hEFO1p is a unique fusion of linker histone and cohesion establishment domains . hEFO1p is widely expressed among the tissues tested . Consistent with a role in chromosome segregation, hEFO1p localizes exclusively to the nucleus when expressed in HeLa tissue culture cells . Moreover, biochemical analyses reveal that hEFO1p exhibits acetyltransferase activity . These findings document the first characterization of a novel human acetyltransferase, hEFO1p, that is comprised of both linker histone and Ctf7p/Eco1p domains.

Plant Physiol, 2003 Nov, 133(3), 1220 - 8 Epub 2003 Oct 23.
Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis; Liu LH et al.; Urea is the major nitrogen (N) form supplied as fertilizer in agricultural plant production and also an important N metabolite in plants . Because urea transport in plants is not well understood, the aim of the present study was to isolate urea transporter genes from the model plant Arabidopsis . Using heterologous complementation of a urea uptake-defective yeast (Saccharomyces cerevisiae) mutant allowed to isolate AtTIP1;1, AtTIP1;2, AtTIP2;1, and AtTIP4;1 from a cDNA library of Arabidopsis . These cDNAs encode channel-like tonoplast intrinsic proteins (TIPs) that belong to the superfamily of major intrinsic proteins (or aquaporins) . All four genes conferred growth of a urea uptake-defective yeast mutant on 2 mm urea in a phloretin-sensitive and pH-independent manner . Uptake studies using 14C-labeled urea into AtTIP2;1-expressing Xenopus laevis oocytes demonstrated that AtTIP2;1 facilitated urea transport also in a pH-independent manner and with linear concentration dependency . Expression studies showed that AtTIP1;2, AtTIP2;1, and AtTIP4;1 genes were up-regulated during early germination and under N deficiency in roots but constitutively expressed in shoots . Subcellular localization of green fluorescent protein-fused AtTIPs indicated that AtTIP1;2, AtTIP2;1, and AtTIP4;1 were targeted mainly to the tonoplast and other endomembranes . Thus, in addition to their role as water channels, TIP transporters may play a role in equilibrating urea concentrations between different cellular compartments.

Plant Physiol, 2003 Nov, 133(3), 1198 - 208 Epub 2003 Oct 23.
Engineering of a water-soluble plant cytochrome P450, CYP73A1, and NMR-based orientation of natural and alternate substrates in the active site; Schoch GA et al.; CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants . The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1 . Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L(-1) yeast culture . This water-soluble enzyme was suitable for 1H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site . The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and 1H-NMR analysis . It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor . Paramagnetic relaxation effects of CYP73A1-Fe(III) on the 1H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme . Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds . Position adjustments are thus likely to occur during the later steps of the catalytic cycle.

Nature, 2003 Oct 23, 425(6960), 859 - 64
Targets of the cyclin-dependent kinase Cdk1; Ubersax JA et al.; The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks) . Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates . Despite their importance in cell-cycle control, few Cdk substrates have been identified . Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts . We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner . The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events . Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.

J Biol Chem, 2004 Jan 2, 279(1), 223 - 30 Epub 2003 Oct 21.
Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools; Koc A et al.; The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU) . Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels . When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels . Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU . The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU . The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.

Genetics, 2003 Oct, 165(2), 543 - 54
The Aspergillus nidulans swoC1 mutant shows defects in growth and development; Lin X et al.; Previous work identified swoC1 as a single-gene mutant with defects in polarity establishment . In this study swoC1 was shown to have defects in endocytosis, compartmentation, nuclear distribution, and conidiation . Temperature-shift experiments showed that the swoC1 mutant establishes multiple random sites of germ tube emergence . Surprisingly, these experiments also showed that even a slight delay in polarity establishment causes defects in later vegetative growth and asexual reproduction . The swoC gene was mapped to the centromere of chromosome III and cloned by complementation of the temperature-sensitive phenotype . The predicted SwoCp is homologous to rRNA pseudouridine synthases of yeast (Cbf5p) and humans (Dkc1p) . However, neither rRNA pseudouridine synthesis nor rRNA processing appears to be affected in the swoC1 mutant . The swoC1 mutation occurs in the putative RNA-binding domain upstream of the C terminus, leaving the N-terminal TRUB catalytic domain intact . Interestingly, while deletion of the swoC gene was lethal in A . nidulans, the C terminus, including NLS, microtubule-binding, and coiled-coil domains, was dispensable for growth . SwoCp likely plays an important role in polar growth and nuclear distribution in A . nidulans, functions not yet described for its homologs.

Trends Cell Biol, 2003 Nov, 13(11), 562 - 9
Spindle asymmetry: a compass for the cell; Kusch J et al.; Spatial coordination of the cell-division axis with cellular polarity and/or with the position of neighboring cells is crucial for embryonic development, organogenesis and tissue homeostasis . In most cell types, the position of the mitotic spindle at the onset of anaphase dictates the orientation of the division axis; in unicellular organisms, it plays an important role in chromosome segregation . Cortical factors play a key role in the orientation of the spindle . Recent data from yeast reveal that the spindle does not passively react to cortical signals but actively interprets them to find its correct position . We review the data leading to a "compass model" for spindle positioning and discuss its potential generality.

Trends Cell Biol, 2003 Nov, 13(11), 547 - 50
Stu2p and XMAP215: turncoat microtubule-associated proteins?
Popov AV, Karsenti E.
The dynamics of microtubule-based (MT) cytoskeletons are controlled by a variety of accessory proteins: microtubule-associated proteins (MAPs), which usually stabilize MTs, and microtubule-destabilizers . Two related MAPs, XMAP215 and Stu2p, are known to stabilize MTs . However, recent studies report that these proteins have a MT-destabilizing function as well . Here we discuss the implications of these reports.

Genome Inform Ser Workshop Genome Inform, 2002, 13, 21 - 9
Detection of periodicity in eukaryotic genomes on the basis of power spectrum analysis; Fukushima A et al.; In the present study, we identified periodic patterns in nucleotide sequence, and characterized nucleotide sequences that confer periodicities to Arabidopsis thaliana and Drosophila melanogaster on the basis of a power spectrum method and frequency of nucleotide sequences . To assign regions that contribute to each periodicity we calculated periodic nucleotide distributions by a parameter proposed in the paper . In A . thaliana, we obtained three periodicities (248 bp-, 167 bp-, and 126 bp) in chromosome 3, three peaks (174 bp-, 88 bp-, and 59 bp-period) in chromosome 4, and four periodicities (356 bp, 174 bp, 88 bp, and 59 bp) in chromosome 5 . These are relation to ORF that consists of Gly-rich amino acid sequences including histone protein that consists of Gly-, Ser-, and Ala-rich amino acids residues . For D . melanogaster genome we found that G or C spectral curves have flat region at middle frequency range from f = 10(-4) to 10(-5) (corresponding to cyclic size 1 kb-5 kb), which may be associated with randomness of base sequence composition . This property has not been observed in Saccharomyces cerevisiae, Caenorhabditis elegans, and Homo sapiens yet.

J Biol Chem, 2004 Jan 2, 279(1), 401 - 6 Epub 2003 Oct 21.
Polycomb group suppressor of zeste 12 links heterochromatin protein 1alpha and enhancer of zeste 2; Yamamoto K et al.; Drosophila suppressor of zeste 12 (Su(z)12) is a Polycomb group (PcG) transcriptional repressor and is present in E(z)-ESC, a multiprotein complex with methylation activity specific for lysine 9 and 27 of histone H3 . Although PcG- and heterochromatin-mediated gene silencing have been considered distinct, mutant flies of Su(z)12 showed not only homeotic transformation but also position effect variegation . We now report that the mammalian SU(Z)12 directly interacts with heterochromatin protein 1alpha (HP1alpha) and PcG enhancer of zeste 2 (EZH2), the mammalian counterpart of E(z), in vitro and in vivo . Two distinct domains in SU(Z)12 are involved in these interactions, the region between the zinc finger motif and the VEFS (VRN2-EMF2-FIS2-Su(z)12) box for HP1alpha (amino acid residues 479-536) and the VEFS box for EZH2 (amino acid residues 600-639), which are not mutually exclusive . Interestingly this region of the VEFS box has been shown to be critical for the phenotype of the Su(z)12 mutant fly . In addition SU(Z)12 represses transcription activity in the presence of HP1alpha in a reporter assay . These results provide a molecular explanation for the functional link of these epigenetic silencing processes mediated by Su(z)12.

J Biol Chem, 2004 Jan 2, 279(1), 188 - 96 Epub 2003 Oct 21.
Structural control of endoplasmic reticulum-associated degradation: effect of chemical chaperones on 3-hydroxy-3-methylglutaryl-CoA reductase; Shearer AG et al.; The endoplasmic reticulum (ER) quality control pathway destroys misfolded and unassembled proteins in the ER . Most substrates of this ER-associated degradation (ERAD) pathway are constitutively targeted for destruction through recognition of poorly understood structural hallmarks of misfolding . However, the normal yeast ER membrane protein 3-hydroxy-3-methylglutaryl-CoA reductase (Hmg2p) undergoes ERAD that is physiologically regulated by sterol pathway signals . We have proposed that Hmg2p ERAD occurs by a regulated transition to an ERAD quality control substrate . Consistent with this, we had previously shown that Hmg2p is strongly stabilized by chemical chaperones such as glycerol, which stabilize misfolded proteins . To understand the features of Hmg2p that permit regulated ERAD, we have thoroughly characterized the effects of chemical chaperones on Hmg2p . These agents caused a reversible, immediate, direct change in Hmg2p degradation consistent with an effect on Hmg2p structure . We devised an in vitro limited proteolysis assay of Hmg2p in its native membranes . In vitro, chemical chaperones caused a dramatic, rapid change in Hmg2p structure to a less accessible form . As in the living cell, the in vitro action of chemical chaperones was highly specific for Hmg2p and completely reversible . To evaluate the physiological relevance of this model behavior, we used the limited proteolysis assay to examine the effects of changing in vivo degradation signals on Hmg2p structure . We found that changes similar to those observed with chemical chaperones were brought about by alteration of natural degradation signal . Thus, Hmg2p can undergo significant, reversible structural changes that are relevant to the physiological control of Hmg2p ERAD . These findings support the idea that Hmg2p regulation is brought about by regulated alteration of folding state . Considering the ubiquitous nature of quality control pathways in biology, it may be that this strategy of regulation is widespread.

J Biol Chem, 2004 Jan 23, 279(4), 2962 - 74 Epub 2003 Oct 21.
Acute regulation of Na/H exchanger NHE3 by adenosine A(1) receptors is mediated by calcineurin homologous protein; Di Sole F et al.; Adenosine is an autacoid that regulates renal Na(+) transport . Activation of adenosine A(1) receptor (A(1)R) by N(6)-cyclopentidyladenosine (CPA) inhibits the Na(+)/H(+) exchanger 3 (NHE3) via phospholipase C/Ca(2+)/protein kinase C (PKC) signaling pathway . Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A(1)R-dependent control of NHE3 activity . One binding partner of the NHE family is calcineurin homologous protein (CHP) . We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells . Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation . CPA (10(-6) M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins) . Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3(C terminus); prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3(C terminus) (amino acids 462-552 of opossum NHE3) . The yeast data were confirmed in OK cells where truncated NHE3 (NHE3(delta552)) still shows CPA-stimulated CHP interaction . Overexpression of the polypeptide from the CHP binding region (NHE3(462-552)) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3(Full-length) interaction . Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity . We conclude that CHPNHE3 interaction is regulated by A(1)R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.

J Biol Chem, 2004 Jan 9, 279(2), 848 - 58 Epub 2003 Oct 21.
Regulation of the dual specificity protein phosphatase, DsPTP1, through interactions with calmodulin; Yoo JH et al.; Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells . In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes . However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants . From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates . Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus) . Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme . At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited . Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate . Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.

Hum Mol Genet, 2003 Dec 15, 12(24), 3331 - 42 Epub 2003 Oct 21.
Reduction in frataxin causes progressive accumulation of mitochondrial damage; Karthikeyan G et al.; Frataxin protein controls iron availability in mitochondria and reduced levels lead to the human disease, Friedreich's ataxia (FRDA) . The molecular aspects of disease progression are not well understood . We developed a highly regulatable promoter system for expressing frataxin in yeast to address the consequences of chronically reduced amounts of this protein . Shutting off the promoter resulted in changes normally associated with loss of frataxin including iron accumulation within the mitochondria and the induction of mitochondrial petite mutants . While there was considerable oxidative damage to mitochondrial proteins, the petites were likely due to accumulation of mitochondrial DNA lesions and subsequent DNA loss . Chronically reduced frataxin levels resulted in similar response patterns . Furthermore, nuclear DNA damage was detected in a rad52 mutant, deficient in double-strand break repair . We conclude that reduced frataxin levels, which is more representative of the disease state, results in considerable oxidative damage in both mitochondrial and nuclear DNA.

Annu Rev Cell Dev Biol, 2003, 19, 261 - 86
The COP9 signalosome; Wei N et al.; The COP9 signalosome (CSN) is composed of eight distinct subunits and is highly homologous to the lid sub-complex of the 26S proteasome . CSN was initially defined as a repressor of photomorphogenesis in Arabidopsis, and it has now been found to participate in diverse cellular and developmental processes in various eukaryotic organisms . Recently, CSN was revealed to have a metalloprotease activity centered in the CSN5/Jab1 subunit, which removes the post-translational modification of a ubiquitin-like protein, Nedd8/Rub1, from the cullin component of SCF ubiquitin E3 ligase (i.e., de-neddylation) . In addition, CSN is associated with de-ubiquitination activity and protein kinase activities capable of phosphorylating important signaling regulators . The involvement of CSN in a number of cellular and developmental processes has been attributed to its control over ubiquitin-proteasome-mediated protein degradation.

Anal Chem, 2003 Jul 15, 75(14), 3419 - 28
An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for "bottom-up" proteomics; Brock A et al.; Here we describe a new quadrupole Fourier transform ion cyclotron resonance hybrid mass spectrometer equipped with an intermediate-pressure MALDI ion source and demonstrate its suitability for "bottom-up" proteomics . The integration of a high-speed MALDI sample stage, a quadrupole analyzer, and a FT-ICR mass spectrometer together with a novel software user interface allows this instrument to perform high-throughput proteomics experiments . A set of linearly encoded stages allows sub-second positioning of any location on a microtiter-sized target with up to 1536 samples with micrometer precision in the source focus of the ion optics . Such precise control enables internal calibration for high mass accuracy MS and MS/MS spectra using separate calibrant and analyte regions on the target plate, avoiding ion suppression effects that would result from the spiking of calibrants into the sample . An elongated open cylindrical analyzer cell with trap plates allows trapping of ions from 1000 to 5000 m/z without notable mass discrimination . The instrument is highly sensitive, detecting less than 50 amol of angiotensin II and neurotensin in a microLC MALDI MS run under standard experimental conditions . The automated tandem MS of a reversed-phase separated bovine serum albumin digest demonstrated a successful identification for 27 peptides covering 45% of the sequence . An automated tandem MS experiment of a reversed-phase separated yeast cytosolic protein digest resulted in 226 identified peptides corresponding to 111 different proteins from 799 MS/MS attempts . The benefits of accurate mass measurements for data validation for such experiments are discussed.

Przegl Lek, 2003, 60(4), 218 - 22
{Paracetamol: therapeutic action, pathogenesis and treatment of acute poisonings complicated by severe liver damage}; Kolacinski Z et al.; The biosynthesis of prostaglandins proceeds in the presence of fatty acid cycloxygenases (COX-1, COX-2) . COX-1 is responsible for the synthesis of prostaglandins indispensable for normal homeostasis, while COX-2 regulates local expression of pro-inflammatory prostaglandins . Paracetamol is a selective inhibitor of COX-2 thus having an analgesic and antipyretic potential . The drug is metabolised primarily in the liver . About 5% of the dose transforms into N-acetylo-p-benzoquinoneimine (NAPQI), a highly active compound . Ingestion of a single paracetamol dose higher than 8 g leads to a depletion of hepatic glutathione reserves and a loss of the detoxifying property of the liver . As a result, hepatic necrosis develops . The specific antidote is N-acetylcysteine (NAC) . If applied within 10-15 h since the poisoning it enables complete survival . The efficacy of specific treatment decreases after 24 h but blood paracetamol is an indication for NAC therapy . The surviving patients with advanced paracetamol poisoning require long-lasting conservative treatment with ornithine and phospholipids as well as a light diet.

Nat Genet, 2003 Nov, 35(3), 277 - 86 Epub 2003 Oct 19.
DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray; Ooi SL et al.; We describe a new synthetic lethality analysis by microarray (SLAM) technique that uses approximately 4,600 Saccharomyces cerevisiae haploid deletion mutants with molecular 'bar codes' (TAGs) . We used SGS1 and SRS2, two 3'-->5' DNA helicase genes, as 'queries' to identify their redundant and unique biological functions . We introduced these 'query mutations' into a haploid deletion pool by integrative transformation to disrupt the query gene in every cell, generating a double mutant pool . Optimization of integrative transformation efficiency was essential to the success of SLAM . Synthetic interactions defined a DNA helicase genetic network and predicted a role for SRS2 in processing damaged replication forks but, unlike SGS1, not in rDNA replication, DNA topology or lagging strand synthesis . SGS1 and SRS2 have synthetic defects with MRC1 but not RAD9, suggesting that SGS1 and SRS2 function in a parallel pathway with MRC1 to transduce the DNA replication stress signal to the general DNA damage checkpoint pathway . Both helicase genes have rad51-reversible synthetic defects with 5'-->3' DNA helicase RRM3, suggesting that RRM3 helps prevent formation of toxic recombination intermediates . SLAM detects synthetic lethality efficiently and ranks candidate genetic interactions, making it an especially useful method.

Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12579 - 83 Epub 2003 Oct 17.
Predicting protein functions from redundancies in large-scale protein interaction networks; Samanta MP et al.; Interpreting data from large-scale protein interaction experiments has been a challenging task because of the widespread presence of random false positives . Here, we present a network-based statistical algorithm that overcomes this difficulty and allows us to derive functions of unannotated proteins from large-scale interaction data . Our algorithm uses the insight that if two proteins share significantly larger number of common interaction partners than random, they have close functional associations . Analysis of publicly available data from Saccharomyces cerevisiae reveals >2,800 reliable functional associations, 29% of which involve at least one unannotated protein . By further analyzing these associations, we derive tentative functions for 81 unannotated proteins with high certainty . Our method is not overly sensitive to the false positives present in the data . Even after adding 50% randomly generated interactions to the measured data set, we are able to recover almost all (approximately 89%) of the original associations.

Mol Biol Cell, 2003 Dec, 14(12), 5060 - 8 Epub 2003 Oct 17.
Rap1 affects the length and heterogeneity of human telomeres; Li B et al.; Telomere length is controlled in part by cis-acting negative regulators that limit telomere extension by telomerase . In budding yeast, the major telomere length regulator scRap1 binds to telomeric DNA and acts to inhibit telomere elongation in cis . Because the human Rap1 ortholog hRap1 does not bind to telomeric DNA directly but is recruited to telomeres by TRF2, we examined its role in telomere length control . The data are consistent with hRap1 being a negative regulator of telomere length, indicating functional conservation . Deletion mapping confirmed that hRap1 is tethered to telomeres through interaction of its C terminus with TRF2 . The telomere length phenotypes of hRap1 deletion mutants implicated both the BRCT and Myb domain as protein interaction domains involved in telomere length regulation . By contrast, scRap1 binds to telomeres with its Myb domains and uses its C terminus to recruit the telomere length regulators Rif1 and Rif2 . Together, our data show that although the role of Rap1 at telomeres has been largely conserved, the domains of Rap1 have undergone extensive functional changes during eukaryotic evolution . Surprisingly, hRap1 alleles lacking the BRCT domain diminished the heterogeneity of human telomeres, indicating that hRap1 also plays a role in the regulation of telomere length distribution.

Curr Genet, 2004 Jan, 44(6), 329 - 38 Epub 2003 Oct 17.
Chromosome rearrangements in isolates that escape from het-c heterokaryon incompatibility in Neurospora crassa; Xiang Q et al.; Chromosomal rearrangement is implicated in human cancers and hereditary diseases . Mechanisms generating chromosomal rearrangements may be shared by a variety of organisms . Spontaneous chromosomal rearrangements, especially large deletions, take place at high frequency in isolates that escape from heterokaryon incompatibility in Neurospora crassa . In this study, chromosomal rearrangements were detected in strains that had escaped from het-c heterokaryon incompatibility in N . crassa . A vc1 mutant carried a 20-kbp deletion covering five ORFs . A vc2 mutant carried a complex chromosome rearrangement with an 8-kbp deletion covering three ORFs, a 34-bp deletion and an 80-kbp inversion . The break-points of chromosome rearrangements in the vc1 and vc2 mutants all have direct repeats of 2 bp, similar to the break-points of some chromosome rearrangements associated with human cancer and genetic diseases . An ahc mutant carried a 31-kbp deletion covering at least 11 ORFs and a het-c deletion mutant carried a 7-kbp deletion covering two ORFs . Additional chromosomal rearrangements occurred in these two strains . These results indicate that escape from heterokaryon incompatibility can be used as a model system for chromosome rearrangement and DNA-repair studies . The impact of the chromosomal rearrangements is discussed, especially the deletion of the predicted ORFs on the phenotype of mutants.

Science, 2003 Nov 28, 302(5650), 1533 - 7 Epub 2003 Oct 16.
Bypassing a kinase activity with an ATP-competitive drug; Papa FR et al.; Unfolded proteins in the endoplasmic reticulum cause trans-autophosphorylation of the bifunctional transmembrane kinase Ire1, which induces its endoribonuclease activity . The endoribonuclease initiates nonconventional splicing of HAC1 messenger RNA to trigger the unfolded-protein response (UPR) . We explored the role of Ire1's kinase domain by sensitizing it through site-directed mutagenesis to the ATP-competitive inhibitor 1NM-PP1 . Paradoxically, rather than being inhibited by 1NM-PP1, drug-sensitized Ire1 mutants required 1NM-PP1 as a cofactor for activation . In the presence of 1NM-PP1, drug-sensitized Ire1 bypassed mutations that inactivate its kinase activity and induced a full UPR . Thus, rather than through phosphorylation per se, a conformational change in the kinase domain triggered by occupancy of the active site with a ligand leads to activation of all known downstream functions.

Science, 2003 Oct 17, 302(5644), 449 - 53
A Bayesian networks approach for predicting protein-protein interactions from genomic data; Jansen R et al.; We have developed an approach using Bayesian networks to predict protein-protein interactions genome-wide in yeast . Our method naturally weights and combines into reliable predictions genomic features only weakly associated with interaction (e.g., messenger RNAcoexpression, coessentiality, and colocalization) . In addition to de novo predictions, it can integrate often noisy, experimental interaction data sets . We observe that at given levels of sensitivity, our predictions are more accurate than the existing high-throughput experimental data sets . We validate our predictions with TAP (tandem affinity purification) tagging experiments . Our analysis, which gives a comprehensive view of yeast interactions, is available at genecensus.org/intint.

J Biol Chem, 2003 Dec 26, 278(52), 52461 - 70 Epub 2003 Oct 16.
Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution; Parthasarathy S et al.; Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate . In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution . The crystallographic asymmetric unit contains a dimeric molecule . The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage . The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate . The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations . The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant . Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit . Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.

Genes Dev, 2003 Nov 1, 17(21), 2648 - 63 Epub 2003 Oct 16.
Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8; Henry KW et al.; Gene activation and repression regulated by acetylation and deacetylation represent a paradigm for the function of histone modifications . We provide evidence that, in contrast, histone H2B monoubiquitylation and its deubiquitylation are both involved in gene activation . Substitution of the H2B ubiquitylation site at Lys 123 (K123) lowered transcription of certain genes regulated by the acetylation complex SAGA . Gene-associated H2B ubiquitylation was transient, increasing early during activation, and then decreasing coincident with significant RNA accumulation . We show that Ubp8, a component of the SAGA acetylation complex, is required for SAGA-mediated deubiquitylation of histone H2B in vitro . Loss of Ubp8 in vivo increased both gene-associated and overall cellular levels of ubiquitylated H2B . Deletion of Ubp8 lowered transcription of SAGA-regulated genes, and the severity of this defect was exacerbated by codeletion of the Gcn5 acetyltransferase within SAGA . In addition, disruption of either ubiquitylation or Ubp8-mediated deubiquitylation of H2B resulted in altered levels of gene-associated H3 Lys 4 methylation and Lys 36 methylation, which have both been linked to transcription . These results suggest that the histone H2B ubiquitylation state is dynamic during transcription, and that the sequence of histone modifications helps to control transcription.

Gene, 2003 Oct 16, 316, 1 - 21
Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development; Messenguy F et al.; In all organisms, correct development, growth and function depends on the precise and integrated control of the expression of their genes . Often, gene regulation depends upon the cooperative binding of proteins to DNA and upon protein-protein interactions . Eukaryotes have widely exploited combinatorial strategies to create gene regulatory networks . MADS box proteins constitute the perfect example of cellular coordinators . These proteins belong to a large family of transcription factors present in most eukaryotic organisms and are involved in diverse and important biological functions . MADS box proteins are combinatorial transcription factors in that they often derive their regulatory specificity from other DNA binding or accessory factors . This review is aimed at analyzing how MADS box proteins combine with a variety of cofactors to achieve functional diversity.

Insect Biochem Mol Biol, 2003 Nov, 33(11), 1085 - 97
Action pattern, specificity, lytic activities, and physiological role of five digestive beta-glucanases isolated from Periplaneta americana; Genta FA et al.; Three laminarinases (LAM, LIC 1, and LIC 2) and two cellulases (CEL 1 and CEL 2) were purified to homogeneity from Periplaneta americana midguts . These beta-glucanases are secreted by salivary glands, stabilized by calcium ions, and have pH optima around 6 . LAM (46 kDa) is active only on laminarin, native or with oxidized ends, and so it is an endo-beta-1,3-glucanase (EC 3.2.1.39) . It processively releases mainly glucose from laminarin and shows lytic activity on fungal cells . LIC 1 (25 kDa) is an endo-beta-1,3(4)-glucanase (EC 3.2.1.6.), because it cleaves internal bonds on both laminarin and lichenin . LIC 1 lyses fungal cells and apparently have high affinity for sequences of cellotetraoses linked by beta-1,3 links, releasing cellotetraose from lichenin . The reaction catalyzed by LIC 1 is not in rapid equilibrium, as suggested by activity-pH data . These data also showed that a group in LIC 1 with pK=4.9 is necessary for substrate binding . LIC 2 (23 kDa) seems to be similar to LIC 1 . The laminarinases are inactivated by carbodiimide, suggesting the presence of a carboxyl group involved in catalysis . LAM and LIC 2 are inhibited by excess laminarin as substrate . CEL 1 (72 kDa) and CEL 2 (73 kDa) quickly decrease the molecular weight of lichenin used as substrate . Therefore, they are endo-beta-1,4-glucanases (EC 3.2.1.4) . Both CEL 1 and CEL 2 are also active on crystalline cellulose . The specificities of P . americana beta-glucanases agree with the omnivorous detritus-feeding habit of this insect, as they are able to attack plant (CEL 1, CEL 2, LIC 1 and LIC 2) and fungal (LIC 1 and LAM) cell walls.

Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 13042 - 7 Epub 2003 Oct 15.
Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis; Ikonen M et al.; Insulin-like growth factor-binding protein-3 (IGFBP-3) regulates IGF bioactivity and also independently modulates cell growth and survival . By using a yeast two-hybrid screen to identify IGFBP-3-interacting proteins, we cloned humanin (HN) as an IGFBP-3-binding partner . HN is a 24-aa peptide that has been shown to specifically inhibit neuronal cell death induced by familial Alzheimer's disease mutant genes and amyloid-beta (Abeta) . The physical interaction of HN with IGFBP-3 was determined to be of high affinity and specificity and was confirmed by yeast mating, displaceable pull-down experiments with (His)-6-tagged HN, and ligand blot experiments . Co-immunoprecipitation of IGFBP-3 and HN from mouse testes confirmed the interaction in vivo . In cross-linking experiments, HN bound IGFBP-3 but did not compete with IGF-I-IGFBP-3 binding; competitive ligand dot blot experiments revealed the 18-aa heparin-binding domain of IGFBP-3 as the binding site for HN . Alanine scanning determined that F6A-HN mutant does not bind IGFBP-3 . HN but not F6A-HN inhibited IGFBP-3-induced apoptosis in human glioblastoma-A172 . In contrast, HN did not suppress IGFBP-3 response in SH-SY5Y neuroblastoma and mouse cortical primary neurons . In primary neurons, IGFBP-3 markedly potentiated HN rescue ability from Abeta1-43 toxicity . In summary, we have identified an interaction between the survival peptide HN and IGFBP-3 that is pleiotrophic in nature and is capable of both synergistic and antagonistic interaction . This interaction may prove to be important in neurological disease processes and could provide important targets for drug development.

RNA, 2003 Nov, 9(11), 1383 - 99
RNA-protein interactions promote asymmetric sorting of the ASH1 mRNA ribonucleoprotein complex; Gonsalvez GB et al.; In Saccharomyces cerevisiae, ASH1 mRNA is localized to the tip of daughter cells during anaphase of the cell cycle . ASH1 mRNA localization is dependent on four cis-acting localization elements as well as Myo4p, She2p, and She3p . Myo4p, She2p, and She3p are hypothesized to form a heterotrimeric protein complex that directly transports ASH1 mRNA to daughter cells . She2p is an RNA-binding protein that directly interacts with ASH1 cis-acting localization elements and associates with She3p . Here we report the identification of seven She2p mutants-N36S, R43A, R44A, R52A, R52K, R63A, and R63K-that result in the delocalization of ASH1 mRNA . These mutants are defective for RNA-binding activity but retain the ability to interact with She3p, indicating that a functional She2p RNA-binding domain is not a prerequisite for association with She3p . Furthermore, the nuclear/cytoplasmic distribution for the N36S and R63K She2p mutants is not altered, indicating that nuclear/cytoplasmic trafficking of She2p is independent of RNA-binding activity . Using the N36S and R63K She2p mutants, we observed that in the absence of She2p RNA-binding activity, neither Myo4p nor She3p is asymmetrically sorted to daughter cells . However, in the absence of She2p, Myo4p and She3p can be asymmetrically segregated to daughter cells by artificially tethering mRNA to She3p, implying that the transport and/or anchoring of the Myo4p/She3p complex is dependent on the presence of associated mRNA.

J Biol Chem, 2003 Dec 26, 278(52), 52873 - 80 Epub 2003 Oct 15.
Cardiolipin stabilizes respiratory chain supercomplexes; Pfeiffer K et al.; Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipin-deficient strain . Reconstitution with cardiolipin largely restored wild-type stability . The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c1 and tightly bound cardiolipin . Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted . An additional marked effect of cardiolipin concerns cytochrome c oxidase . We show that a cardiolipin-deficient strain harbored almost inactive resting cytochrome c oxidase in the membrane . Transition to the fully active pulsed state occurred on a minute time scale.

J Biochem (Tokyo), 2003 Sep, 134(3), 447 - 57
Identification and characterization of a Xenopus homolog of Dbf4, a regulatory subunit of the Cdc7 protein kinase required for the initiation of DNA replication; Furukohri A et al.; Dbf4 is a regulatory subunit for the Cdc7 protein kinase that is required for the initiation of eukaryotic DNA replication, but the precise roles of Dbf4-Cdc7 remain to be determined . Here we identified a Xenopus homolog of Dbf4 (XDbf4) and characterized XDbf4 and Xenopus Cdc7 (XCdc7) in Xenopus egg extracts . XDbf4 formed a complex with XCdc7 in egg extracts and activated XCdc7 kinase activity in vitro . In contrast with Dbf4 in yeast and mammalian cultured cells, the XDbf4 levels in egg extracts did not change during the cell cycle progression . XDbf4 was a phosphoprotein in interphase extracts, and was apparently hyperphosphorylated in cytostatic factor (CSF)-mediated, metaphase-arrested extracts and in mitotic extracts . However, the hyperphosphorylation of XDbf4 did not seem to affect the level of kinase activation, or chromatin binding of the XDbf4-XCdc7 complex . Upon release from CSF-arrest, XDbf4 was partially dephosphorylated and bound to chromatin . Interestingly, XDbf4 was loaded onto chromatin before XCdc7 during DNA replication in egg extracts . These results suggest that the function of XDbf4-XCdc7 during the early embryonic cell cycle is regulated in a manner distinct from that during the somatic cell cycle.

Curr Biol, 2003 Oct 14, 13(20), 1820 - 3
Formin leaky cap allows elongation in the presence of tight capping proteins; Zigmond SH et al.; Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring . FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro . It also binds to the filament barbed end where it appears to act as a "leaky" capper, slowing both polymerization and depolymerization by approximately 50% . We now find that FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end . We also find that FH1FH2 forms a tetramer . The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper . This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin . The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates.

Mol Cell Biol, 2003 Nov, 23(21), 7887 - 901
In vivo evidence that defects in the transcriptional elongation factors RPB2, TFIIS, and SPT5 enhance upstream poly(A) site utilization; Cui Y et al.; While a number of proteins are involved in elongation processes, the mechanism for action of most of these factors remains unclear primarily because of the lack of suitable in vivo model systems . We identified in yeast several genes that contain internal poly(A) sites whose full-length mRNA formation is reduced by mutations in RNA polymerase II subunit RPB2, elongation factor SPT5, or TFIIS . RPB2 and SPT5 defects also promoted the utilization of upstream poly(A) sites for genes that contain multiple 3' poly(A) signaling sequences, supporting a role for elongation in differential poly(A) site choice . Our data suggest that elongation defects cause increased transcriptional pausing or arrest that results in increased utilization of internal or upstream poly(A) sites . Transcriptional pausing or arrest can therefore be visualized in vivo if a gene contains internal poly(A) sites, allowing biochemical and genetic study of the elongation process.

Mol Cell Biol, 2003 Nov, 23(21), 7818 - 28
Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space; Frazier AE et al.; The mitochondrial inner membrane contains numerous multispanning integral proteins . The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane . While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane . We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins . The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate . Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes . Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane . We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.

Mol Cell Biol, 2003 Nov, 23(21), 7809 - 17
Post-TATA binding protein recruitment clearance of Gcn5-dependent histone acetylation within promoter nucleosomes; Topalidou I et al.; Transcriptional activation of eukaryotic genes often requires the function of histone acetyltransferases (HATs), which is expected to result in the hyperacetylation of histones within promoter nucleosomes . In this study we show that, in Saccharomyces cerevisiae, the steady-state levels of Gcn5-dependent histone acetylation within a number of transcriptionally active promoters are inversely related to the rate of transcription . High acetylation levels were measured only when transcription was attenuated either by TATA element mutations or in a strain carrying a temperature-sensitive protein component of RNA polymerase II . In addition, we show that in one case the low levels of histone acetylation depend on the function of the Rpd3 histone deacetylase . These results point to the existence of an unexpected interplay of two opposing histone-modifying activities which operate on promoter nucleosomes following the initiation of RNA synthesis . Such interplay could ensure rapid turnover of chromatin acetylation states in continuously reprogrammed transcriptional systems.

Mol Cell Biol, 2003 Nov, 23(21), 7611 - 27
Quantitative analysis of CBP- and P300-induced histone acetylations in vivo using native chromatin; McManus KJ et al.; In vivo, histone tails are involved in numerous interactions, including those with DNA, adjacent histones, and other, nonhistone proteins . The amino termini are also the substrates for a number of enzymes, including histone acetyltransferases (HATs), histone deacetylases, and histone methyltransferases . Traditional biochemical approaches defining the substrate specificity profiles of HATs have been performed using purified histone tails, recombinant histones, or purified mononucleosomes as substrates . It is clear that the in vivo presentation of the substrate cannot be accurately represented by using these in vitro approaches . Because of the difficulty in translating in vitro results into in vivo situations, we developed a novel single-cell HAT assay that provides quantitative measurements of endogenous HAT activity . The HAT assay is performed under in vivo conditions by using the native chromatin structure as the physiological substrate . The assay combines the spatial resolving power of laser scanning confocal microscopy with simple statistical analyses to characterize CREB binding protein (CBP)- and P300-induced changes in global histone acetylation levels at specific lysine residues . Here we show that CBP and P300 exhibit unique substrate specificity profiles, consistent with the developmental and functional differences between the two HATs.

Mol Cell Biol, 2003 Nov, 23(21), 7566 - 84
Pressure-induced differential regulation of the two tryptophan permeases Tat1 and Tat2 by ubiquitin ligase Rsp5 and its binding proteins, Bul1 and Bul2; Abe F et al.; Tryptophan uptake appears to be the Achilles' heel in yeast physiology, since under a variety of seemingly diverse toxic conditions, it becomes the limiting factor for cell growth . When growing cells of Saccharomyces cerevisiae are subjected to high hydrostatic pressure, tryptophan uptake is down-regulated, leading to cell cycle arrest in the G(1) phase . Here we present evidence that the two tryptophan permeases Tat1 and Tat2 are differentially regulated by Rsp5 ubiquitin ligase in response to high hydrostatic pressure . Analysis of high-pressure growth mutants revealed that the HPG1 gene was allelic to RSP5 . The HPG1 mutation or the bul1Delta bul2Delta double mutation caused a marked increase in the steady-state level of Tat2 but not of Tat1, although both permeases were degraded at high pressure in an Rsp5-dependent manner . There were marked differences in subcellular localization . Tat1 localized predominantly in the plasma membrane, whereas Tat2 was abundant in the internal membranes . Moreover, Tat1 was associated with lipid rafts, whereas Tat2 localized in bulk lipids . Surprisingly, Tat2 became associated with lipid rafts upon the occurrence of a ubiquitination defect . These results suggest that ubiquitination is an important determinant of the localization and regulation of these tryptophan permeases . Determination of the activation volume (DeltaV( not equal )) for Tat1- and Tat2-mediated tryptophan uptake (89.3 and 50.8 ml/mol, respectively) revealed that both permeases are highly sensitive to membrane perturbation and that Tat1 rather than Tat2 is likely to undergo a dramatic conformational change during tryptophan import . We suggest that hydrostatic pressure is a unique tool for elucidating the dynamics of integral membrane protein functions as well as for probing lipid microenvironments where they localize.

J Biol Chem, 2004 Jan 2, 279(1), 348 - 55 Epub 2003 Oct 13.
Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library; Shiraishi K et al.; Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products . Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change . To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae . We isolated 142 ts p53 mutants, including 131 unreported ts mutants . These mutants clustered in beta-strands in the DNA-binding domain, particularly in one of the two beta-sheets of the protein, and 15 residues (Thr155, Arg158, Met160, Ala161, Val172, His214, Ser215, Pro223, Thr231, Thr253, Ile254, Thr256, Ser269, Glu271, and Glu285) were ts hot spots . Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells . The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies . These results indicated that the intramolecular beta-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.

Biochem Biophys Res Commun, 2003 Oct 31, 310(4), 1175 - 80
Gcn4 negatively regulates expression of genes subjected to nitrogen catabolite repression; Sosa E et al.; It has been considered that three key elements participate in nitrogen catabolite repression (NCR) of Saccharomyces cerevisiae: the GLN3 and GAT1/NIL1-encoded transcriptional activators and their negative regulator Ure2 . The fact that expression of various NCR-sensitive genes is not derepressed in the absence of Ure2 has led to the proposition that there must exist a protein with a similar function to that of Ure2 . The results presented in this paper show that various NCR-sensitive genes are derepressed through GLN3-mediated transcriptional activation in a gcn4Delta mutant . This effect is additive to that exerted by the lack of Ure2 and to that evoked in rapamycin-treated cultures . Our results uncover the fact that NCR is not solely achieved through the action of Gln3, Gat1, and Ure2 . Since Gcn4 regulates the expression of a broad spectrum of genes, the lack of this transcriptional activator could prevent the expression of a potential Gln3 antagonist . Alternatively, Gcn4 could directly hinder Gln3 functioning.

Plant Mol Biol, 2003 Jul, 52(5), 1077 - 88
A plasma membrane zinc transporter from Medicago truncatula is up-regulated in roots by Zn fertilization, yet down-regulated by arbuscular mycorrhizal colonization; Burleigh SH et al.; Here we present a Zn transporter cDNA named MtZIP2 from the model legume Medicago truncatula . MtZIP2 encodes a putative 37 kDa protein with 8-membrane spanning domains and has moderate amino acid identity with the Arabidopsis thaliana Zn transporter AtZIP2p . MtZIP2 complemented a Zn-uptake mutant of yeast implying that the protein encoded by this gene can transport Zn across the yeast's plasma membrane . The product of a MtZIP2-GFP fusion construct introduced into onion cells by particle bombardment likewise localized to the plasma membrane . The MtZIP2 gene was expressed in roots and stems, but not in leaves of M . truncatula and, in contrast to all other plant Zn transporters characterized thus far, MtZIP2 was up-regulated in roots by Zn fertilization . Expression was highest in roots exposed to a toxic level of Zn . MtZIP2 expression was also examined in the roots of M . truncatula when colonized by the obligate plant symbiont, arbuscular mycorrhizal (AM) fungi, since AM fungi are renowned for their ability to supply plants with mineral nutrients, including Zn . Expression was down-regulated in the roots of the mycorrhizal plants and was associated with a reduced level of Zn within the host plant tissues.

Plant Mol Biol, 2003 Jul, 52(5), 1051 - 62
An antirrhinum ternary complex factor specifically interacts with C-function and SEPALLATA-like MADS-box factors; Causier B et al.; The development of floral reproductive organs requires the activity of plant MADS-box transcription factors (MBFs) belonging to the C function . The C function can only operate within a floral context, specified by MBFs belonging to the SEPALLATA class of proteins . Here we describe the specific interaction between a novel protein, MIP1, and C-function and SEPALLATA (SEP)-like MBFs . MIP1 is the first member of a new class of proteins unique to plants . None of the family members have yet been assigned a function . Motif searches reveal a leucine zipper domain within a conserved N-terminal region of MIP1 . The leucine zipper lies within a region sufficient for interaction with plant MBFs . MIP1 interacts with a domain of plant MBFs that is analogous to the domain of animal and yeast MBFs involved in ternary complex formation . The MIP1 protein is predicted to localise to the nucleus and activates yeast reporter genes in vivo . MIP1 is expressed in the fourth whorl of the flower, in an overlapping temporal and spatial expression pattern with the C-function and SEP-like genes . Taken together, this suggests that MIP1 acts as a ternary complex factor specifically with C-function and SEP-like MBFs.

Plant Mol Biol, 2003 Jul, 52(5), 967 - 80
Expression of the cation transporter McHKT1 in a halophyte; Su H et al.; From the ice plant, Mesembryanthemum crystallinum, McHKT1 was isolated encoding a protein 41-61% identical to other plant HKT1-like sequences previously described as potassium or sodium/potassium transporters . McHKT1 acts as a potassium transporter in yeast with specificity similar to that of wheat HKT1 . In Xenopus oocytes it transports cations with a specificity Rb+ > Cs+ > {K+ = Na+ = Li+} . McHKT1 is exclusively localized to the plasma membrane . The isoform isolated is most highly expressed in leaves and is present in stems, flowers and seed pods but absent from the root where, according to immunological data, a second isoform exists which does not cross-hybridize with the leaf form in RNA blots at high stringency . McHKT1 transcript amounts increase during the first 6-10 h of stress and then decline to pre-stress levels with kinetics reminiscent of the initial influx of sodium into this halophyte . Immunocytological localization showed strong signals in the leaf vasculature and surrounding mesophyll cells but low-intensity signals are also detected in other cell types . In roots, McHKT is mainly confined to endodermis and stele . Possible functions of McHKT1 in ion homeostasis in the halophytic ice plant are discussed.

Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12735 - 40 Epub 2003 Oct 13.
A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery; Hitchcock AL et al.; Ubiquitination of membrane-associated proteins can direct their proteasome-mediated degradation or activation at the endoplasmic reticulum (ER), as well as their endocytosis and intracellular sorting . However, the full spectrum of ubiquitinated membrane proteins has not been determined . Here we combined proteomic analysis with yeast genetics to identify 211 ubiquitinated membrane-associated proteins in Saccharomyces cerevisiae and map >30 precise sites of ubiquitination . Major classes of identified ubiquitinated proteins include ER-resident membrane proteins, plasma membrane-localized permeases, receptors, and enzymes, and surprisingly, components of the actin cytoskeleton . By determining the differential abundance of ubiquitinated proteins in yeast mutated for NPL4 and UBC7, which are major components of ER-associated degradation (ERAD), we furthermore were able to classify 83 of these identified ubiquitinated membrane proteins as potential endogenous substrates of the ERAD pathway . These substrates are highly enriched for proteins that localize to or transit through the ER . Interestingly, we also identified novel membrane-bound transcription factors that may be subject to ubiquitin/proteasome-mediated cleavage and activation at the ER membrane.

J Cell Biol, 2003 Oct 13, 163(1), 57 - 69
A role for Yip1p in COPII vesicle biogenesis; Heidtman M et al.; Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases . We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway . Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport . Surprisingly, inhibition is specific to the COPII-dependent budding stage . In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates . Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding . Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation . We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.

J Cell Biol, 2003 Oct 13, 163(1), 21 - 6
Cdc48p is required for the cell cycle commitment point at Start via degradation of the G1-CDK inhibitor Far1p; Fu X et al.; The budding yeast Cdc48p and its mammalian homologue p97 are involved in many important cellular activities . Because previous cdc48 mutants have exclusive G2/M arrest, Cdc48p was thought to play an essential role only during mitosis . We found that Cdc48p is required for the execution of Start (a yeast cell cycle commitment point equivalent to the restriction point in mammalian cells) in both a normal mitotic cell cycle and cell cycle reentry after mating pheromone withdrawal through degradation of the G1-cyclin-dependent kinase inhibitor Far1p . Our work is the first to uncover novel roles of Cdc48p as a critical cell cycle regulator in G1, and to shed new light on cell cycle regulation of Far1p, which is the first cyclin-dependent kinase inhibitor shown to be a substrate of an essential proteolysis event mediated by Cdc48p.

Gene, 2003 Oct 2, 315, 177 - 82
A single amino acid substitution in MSH5 results in DNA alkylation tolerance; Bawa S et al.; DNA alkylation tolerance is a major concern in cancer chemotherapy . It has been suggested that mutations in DNA mismatch repair genes may result in alkylation tolerance . This alkylation tolerant phenotype is often manifested in cells lacking an O(6)-methylguanine DNA methyltransferase (MTase) activity . However, deletion of each mismatch repair gene in the MTase mutant of a model eukaryotic yeast does not result in alkylation tolerance . We previously isolated an alkylation tolerant mutant and mapped the mutation to MSH5 . Here we present evidence that a single point mutation that results in a Y823H amino acid substitution, but not deletion, of the MSH5 gene is responsible for tolerance to killing by DNA alkylating agents . We also find that other preexisting amino acid variations may also enhance alkylation tolerance in the above mutation background . Since MSH5 encodes a protein homologous to DNA mismatch recognition proteins, mismatch repair genes are frequently mutated in cancers cells and, like mismatch repair genes, MSH5 is highly conserved from yeast to human, this observation suggests novel mechanisms of chemotherapeutic drug resistance that may occur in certain human cancer patients.

Gene, 2003 Oct 2, 315, 1 - 13
Roles for BTAF1 and Mot1p in dynamics of TATA-binding protein and regulation of RNA polymerase II transcription; Pereira LA et al.; Regulation of RNA polymerase II (pol II) transcription is a highly dynamic process requiring the coordinated interaction of an array of regulatory proteins . Central to this process is the TATA-binding protein (TBP), the key component of the multiprotein complex TFIID . Interaction of TBP with core promoters nucleates the assembly of the preinitiation complex and subsequent recruitment of pol II . Despite recent advances in our understanding of the dynamic nature of the pol II transcription apparatus, the dynamics of TBP function on pol II promoters has remained largely unexplored . Human BTAF1 (TAF(II)170/TAF-172) and its yeast ortholog, Mot1p, are evolutionarily conserved members of the SNF2-like family of ATPase proteins . Genetic identification of Mot1p as a repressor of pol II transcription was supported by findings that Mot1p and BTAF1 could dissociate TBP from TATA DNA complexes using the energy of ATP hydrolysis . Recent data have revealed new aspects of BTAF1 and Mot1p as positive regulators of TBP function in the pol II system and have described new observations relating to their molecular mechanism of action . We review these data in the context of previous findings with particular attention paid to how human BTAF1 and Mot1p may dynamically regulate TBP function on pol II promoters in cells.

Free Radic Biol Med, 2003 Oct 15, 35(8), 889 - 900
Two redox centers within Yap1 for H2O2 and thiol-reactive chemicals signaling; Azevedo D et al.; The Yap1 transcription factor regulates yeast responses to H2O2 and to several unrelated chemicals and metals . Activation by H2O2 involves Yap1 Cys303-Cys598 intra-molecular disulfide bond formation directed by the H2O2 sensor Orp1/Gpx3 . We show here that the electrophile N-ethylmaleimide activates Yap1 by covalent modification of Yap1 C-terminal Cys598, Cys620, and Cys629, in an Orp1 and Yap1-oxidation-independent way, thus establishing an alternate and distinct mode of Yap1 activation . We also show that menadione, a superoxide anion generator and a highly reactive electrophile, operates both modes of Yap1 activation . Further, the Yap1 C-terminal domain reactivity towards other electrophiles (4-hydroxynonenal, iodoacetamide) and metals (cadmium, selenium) suggests a common mechanism for sensing thiol reactive chemicals, involving thiol chemical modification . We propose that Yap1 has two distinct molecular redox centers, one triggered by ROS (hydroperoxides and the superoxide anion) and the other by chemicals with thiol reactivity (electrophiles and divalent heavy metals cations) . These data indicate that yeast cells cannot sense these compounds through the same molecular devices, albeit they are all electrophilic.

Biochemistry, 2003 Oct 21, 42(41), 11982 - 91
The redox domain of the Yap1p transcription factor contains two disulfide bonds; Wood MJ et al.; The subcellular localization of the Saccharomyces cerevisiae transcription factor Yap1p is regulated by oxidation and reduction . We purified Yap1p from yeast and characterized its properties in vitro . Electrophoretic mobility shift assays showed that the purified protein can specifically bind the TRX2 target promoter . Yap1p was purified under reducing conditions, but removal of reducing agents resulted in the formation of an oxidized Yap1p species with properties similar to in vivo oxidized Yap1p . MALDI-TOF mass spectrometry analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629 . A stable domain of approximately 15 kDa was detected upon limited proteolysis of oxidized but not reduced Yap1p . This Yap1p protease resistant domain was purified, and MALDI-TOF mass spectrometry analysis showed that it was comprised of two separate cysteine-containing peptides of Yap1p . These peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds . Taken together, these data suggest that the domain that controls Yap1p subcellular localization is modular and contains a redox center comprised of four cysteine residues.

Nat Biotechnol, 2003 Nov, 21(11), 1337 - 42 Epub 2003 Oct 12.
Computational discovery of gene modules and regulatory networks; Bar-Joseph Z et al.; We describe an algorithm for discovering regulatory networks of gene modules, GRAM (Genetic Regulatory Modules), that combines information from genome-wide location and expression data sets . A gene module is defined as a set of coexpressed genes to which the same set of transcription factors binds . Unlike previous approaches that relied primarily on functional information from expression data, the GRAM algorithm explicitly links genes to the factors that regulate them by incorporating DNA binding data, which provide direct physical evidence of regulatory interactions . We use the GRAM algorithm to describe a genome-wide regulatory network in Saccharomyces cerevisiae using binding information for 106 transcription factors profiled in rich medium conditions data from over 500 expression experiments . We also present a genome-wide location analysis data set for regulators in yeast cells treated with rapamycin, and use the GRAM algorithm to provide biological insights into this regulatory network

Inflamm Bowel Dis, 2003 Sep, 9(5), 324 - 31
Indeterminate colitis; Geboes K et al.; A diagnosis of Crohn's disease (CD) and ulcerative colitis (UC) is based on a combination of clinical, histologic, endoscopic, and radiologic data . The distinction between UC and CD can be difficult because of the lack of a differentiating single gold standard . Indeterminate colitis (IC) was introduced by pathologists for the diagnosis of surgical colectomy specimens showing an overlap between the features of UC and CD . The diagnosis of IC was based on macroscopic and microscopic features . The term indeterminate colitis is in recent years more widely applied to include all cases with endoscopic, radiographic, and histologic evidence of chronic inflammatory bowel disease confined to the colon, but without fulfilment of diagnostic criteria for UC and CD . As for UC and CD, the diagnosis of IC has therefore become a clinicopathologic diagnosis . IC is generally considered to be a temporary diagnosis . The clinical characteristics of patients with IC are, however, somewhat different from the characteristics of those with UC . Furthermore, serologic markers such as perinuclear antineutrophil cytoplasmic antibody and anti-Saccharomyces cerevisiae, which are strongly linked with UC and CD, are both negative in a subset of patients with IC . Therefore, the possibility that IC could be a separate entity must be investigated.

Plant Physiol, 2003 Oct, 133(2), 919 - 29
Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase; Vanoosthuyse V et al.; Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily . Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK . A B . oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK . Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals . A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain . The binding site is conserved and functional in several other members of the PRK family . The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.

Proc Natl Acad Sci U S A, 2003 Oct 28, 100(22), 12630 - 5 Epub 2003 Oct 10.
AIPL1, a protein implicated in Leber's congenital amaurosis, interacts with and aids in processing of farnesylated proteins; Ramamurthy V et al.; The most common form of blindness at birth, Leber's congenital amaurosis (LCA), is inherited in an autosomal recessive fashion . Mutations in six different retina-specific genes, including a recently discovered gene, AIPL1, have been linked to LCA in humans . To understand the molecular basis of LCA caused by aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) mutations, and to elucidate the normal function of AIPL1, we performed a yeast two-hybrid screen using AIPL1 as bait . The screen demonstrated that AIPL1 interacts specifically with farnesylated proteins . Mutations in AIPL1 linked to LCA compromise this activity . These findings suggest that the essential function of AIPL1 within photoreceptors requires interactions with farnesylated proteins . Analysis of isoprenylation in cultured human cells shows that AIPL1 enhances the processing of farnesylated proteins . Based on these findings, we propose that AIPL1 interacts with farnesylated proteins and plays an essential role in processing of farnesylated proteins in retina.

Bioinformatics, 2003 Oct 12, 19(15), 1952 - 63
Background rareness-based iterative multiple sequence alignment algorithm for regulatory element detection; Narasimhan C et al.; MOTIVATION: Experimental methods capable of generating sets of co-regulated genes have become commonplace, however, recognizing the regulatory motifs responsible for this regulation remains difficult . As a result, computational detection of transcription factor binding sites in such data sets has been an active area of research . Most approaches have utilized either Gibbs sampling or greedy strategies to identify such elements in sets of sequences . These existing methods have varying degrees of success depending on the strength and length of the signals and the number of available sequences . We present a new deterministic iterative algorithm for regulatory element detection based on a Markov chain background . As in other methods, sequences in the entire genome and the training set are taken into account in order to discriminate against commonly occurring signals and produce patterns, which are significant in the training set . RESULTS: The results of the algorithm compare favorably with existing tools on previously known and newly compiled data sets . The iteration based search appears rather rigorous, not only finding the binding sites, but also showing how the binding site stands out from genomic background . The approach used to score the results is critical and a discussion of various scoring schemes and options is also presented . Benchmarking of several methods shows that while most tools are good at detecting strong signals, Gibbs sampling algorithms give inconsistent results when the regulatory element signal becomes weak . A Markov chain based background model alleviates the drawbacks of MAP (maximum a posteriori log likelihood) scores . AVAILABILITY: Available on request from the authors . SUPPLEMENTARY INFORMATION: Data and the results presented in this paper are available on the web at http://compbio.ornl.gov/mira/index.html

Bioinformatics, 2003 Oct 12, 19(15), 1917 - 26
Prediction of regulatory networks: genome-wide identification of transcription factor targets from gene expression data; Qian J et al.; MOTIVATION: Defining regulatory networks, linking transcription factors (TFs) to their targets, is a central problem in post-genomic biology . One might imagine one could readily determine these networks through inspection of gene expression data . However, the relationship between the expression timecourse of a transcription factor and its target is not obvious (e.g . simple correlation over the timecourse), and current analysis methods, such as hierarchical clustering, have not been very successful in deciphering them . RESULTS: Here we introduce an approach based on support vector machines (SVMs) to predict the targets of a transcription factor by identifying subtle relationships between their expression profiles . In particular, we used SVMs to predict the regulatory targets for 36 transcription factors in the Saccharomyces cerevisiae genome based on the microarray expression data from many different physiological conditions . We trained and tested our SVM on a data set constructed to include a significant number of both positive and negative examples, directly addressing data imbalance issues . This was non-trivial given that most of the known experimental information is only for positives . Overall, we found that 63% of our TF-target relationships were confirmed through cross-validation . We further assessed the performance of our regulatory network identifications by comparing them with the results from two recent genome-wide ChIP-chip experiments . Overall, we find the agreement between our results and these experiments is comparable to the agreement (albeit low) between the two experiments . We find that this network has a delocalized structure with respect to chromosomal positioning, with a given transcription factor having targets spread fairly uniformly across the genome . AVAILABILITY: The overall network of the relationships is available on the web at http://bioinfo.mbb.yale.edu/expression/echipchip

Eukaryot Cell, 2003 Oct, 2(5), 1099 - 114
Posttranslational modifications required for cell surface localization and function of the fungal adhesin Aga1p; Huang G et al.; Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts . Adhesins are cell surface proteins that mediate these different cell adhesion interactions . In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation . The processing of some fungal GPI anchors, creating links to cell wall beta-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions . By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function . Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins . A portion of the cellular pool of Aga1p was determined to be cell wall resident . Aga1p and the alpha-agglutinin Agalpha1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATalpha cells for single mutants but in both cell types when both genes are absent . We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity . Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.

Eukaryot Cell, 2003 Oct, 2(5), 1046 - 52
Role of second-largest RNA polymerase I subunit Zn-binding domain in enzyme assembly; Naryshkina T et al.; The second-largest subunits of eukaryal RNA polymerases are similar to the beta subunits of prokaryal RNA polymerases throughout much of their lengths . The second-largest subunits from eukaryal RNA polymerases contain a four-cysteine Zn-binding domain at their C termini . The domain is also present in archaeal homologs but is absent from prokaryal homologs . Here, we investigated the role of the C-terminal Zn-binding domain of Rpa135, the second-largest subunit of yeast RNA polymerase I . Analysis of nonfunctional Rpa135 mutants indicated that the Zn-binding domain is required for recruitment of the largest subunit, Rpa190, into the RNA polymerase I complex . Curiously, the essential function of the Rpa135 Zn-binding domain is not related to Zn(2+) binding per se, since replacement of only one of the four cysteine residues with alanine led to the loss of Rpa135 function . Even more strikingly, replacement of all four cysteines with alanines resulted in functional Rpa135.

Eukaryot Cell, 2003 Oct, 2(5), 937 - 48
Regulation of ENA1 Na(+)-ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway; Ruiz A et al.; Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na(+)-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes . However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain . We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at -751 to -667, containing a calcineurin-dependent response element (CDRE), and one downstream region (-573 to -490) whose activity responds to intracellular alkalinization . In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene . The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity . In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant . It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH . However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect . In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants.

Eukaryot Cell, 2003 Oct, 2(5), 930 - 6
Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p; Goehring AS et al.; Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins . Urm1p conjugation has been implicated in the budding process and in nutrient sensing . Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p . The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p . Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response . Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates . These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.

Science, 2003 Oct 10, 302(5643), 240 - 1
Genomics . Microarrays--guilt by association; Quackenbush J; DNA microarray analysis has provided a wealth of data on global patterns of gene expression but has yet to deliver on its early promise of identifying networks of interacting gene products . In his Perspective, Quackenbush discusses new work (Stuart et al.) that uses evolutionary conservation of gene expression patterns in yeast, worm, fruit fly, and human in an attempt to identify functionally related groups of genes.

Plant Physiol, 2003 Nov, 133(3), 1170 - 80 Epub 2003 Oct 09.
Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation; Rhee SY et al.; Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development . Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging . QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica) . The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity . In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings . Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage . Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.

Plant Physiol, 2003 Nov, 133(3), 1360 - 6 Epub 2003 Oct 09.
PRT1 of Arabidopsis is a ubiquitin protein ligase of the plant N-end rule pathway with specificity for aromatic amino-terminal residues; Stary S et al.; The gene PRT1 of Arabidopsis, encoding a 45-kD protein with two RING finger domains, is essential for the degradation of F-dihydrofolate reductase, a model substrate of the N-end rule pathway of protein degradation . We have determined the function of PRT1 by expression in yeast (Saccharomyces cerevisiae) . PRT1 can act as a ubiquitin protein ligase in the heterologous host . The identified substrates of PRT1 have an aromatic residue at their amino-terminus, indicating that PRT1 mediates degradation of N-end rule substrates with aromatic termini but not of those with aliphatic or basic amino-termini . Expression of model substrates in mutant and wild-type plants confirmed this substrate specificity . A ligase activity exclusively devoted to aromatic amino-termini of the N-end rule pathway is apparently unique to plants . The results presented also imply that other known substrates of the plant N-end rule pathway are ubiquitylated by one or more different ubiquitin protein ligases.

Mol Biol Cell, 2003 Nov, 14(11), 4342 - 51 Epub 2003 Jul 25.
Interaction with Tap42 is required for the essential function of Sit4 and type 2A phosphatases; Wang H et al.; In Saccharomyces cerevisiae, Pph21 and Pph22 are the two catalytic subunits of type 2A phosphatase (PP2Ac), and Sit4 is a major form of 2A-like phosphatase . The function of these phosphatases requires their association with different regulatory subunits . In addition to the conventional regulatory subunits, namely, the A and B subunits for Pph21/22 and the Sap proteins for Sit4, these phosphatases have been found to associate with a protein termed Tap42 . In this study, we demonstrated that Sit4 and PP2Ac interact with Tap42 via an N-terminal domain that is conserved in all type 2A and 2A-like phosphatases . We found that the Sit4 phosphatase in the sit4-102 strain contains a reverse-of-charge amino acid substitution within its Tap42 binding domain and is defective for formation of the Tap42-Sit4 complex . Our results suggest that the interaction with Tap42 is required for the activity as well as for the essential function of Sit4 and PP2Ac . In addition, we showed that Tap42 is able to interact with two other 2A-like phosphatases, Pph3 and Ppg1.

Mol Biol Cell, 2003 Nov, 14(11), 4486 - 98 Epub 2003 Aug 07.
The role of the polo kinase Cdc5 in controlling Cdc14 localization; Visintin R et al.; In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis . Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus . During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks . The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase . Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus . Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus . We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1 . Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation . Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.

J Biol Chem, 2003 Dec 26, 278(52), 52629 - 40 Epub 2003 Oct 09.
Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein; Al-Shawi MK et al.; ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited . To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates . We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R . A., Omote, H., Nakamoto, R . K., and Al-Shawi, M . K . (2000) Arch . Biochem . Biophys . 376, 34-46) as well as Chinese hamster Pgp (PGP1) . Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states . Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed . Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity . We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates . Rather, it is an intrinsic mechanistic property of the enzyme . We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state . The overall rate-limiting step of Pgp during transport is a carrier reorientation step . Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency . The drug inhibition phase was associated with low affinity drug-binding sites . These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A . E., Al-Shawi, M . K., and Urbatsch, I . L . (1995) FEBS Lett . 377, 285-289) . A new kinetic model of drug transport is presented.

Dev Biol, 2003 Oct 15, 262(2), 294 - 302
The CBP coactivator functions both upstream and downstream of Dpp/Screw signaling in the early Drosophila embryo; Lilja T et al.; The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator . A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-beta molecules Dpp and Screw in the early embryo . We show that the expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos . This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell . Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression . At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression . In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad . It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes . These results highlight the exceptional sensitivity of components in the TGF-beta signaling pathway to a decline in CBP concentration.

FEBS Lett, 2003 Oct 9, 553(1-2), 163 - 6
Control of mitochondrial protein import by pH; Grigoriev SM et al.; Protein import into mitochondria is inhibited by protons (IC(50) pH 6.5) . The channels of the import machinery were examined to further investigate this pH dependence . TOM and TIM23 are the protein translocation channels of the mitochondrial outer and inner membranes, respectively, and their single channel behaviors at various pHs were determined using patch-clamp techniques . While not identical, increasing H(+) concentration decreases the open probability of both TIM23 and TOM channels . The pattern of the pH dependences of protein import and channel properties suggests TIM23 open probability can limit import of nuclear-encoded proteins into the matrix of yeast mitochondria.

Mol Cell, 2003 Aug, 12(2), 437 - 47
Binding of TFIIB to RNA polymerase II: Mapping the binding site for the TFIIB zinc ribbon domain within the preinitiation complex; Chen HT et al.; RNA polymerase II (Pol II) is recruited to promoters by interaction with general transcription factors . The zinc ribbon domain of the general factor TFIIB is essential for Pol II recruitment . Site-specific photocrosslinking and directed hydroxyl radical probing were used to map the location of the TFIIB zinc ribbon domain on Pol II within the transcription preinitiation complex (PIC) . These results, along with mutational analysis, suggest that in the PIC, the TFIIB ribbon domain interacts with a surface of the Pol II Dock domain where it overlaps the RNA exit point . This surface is best conserved in polymerases that require a TFIIB-like factor . Our results suggest a general mechanism for interaction of TFIIB-like factors and RNA polymerases and a mechanism for the function of the ribbon domain.

Mol Cell, 2003 Aug, 12(2), 401 - 11
Retrograde signaling is regulated by the dynamic interaction between Rtg2p and Mks1p; Liu Z et al.; Activation of retrograde signaling (RS) by mitochondrial dysfunction or by inhibition of TOR kinases in yeast results in nuclear accumulation of the transcription factors, Rtg1p and Rtg3p . This process requires Rtg2p, a novel cytoplasmic protein with an N-terminal ATP binding domain . We show that Rtg2p controls RS by reversibly binding a negative regulator, Mks1p . The inhibitory form of Mks1p is phosphorylated and complexed with the 14-3-3 proteins, Bmh1p and Bmh2p, which are also negative regulators of RS . A hypophosphorylated form of Mks1p bound to Rtg2p is inactive . Point mutations in the Rtg2p ATP binding domain simultaneously block RS and Mks1p-Rtg2p interaction . We propose that activation of RS via mitochondrial dysfunction and TOR inhibition intersect at the Rtg2p-Mks1p switch.

Dev Cell, 2003 Oct, 5(4), 583 - 94
ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains; Mironov AA et al.; Protein transport between the ER and the Golgi in mammalian cells occurs via large pleiomorphic carriers, and most current models suggest that these are formed by the fusion of small ER-derived COPII vesicles . We have examined the dynamics and structural features of these carriers during and after their formation from the ER by correlative video/light electron microscopy and tomography . We found that saccular carriers containing either the large supramolecular cargo procollagen or the small diffusible cargo protein VSVG arise through cargo concentration and direct en bloc protrusion of specialized ER domains in the vicinity of COPII-coated exit sites . This formation process is COPII dependent but does not involve budding and fusion of COPII-dependent vesicles . Fully protruded saccules then move centripetally, evolving into one of two types of carriers (with distinct kinetic and structural features) . These findings provide an alternative framework for analysis of ER-to-Golgi traffic.

J Biol Chem, 2004 Feb 6, 279(6), 4450 - 8 Epub 2003 Oct 08.
Exploratory and confirmatory gene expression profiling of mac1Delta; De Freitas JM et al.; Exploratory outlier identification methods and confirmatory gene expression studies showed induction of the iron regulon in Saccharomyces cerevisiae lacking Mac1p, a copper-responsive transcription factor . The Aft1p/Aft2p binding motif was the most discriminating motif between up- and down-regulated genes, and we identified new genes potentially regulated by Aft1p/Aft2p . In addition, multiple genes encoding proteins containing Fe-S clusters were down-regulated suggesting metabolic reorganization to conserve iron in mac1Delta . Null mutants of each of the differentially expressed genes were characterized for copper- or iron-related phenotypes . New or additional support for a role in copper and iron homeostasis is provided in this study for the gene products of AKR1, MRS4, PCA1, SSU1, TIS11, YBR047W, YHL035C, YHR045W, YLR047C, YLR126C, and YTP1.

J Neurosci, 2003 Oct 8, 23(27), 9133 - 45
sup-9, sup-10, and unc-93 may encode components of a two-pore K+ channel that coordinates muscle contraction in Caenorhabditis elegans; de la Cruz IP et al.; Genetic studies of sup-9, unc-93, and sup-10 strongly suggest that these genes encode components of a multi-subunit protein complex that coordinates muscle contraction in Caenorhabditis elegans . We cloned sup-9 and sup-10 and found that they encode a two-pore K+ channel and a novel transmembrane protein, respectively . We also found that UNC-93 and SUP-10 colocalize with SUP-9 within muscle cells, and that UNC-93 is a member of a novel multigene family that is conserved among C . elegans, Drosophila, and humans . Our results indicate that SUP-9 and perhaps other two-pore K+ channels function as multiprotein complexes, and that UNC-93 and SUP-10 likely define new classes of ion channel regulatory proteins.

Bioinformatics, 2003 Oct, 19 Suppl 2, II227 - II236
Estimating gene networks from gene expression data by combining Bayesian network model with promoter element detection; Tamada Y et al.; We present a statistical method for estimating gene networks and detecting promoter elements simultaneously . When estimating a network from gene expression data alone, a common problem is that the number of microarrays is limited compared to the number of variables in the network model, making accurate estimation a difficult task . Our method overcomes this problem by integrating the microarray gene expression data and the DNA sequence information into a Bayesian network model . The basic idea of our method is that, if a parent gene is a transcription factor, its children may share a consensus motif in their promoter regions of the DNA sequences . Our method detects consensus motifs based on the structure of the estimated network, then re-estimates the network using the result of the motif detection . We continue this iteration until the network becomes stable . To show the effectiveness of our method, we conducted Monte Carlo simulations and applied our method to Saccharomyces cerevisiae data as a real application . Contact: tamada@ims.u-tokyo.ac.jp

Eur J Cell Biol, 2003 Aug, 82(8), 401 - 10
Targeting of the human adrenoleukodystrophy protein to the peroxisomal membrane by an internal region containing a highly conserved motif; Landgraf P et al.; In this study we addressed the targeting requirements of peroxisomal ABC transporters, in particular the human adrenoleukodystrophy protein . This membrane protein is defective or missing in X-linked adrenoleukodystrophy, a neurodegenerative disorder predominantly presenting in childhood . Using adrenoleukodystrophy protein deletion constructs and green fluorescent protein fusion constructs we identified the amino acid regions 1-110 and 67-164 to be sufficient for peroxisomal targeting . However, the minimal region shared by these constructs (amino acids 67-110) is not sufficient for peroxisomal targeting by itself . Additionally, the NH2-terminal 66 amino acids enhance targeting efficiency . Green fluorescent protein-labeled fragments of human peroxisomal membrane protein 69 and Saccharomyces cerevisiae Pxa1 corresponding to the amino acid 67-164 adrenoleukodystrophy protein region were also directed to the mammalian peroxisome . The required region contains a 14-amino-acid motif (71-84) conserved between the adrenoleukodystrophy protein and human peroxisomal membrane protein 69 and yeast Pxa1 . Omission or truncation of this motif in the adrenoleukodystrophy protein abolished peroxisomal targeting . The single amino acid substitution L78F resulted in a significant reduction of targeting efficiency . The in-frame deletion of three amino acids (del78-80LLR) within the proposed targeting motif in two patients suffering from X-linked adrenoleukodystrophy resulted in the mislocalization of a green fluorescent protein fusion protein to nucleus, cytosol and mitochondria . Our data define the targeting region of human adrenoleukodystrophy protein containing a highly conserved 14-amino-acid motif.

EMBO J, 2003 Oct 15, 22(20), 5612 - 21
Requirement of the MRN complex for ATM activation by DNA damage; Uziel T et al.; The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs) . In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network . ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition . The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs . Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively . The order in which ATM and MRN act in the early phase of the DSB response is unclear . Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways . Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.

EMBO J, 2003 Oct 15, 22(20), 5403 - 11
Mechanisms underlying excitatory effects of group I metabotropic glutamate receptors via inhibition of 2P domain K+ channels; Chemin J et al.; Group I metabotropic glutamate receptors (mGluRs) are implicated in diverse processes such as learning, memory, epilepsy, pain and neuronal death . By inhibiting background K(+) channels, group I mGluRs mediate slow and long-lasting excitation . The main neuronal representatives of this K(+) channel family (K(2P) or KCNK) are TASK and TREK . Here, we show that in cerebellar granule cells and in heterologous expression systems, activation of group I mGluRs inhibits TASK and TREK channels . D-myo-inositol-1,4,5-triphosphate and phosphatidyl-4,5-inositol-biphosphate depletion are involved in TASK channel inhibition, whereas diacylglycerols and phosphatidic acids directly inhibit TREK channels . Mechanisms described here with group I mGluRs will also probably stand for many other receptors of hormones and neurotransmitters.

EMBO J, 2003 Oct 15, 22(20), 5370 - 81
Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex; Chacinska A et al.; Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex) . Little is known about the functional cooperation of these translocases . We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS) . The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex . Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex . Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex . Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.

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