S. cerevisiae

Mol Cell Biol, 2004 Jul, 24(14), 6184 - 93
mRNA capping enzyme activity is coupled to an early transcription elongation; Kim HJ et al.; One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect . This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity . However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure . Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation . First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II . Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff . Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientation-dependent manner . Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription . Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further.

FEBS Lett, 2004 Jul 2, 569(1-3), 140 - 8
A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens; Bernard C et al.; Thlaspi caerulescens exhibits a unique capacity for cadmium tolerance and accumulation . We investigated the molecular basis of this exceptional Cd(2+) tolerance by screening for T . caerulescens genes, which alleviate Cd(2+) toxicity upon expression in Saccharomyces cerevisiae . This allowed for the isolation of a cDNA encoding a peptide with homology to the C-terminal part of a heavy metal ATPase . The corresponding TcHMA4 full-length sequence was isolated from T . caerulescens and compared to its homolog from Arabidopsis thaliana (AtHMA4) . Expression of TcHMA4 and AtHMA4 cDNAs conferred Cd sensitivity in yeast, while expression of TcHMA4-C and AtHMA4-C cDNAs encoding the C-termini of, respectively, TcHMA4 and AtHMA4 conferred Cd tolerance . Moreover, heterologous expression in yeast suggested a higher Cd binding capacity of TcHMA4-C compared to AtHMA4-C . In planta, both HMA4 genes were expressed at a higher level in roots than in shoots . However, TcHMA4 shows a much higher constitutive expression than AtHMA4 . Our data indicate that HMA4 could be involved in Cd(2+) transport and possibly in the Cd hyperaccumulation character.

FEBS Lett, 2004 Jul 2, 569(1-3), 89 - 93
The Arabidopsis thaliana chloroplast inner envelope protein ARTEMIS is a functional member of the Alb3/Oxa1/YidC family of proteins; Funes S et al.; The Arabidopsis thaliana protein ARTEMIS is an integral component of the chloroplast inner envelope required for chloroplast division . It contains a domain of significant homology to members of the Alb3/Oxa1/YidC protein family . Here, we show that upon expression in yeast mitochondria, ARTEMIS can partially take over the function of yeast Oxa1 in the insertion and assembly of mitochondrial membrane proteins . This identifies ARTEMIS as a functional member of the Alb3/Oxa1/YidC protein family and suggests the existence of a novel protein sorting pathway in chloroplasts which integrates polypeptides from the stroma into the inner envelope by an evolutionary conserved process.

FEBS Lett, 2004 Jul 2, 569(1-3), 65 - 9
The mitochondrial ABC transporter Atm1p functions as a homodimer; Chloupkova M et al.; The ATP-binding cassette (ABC) transporters constitute one of the largest families of proteins in evolution . The ATM1 gene of the yeast Saccharomyces cerevisiae encodes an ABC protein, which is localized to the mitochondrial inner membrane . A deletion of ATM1 results in the accumulation of up to a 30-fold excess of mitochondrial iron, loss of mitochondrial cytochromes and abnormalities of cytosolic iron metabolism . In this study, we have evaluated the role of conserved sequence elements in Atm1p in its function and dimerization in vivo . We report that conserved residues in the Walker A and B motifs of the nucleotide binding domain, which are required for ATP binding and hydrolysis, are essential for Atm1p function . In addition, we provide evidence that ATP binding is important for Atm1p dimerization.

Mol Cell, 2004 Jul 2, 15(1), 107 - 16
The Ras/PKA signaling pathway directly targets the Srb9 protein, a component of the general RNA polymerase II transcription apparatus; Chang YW et al.; RNA polymerase II transcription is a complex process that is controlled at multiple levels . The data presented here add to this repertoire by showing that signal transduction pathways can directly regulate gene expression by targeting components of the general RNA polymerase II apparatus . In particular, this study shows that the Ras/PKA signaling pathway in Saccharomyces cerevisiae regulates the activity of the Srb complex, a regulatory group of proteins that is part of the RNA polymerase II holoenzyme . Genetic and biochemical data indicate that Srb9p is a substrate for PKA and that this phosphorylation modulates the activity of the Srb complex . The Srb complex, like many components of the RNA II polymerase machinery, is responsible for regulating the expression of a relatively large number of genes . Thus, this type of a transcriptional control mechanism would provide the cell with an efficient way of bringing about broad changes in gene expression.

Mol Cell, 2004 Jul 2, 15(1), 95 - 105
Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation; Schaffar G et al.; The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions . These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP . We show, in vitro and in cells, that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner . FRET experiments indicate that these toxic forms are generated through a conformational rearrangement in huntingtin . Interaction of toxic huntingtin with the benign polyQ repeat of TBP structurally destabilizes the transcription factor, independent of the formation of insoluble coaggregates . Hsp70/Hsp40 chaperones interfere with the conformational change in mutant huntingtin and inhibit the deactivation of TBP . These results outline a molecular mechanism of cellular toxicity in polyQ disease and can explain the beneficial effects of molecular chaperones.

Mol Cell, 2004 Jul 2, 15(1), 69 - 81
Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1; Martinez-Campa C et al.; Acetylation of histone tails plays a key role in chromatin dynamics and is associated with the potential for gene expression . We show here that a 2-3 bp mispositioning of the nucleosome covering the TATA box at PHO5 induces a dependency on the acetylatable lysine residues of the histone H4 N-terminal region and on the TFIID-associated bromodomain factor Bdf1 . This dependency arises either through fusion of the PHO5 promoter to a lacZ reporter or by mutation of the TATA box in the natural gene . The results suggest that promoters in which the TATA box is either absent or poorly accessible on the surface of a nucleosome may compensate by using Bdf1 bromodomains and acetylated H4 tails to anchor TFIID to the promoter during the initial stages of transcription activation . We propose that nucleosome positioning at the nucleotide level provides a subtle, but highly effective, mechanism for gene regulation.

Mol Cell, 2004 Jul 2, 15(1), 5 - 15
Targeted mRNA degradation by deadenylation-independent decapping; Badis G et al.; Modulating the rate of mRNA degradation is a fast and efficient way to control gene expression . In a yeast strain deleted of EDC3, a component of the decapping machinery conserved in eukaryotes, the transcript coding the ribosomal protein Rps28b is specifically stabilized, as demonstrated by microarray and time course experiments . This stabilization results from the loss of RPS28B autoregulation, which occurs at the level of mRNA decay . Using mutants of the major deadenylase, we show that this regulation occurs at the level of decapping and bypasses deadenylation . Rps28b interacts with a conserved hairpin structure within the 3'UTR of its own mRNA and with components of the decapping machinery, including Edc3 . We conclude that Rps28b, in the presence of Edc3, directly recruits the decapping machinery on its own mRNA . These findings show that specific modulation of the decapping efficiency on natural transcripts can control mRNA turnover.

Mol Cell, 2004 Jul 2, 15(1), 1 - 2
Regulation of mRNA decay: decapping goes solo; Jacobson A; In this issue of Molecular Cell, Badis et al . demonstrate that autoregulated destabilization of the RPS28B mRNA is mediated by Edc3p interaction with an Rps28p/3'UTR mRNP complex . Subsequent recruitment of Dcp1p/Dcp2p to the complex appears to ensure rapid, deadenylation-independent decapping of the mRNA.

Mol Microbiol, 2004 Jul, 53(1), 335 - 44
Activation of pleiotropic drug resistance by the J-protein and Hsp70-related proteins, Zuo1 and Ssz1; Eisenman HC et al.; Ssz1 (Pdr13) and Zuo1, members of the Hsp70 and J-protein molecular chaperone families, respectively, form a heterodimer and function on the ribosome with the Hsp70, Ssb, presumably assisting folding of newly synthesized polypeptides . As it has also been reported that Ssz1 induces pleiotropic drug resistance (PDR) when overexpressed, a possible role for Zuo1 in PDR was investigated . The C-terminal domain of Zuo1, which is dispensable for Zuo1's chaperone function on the ribosome, is both necessary and sufficient for PDR induction by Zuo1 . A single domain of Ssz1, the N-terminal ATPase domain, is sufficient for PDR induction as well, indicating that Ssz1 does not function as a chaperone in PDR . No role for Ssb was found in PDR; overexpression did not affect PDR, nor was its presence required for Ssz1's or Zuo1's effect on PDR . As our results also indicate that Ssz1 and Zuo1 must be free of ribosomes to induce PDR, we propose that Ssz1's and Zuo1's function in PDR is distinct from their role as ribosome-associated co-chaperones and may be regulatory in nature.

Biochemistry, 2004 Jul 6, 43(26), 8579 - 89
Identification of DNA 3'-phosphatase active site residues and their differential role in DNA binding, Mg2+ coordination, and catalysis; Deshpande RA et al.; DNA 3'-phosphatase (Tpp1) from Saccharomyces cerevisiae, a homologue of human polynucleotide kinase/3'-phosphatase, has been shown to participate in DNA damage repair by removing 3'-phosphate blocking lesions . Tpp1 shows similarity to the l-2-haloacid dehalogenase superfamily of enzymes . By comparison to phosphoserine phosphatase, a well-studied member of this family, we designed conservative and nonconservative substitutions of likely active site residues of Tpp1 and tested them in a variety of assays . From the loss or impairment of activity, we identified D35, D37, T39, S88, K170, D206, and D218 as being involved in Tpp1 catalysis . D35 and K170 were the most critical since maximum inactivation was seen with even conservative mutations . Tpp1 bound DNA through its active site in a Mg(2+)-dependent manner and exhibited a preference for dsDNA . Although Tpp1 bound more strongly to DNA with a free 3' terminus, it also bound well to covalently closed DNA, suggesting a possible lesion scanning mechanism . DNA binding studies further indicated that Tpp1 coordinates Mg(2+) through D35 and D206 and contacts the DNA 3' end through D37 . The removal of 3'-phosphate involved a phospho-Tpp1 intermediate, and our results support D35 as being the point of covalent attachment . On the basis of these similarities in mutant phenotypes of Tpp1 and phosphoserine phosphatase, we propose a reaction mechanism for Tpp1 which explains its strict phosphate specificity.

Biochemistry, 2004 Jul 6, 43(26), 8568 - 78
Sml1p is a dimer in solution: characterization of denaturation and renaturation of recombinant Sml1p; Gupta V et al.; Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity . During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death . Structural characterization of Sml1p is an important step in understanding the regulation of RNR . Until now the oligomeric state of Sml1p was unknown . Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence . To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine . Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond . Further studies of several truncated Sml1p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization . Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles . It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.

Biochemistry, 2004 Jul 6, 43(26), 8447 - 58
raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences; Vallee-Belisle A et al.; Recent experimental and theoretical studies in protein folding suggest that the rates and underlying mechanisms by which proteins attain the native state are largely determined by the topological complexity of a specific fold rather than by the fine details of the amino acid sequences . However, such arguments are based upon the examination of a limited number of protein folds . To test this view, we sought to investigate whether proteins belonging to the ubiquitin superfamily display similar folding behavior . To do so, we compared the folding-unfolding transitions of mammalian ubiquitin (mUbi) with those of its close yeast homologue (yUbi), and to those of the structurally related Ras binding domain (RBD) of the serine/threonine kinase raf that displays no apparent sequence homology with the ubiquitin family members . As demonstrated for mUbi {Krantz, B . A., and Sosnick, T . R . (2000) Biochemistry 39, 11696-11701}, we show that a two-state transition model with no burst phase intermediate can describe folding of both yUbi and raf RBD . We further demonstrate that (1) all three proteins refold at rates that are within 1 order of magnitude (1800, 1100, and 370 s(-1) for mUbi, raf RBD, and yUbi, respectively), (2) both mUbi and raf RBD display similar refolding heterogeneity, and (3) the folding free energy barriers of both mUbi and raf RBD display a similar temperature dependence and sensitivity to a stabilizing agent or to mutations of a structurally equivalent central core residue . These findings are consistent with the view that rates and mechanisms for protein folding depend mostly on the complexity of the native structure topology rather than on the fine details of the amino acid sequence.

Mol Genet Genomics, 2004 Aug, 272(1), 47 - 56 Epub 2004 Jun 19.
DIRS retroelements in arthropods: identification of the recently active TcDirs1 element in the red flour beetle Tribolium castaneum; Goodwin TJ et al.; Members of the DIRS family of retrotransposons differ from most other known retrotransposons in that they encode a tyrosine recombinase (YR), a type of enzyme frequently involved in site-specific recombination . This enzyme is believed to insert the extrachromosomal DNA intermediate of DIRS element retrotransposition into the host genome . DIRS elements have been found in plants, a slime mold, fungi, and a variety of animals including vertebrates, echinoderms and nematodes . They have a somewhat patchy distribution, however, apparently being absent from a number of model organisms such as Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster . In this report we describe the first DIRS retroelement to be identified in an arthropod . This element, TcDirs1, was found in the red flour beetle Tribolium castaneum (Coleoptera) . It is generally similar in sequence and structure to several previously described members of the DIRS group: it is bordered by inverted terminal repeats and it has a similar set of protein-coding domains (Gag, reverse transcriptase/ribonuclease H, and the YR), although these are arranged in a novel fashion . TcDirs1 elements exhibit several features indicative of recent activity, such as intact coding regions, a high level of sequence similarity between distinct elements and polymorphic insertion sites . Given their presence in an experimentally tractable host, these potentially active elements might serve as useful models for the study of DIRS element retrotransposition . An element closely related to TcDirs1 was also detected in sequences from a second arthropod, the honey bee Apis mellifera (Hymenoptera), suggesting that these retrotransposons are long-term residents of arthropod genomes.

Mol Genet Genomics, 2004 Jul, 271(6), 651 - 7 Epub 2004 Jun 18.
Interaction of FIE, a polycomb protein, with pRb: a possible mechanism regulating endosperm development; Mosquna A et al.; Inactivation of the Arabidopsis protein FERTILIZATION INDEPENDENT ENDOSPERM (FIE) induces division of the central cell of the embryo sac, leading to endosperm development in the absence of fertilization . The mechanism whereby FIE regulates this process is unknown . We postulated that activation of central cell division in fie mutant plants might involve the retinoblastoma protein (pRb), a cell cycle regulatory element . Pull-down and surface plasmon resonance assays demonstrated that FIE interacts in-vitro with the pRb homologues from Arabidopsis (AtRb), maize (ZmRb) and human (HuRb) . The interaction of FIE with ZmRB and HuRb in the yeast two-hybrid system supports the possibility that a FIE-pRb interaction may occur also in planta . Mutational analysis showed that this interaction does not occur via the LxCxE motif of the FIE protein nor via the pocket B domain of pRb . These results suggest that FIE may inhibit premature division of the central cell of the embryo sac, at least partly, through interaction with pRb, and suppression of pRb-regulated genes.

Nat Genet, 2004 Jul, 36(7), 707 - 13 Epub 2004 Jun 27.
Identification of TFB5, a new component of general transcription and DNA repair factor IIH; Ranish JA et al.; We previously described the use of quantitative proteomics to study macromolecular complexes . Applying the method to analyze a yeast RNA polymerase II preinitiation complex, we identified a new 8-kDa protein, encoded by the uncharacterized open reading frame YDR079c-a, as a potential new component of the preinitiation complex . Here we show that YDR079c-a is a bona fide component of polymerase II preinitiation complexes and investigate its role in transcription . YDR079c-a is recruited to promoters both in vivo and in vitro and is required for efficient transcription in vitro and for normal induction of GAL genes . In addition, YDR079c-a is a core component of general transcription and DNA repair factor IIH and is required for efficient recruitment of TFIIH to a promoter . Yeast lacking YDR079c-a grow slowly, and, like strains carrying mutations in core TFIIH subunits, are sensitive to ultraviolet radiation . YDR079c-a is conserved throughout evolution, and mutations in the human ortholog account for a DNA repair-deficient form of the tricothiodystrophy disorder called TTD-A(2) . The identification of a new, evolutionarily conserved, core TFIIH subunit is essential for our understanding of TFIIH function in transcription, DNA repair and human disease.

Methods Mol Biol, 2004, 281, 189 - 98
Functional analysis of APC-Cdh1; Sudo T et al.; To maintain genomic integrity against various kinds of genotoxic stress, cells have multiple checkpoints in the cell cycle . When one of the cell cycle events, such as DNA synthesis, DNA repair, and chromosomal segregation, has not been successfully completed, checkpoints will delay progression until the step is correctly accomplished, and only then will they relieve the arrest to allow the cell to move to the next phase . Cells lacking functional checkpoints display genomic aberrations, resulting in the acquisition of phenotypic changes of cancer cells . Anaphase-promoting complex (APC) is activated by two regulatory proteins: Cdc20 and Cdh1 . In yeast and Drosophila, Cdh1-dependent APC (APC-Cdh1) activity targets mitotic cyclins from the end of mitosis to the G1 phase . Loss of Cdh1 induces unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27Kip1 . Furthermore, Cdh1-deficient DT40 cells fail to maintain DNA damage-induced G2 arrest, and Cdh1-APC is activated by X-irradiation-induced DNA damage . Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest . In light of the differences between normal and cancer cells, checkpoints can be ideal targets for developmental cancer therapeutics . In this chapter, we describe how to analyze three checkpoints (spindle assembly checkpoint, rapamycin-induced G1 checkpoint, and DNA damage-induced G2 checkpoint) in conjunction with cell synchronization.

Fungal Genet Biol, 2004 Aug, 41(8), 735 - 53
Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis; Li R et al.; Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungal pathogen capable of infecting a wide range of plants . To identify genes involved in fungal development and pathogenesis we generated 2232 expressed sequence tags (ESTs) from two cDNA libraries constructed using either mycelia grown in pectin medium or tissues from infected Brassica napus stems . A total of 774 individual fungal genes were identified of which 39 were represented only among the infected plant EST collection . Annotation of 534 unigenes was possible following the categories applied to Saccharomyces cerevisiae and the Universal Gene Ontology scheme . cDNAs were identified that encoded potential pathogenicity factors including four endopolygalacturonases, two exopolygalacturonases, and several metabolite transporters . The potential role of these genes, as well as those encoding signal transduction factors, in the infection process is discussed.

J Biol Chem, 2004 Sep 3, 279(36), 38047 - 54 Epub 2004 Jun 24.
The presequence translocase-associated protein import motor of mitochondria . Pam16 functions in an antagonistic manner to Pam18; Li Y et al.; Transport of preproteins into the mitochondrial matrix requires the presequence translocase of the inner membrane (TIM23 complex) and the presequence translocase-associated motor (PAM) . The motor consists of five essential subunits, the mitochondrial heat shock protein 70 (mtHsp70) and four cochaperones, the nucleotide exchange-factor Mge1, the translocase-associated fulcrum Tim44, the J-protein Pam18, and Pam16 . Pam16 forms a complex with Pam18 and displays similarity to J-proteins but lacks the canonical tripeptide motif His-Pro-Asp (HPD) . We report that Pam16 does not function as a typical J-domain protein but, rather, antagonizes the function of Pam18 . Pam16 specifically inhibits the Pam18-mediated stimulation of the ATPase activity of mtHsp70 . The inclusion of the HPD motif in Pam16 does not confer the ability to stimulate mtHsp70 activity . Pam16-HPD fully substitutes for wild-type Pam16 in vitro and in vivo but is not able to replace Pam18 . Pam16 represents a new type of cochaperone that controls the stimulatory effect of the J-protein Pam18 and regulates the interaction of mtHsp70 with precursor proteins during import into mitochondria.

J Anim Sci, 2004 Jun, 82(6), 1882 - 91
Dietary supplementation with phosphorylated mannans improves growth response and modulates immune function of weanling pigs; Davis ME et al.; Phosphorylated mannans derived from the yeast cell wall of Saccharomyces cerevisiae may beneficially modulate immune function in the weanling pig, possibly providing an alternative to the use of dietary growth-promoting antibiotics . Therefore, in this study, 32 pigs averaging 19 d of age and 5.7 +/- 0.2 kg initial BW were randomly assigned to 16 pens in an environmentally controlled nursery to determine the effects of dietary supplementation with phosphorylated mannans on growth and immune function . Average daily gain and G:F ratio increased (P < 0.05) when pigs were fed diets supplemented with mannans from d 0 to 14 after weaning and in the overall experiment . Percentage of neutrophils was lower (P < 0.08) and percentage of lymphocytes was higher (P < 0.05) in blood from pigs fed mannans than when pigs were fed the basal diet . Lamina propria macrophages isolated from pigs fed diets containing mannans phagocytosed a greater (P < 0.05) number of sheep red blood cells (2.63 +/- 0.11) than did lamina propria macrophages isolated from pigs fed the basal diet (2.31 +/- 0.11) . On d 19 after weaning, pigs fed diets supplemented with mannans tended to have a greater (P < 0.10) percentage of CD14+ lamina propria leukocytes than did pigs fed the basal diet . On d 21 following weaning, the percentage of CD14+MHCII+ leukocytes isolated from lamina propria tissue tended (P < 0.10) to be lower when pigs were fed mannans than when pigs were fed the basal diet . Pigs fed diets containing mannans had a lower (P < 0.05) ratio of CD3+CD4+:CD3+CD8+ T lymphocytes isolated from jejunal lamina propria tissue only on d 21 after weaning compared with pigs fed the basal diet . Supplementation of mannans in the diets of weanling pigs improved gain and efficiency, and intermittently affected selected components of the young pigs' immune function both systemically and enterically.

Cell Struct Funct, 1999 Oct, 24(5), 365 - 72
Gamma-tubulin at ten: progress and prospects; Oakley BR et al.; The existence of gamma-tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in gamma-tubulin research and to discuss some of the unanswered questions about gamma-tubulin function . gamma-Tubulin is ubiquitous in eukaryotes and is generally quite conserved . Two highly divergent gamma-tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans . Several organisms have two gamma-tubulin genes . In Drosophila melanogaster, the two gamma-tubulins differ significantly in sequence and expression pattern . In other organisms the two gamma-tubulins are almost identical and expression patterns have not been determined . gamma-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle . gamma-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date . In animal cells, complexes containing gamma-tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubules . In spite of the considerable progress that has been made in gamma-tubulin research important questions remain to be answered . The exact mechanisms of microtubule nucleation by gamma-tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from gamma-tubulin complexes is regulated . Finally, there is evidence that gamma-tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated.

EMBO J, 2004 Jul 21, 23(14), 2872 - 81 Epub 2004 Jun 24.
Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS; Furukawa Y et al.; The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) has the distinction of being one of the most abundant disulfide-containing protein known in the eukaryotic cytosol; however, neither catalytic nor physiological roles for the conserved disulfide are known . Here we show that the disulfide status of Saccharomyces cerevisiae SOD1 significantly affects the monomer-dimer equilibrium, the interaction with the copper chaperone CCS, and the activity of the enzyme itself . Disulfide formation in SOD1 by O(2) is slow but is greatly accelerated by the Cu-bound form of CCS (Cu-CCS) in vivo and in vitro even in the presence of excess reductants; once formed, this disulfide is kinetically stable . Biochemical assays reveal that Cu-CCS facilitates Cys oxidation and disulfide isomerization in the stepwise conversion of the immature form of the enzyme to the active state . The immature form of SOD1 is most susceptible to oxidative insult and to aggregation reminiscent of that observed in amyotrophic lateral sclerosis . Thus Cu-CCS mediation of correct disulfide formation in SOD1 is important for regulation of enzyme activity and for prevention of misfolding or aggregation.

EMBO J, 2004 Jul 21, 23(14), 2862 - 71 Epub 2004 Jun 24.
A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila; Temme C et al.; The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae . We have identified homologs for almost all subunits of this complex in the Drosophila genome . Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity . In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci . Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails . Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock . Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation . A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit . The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W372 - 4
MITOPRED: a web server for the prediction of mitochondrial proteins; Guda C et al.; MITOPRED web server enables prediction of nucleus-encoded mitochondrial proteins in all eukaryotic species . Predictions are made using a new algorithm based primarily on Pfam domain occurrence patterns in mitochondrial and non-mitochondrial locations . Pre-calculated predictions are instantly accessible for proteomes of Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila, Homo sapiens, Mus musculus and Arabidopsis species as well as all the eukaryotic sequences in the Swiss-Prot and TrEMBL databases . Queries, at different confidence levels, can be made through four distinct options: (i) entering Swiss-Prot/TrEMBL accession numbers; (ii) uploading a local file with such accession numbers; (iii) entering protein sequences; (iv) uploading a local file containing protein sequences in FASTA format . Automated updates are scheduled for the pre-calculated prediction database so as to provide access to the most current data . The server, its documentation and the data are available from http://mitopred.sdsc.edu.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W176 - 80
Gene2Oligo: oligonucleotide design for in vitro gene synthesis; Rouillard JM et al.; There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis . Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR . Ideally, such oligonucleotides should represent both strands of the final DNA molecule . They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly . This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set . Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences . We have developed Gene2Oligo , a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands . The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis . We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W83 - 8
PathBLAST: a tool for alignment of protein interaction networks; Kelley BP et al.; PathBLAST is a network alignment and search tool for comparing protein interaction networks across species to identify protein pathways and complexes that have been conserved by evolution . The basic method searches for high-scoring alignments between pairs of protein interaction paths, for which proteins of the first path are paired with putative orthologs occurring in the same order in the second path . This technique discriminates between true- and false-positive interactions and allows for functional annotation of protein interaction pathways based on similarity to the network of another, well-characterized species . PathBLAST is now available at as a web-based query . In this implementation, the user specifies a short protein interaction path for query against a target protein-protein interaction network selected from a network database . PathBLAST returns a ranked list of matching paths from the target network along with a graphical view of these paths and the overlap among them . Target protein-protein interaction networks are currently available for Helicobacter pylori, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster . Just as BLAST enables rapid comparison of protein sequences between genomes, tools such as PathBLAST are enabling comparative genomics at the network level.

Am J Pathol, 2004 Jul, 165(1), 107 - 13
The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status; West RB et al.; We recently characterized gene expression patterns in gastrointestinal stromal tumors (GISTs) using cDNA microarrays, and found that the gene FLJ10261 (DOG1, discovered on GIST-1), encoding a hypothetical protein, was specifically expressed in GISTs . The immunoreactivity of a rabbit antiserum to synthetic DOG1 peptides was assessed on two soft tissue tumor microarrays . The tissue microarrays included 587 soft tissue tumors, with 149 GISTs, including 127 GIST cases for which the KIT and PDGFRA mutation status was known . Immunoreactivity for DOG1 was found in 136 of 139 (97.8%) of scorable GISTs . All seven GIST cases with a PDGFRA mutation were DOG1-positive, while most of these failed to react for KIT . The immunohistochemical findings were confirmed with in situ hybridization probes for DOG1, KIT, and PDGFRA . Other neoplasms in the differential diagnosis of GIST, including desmoid fibromatosis (0 of 17) and Schwannoma (0 of 3), were immunonegative for DOG1 . Only 4 of 438 non-GIST cases were immunoreactive for DOG1 . DOG1, a protein of unknown function, is expressed strongly on the cell surface of GISTs and is rarely expressed in other soft tissue tumors . Reactivity for DOG1 may aid in the diagnosis of GISTs, including PDGFRA mutants that fail to express KIT antigen, and lead to appropriate treatment with imatinib mesylate, an inhibitor of the KIT tyrosine kinase.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2483 - 9
Candida glabrata erg1 mutant with increased sensitivity to azoles and to low oxygen tension; Tsai HF et al.; A Candida glabrata erg1 (Cgerg1) mutant, CgTn201S, was identified by transposon mutagenesis and by increased fluconazole susceptibility . CgERG1 encodes a 489-amino-acid protein which, on the basis of its homology with Saccharomyces cerevisiae ERG1, is a squalene epoxidase essential for ergosterol synthesis . Interruption following codon 475 of CgErg1p decreased the ergosterol content by 50%; caused accumulation of the squalene precursor; increased the levels of susceptibility to fluconazole, itraconazole, and terbinafine; increased the level of resistance to amphotericin B; increased the levels of rhodamine 6G and {(3)H}-fluconazole uptake; reduced the level of growth; and blocked growth under conditions of low oxygen tension . In addition, CgTn201S efficiently took up exogenous cholesterol from cholesterol-containing serum . Cholesterol constituted 34% of the extractable sterols in CgTn201S when it was grown aerobically on serum-containing medium . Under the same conditions, C . albicans contained only 0.1 to 1.2% cholesterol . Exogenous sterols also restored growth under conditions of low oxygen tension . Finally, complementation of the Cgerg1 mutation restored the levels of {(3)H}fluconazole uptake and drug susceptibility to wild-type levels.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2409 - 14
Cloning of S-adenosyl-L-methionine:C-24-Delta-sterol-methyltransferase (ERG6) from Leishmania donovani and characterization of mRNAs in wild-type and amphotericin B-Resistant promastigotes; Pourshafie M et al.; The 24-alkylated sterols have been shown previously to be absent in membranes of amphotericin B (AmB)-resistant Leishmania donovani promastigotes, suggesting that the S- adenosyl-l-methionine:C-24-Delta-sterol-methyltransferase (SCMT or ERG6) was not functional or not expressed in AmB-resistant (AmB-R) parasites . From an L . donovani wild-type clone, we cloned two cDNAs with an identical open reading frame encoding a putative SCMT, the enzyme responsible for a first sterol methylation at the C-24 position . The two cDNAs differed by their 3'-untranslated region (3'-UTR) and 5'-UTR sequences . One transcript (A) had a normal structure with a spliced leader and was highly expressed in normal cells but absent in AmB-R cells . The other (B), which did not possess the spliced leader sequence, was weakly expressed in normal cells but strongly expressed in AmB-R cells . As a functional test, ERG6 null mutant Saccharomyces cerevisiae yeasts were transformed using the pYES2.1 TOPO TA expression vector containing the candidate SCMT1/ERG6 coding sequence cloned from L . donovani . The transformed yeasts exhibited C-24 alkylated sterol expression, mainly ergosterol, within their membranes, proving that the isolated cDNA encodes on a SCMT responsible for sterol methylation . In AmB-R L . donovani promastigotes, the absence of the normal transcript (A) and the expression of an abnormal species (B) devoid of a spliced leader could explain the absence of sterol methylation in these cells . Further studies using a homologous system will allow us to draw conclusions about the relationship between SCMT expression and AmB resistance in Leishmania.

Electrophoresis, 2004 Jun, 25(12), 1765 - 71
Chamber with modifiable inner width for performing transversal alternating field electrophoresis in variable numbers of minigels; Riveron AM et al.; We present a transversal alternating field electrophoresis chamber that allows modifiable inner widths to accommodate low- or high-throughput formats, with 7.8 cm opposite electrode separation and 30.4 cm electrode length . Removable slotted sheets divide the chamber into four smaller compartments, each one supporting a minigel of 3.85 cm in length and 7.1 cm in width . Replacements of slotted sheets with solid dielectric blocks with the sizes and shapes of compartments permit to occlude chamber compartments, changing from 4 to 1 the numbers of minigels per run, from 88 to 13 the maximum numbers of samples, and from 1688 to 422 mL the volume of buffer poured into the chamber . Saccharomyces cerevisiae chromosomes gave its characteristic DNA band pattern in all compartments, whereas migrations of DNA molecules are not affected by the occlusion of compartments.

Mol Pharmacol, 2004 Jul, 66(1), 153 - 60
Enhanced 7-ethyl-10-hydroxycamptothecin (SN-38) lethality by methylselenocysteine is associated with Chk2 phosphorylation at threonine-68 and down-regulation of Cdc6 expression; Yin MB et al.; Methylselenocysteine (MSC) is an organic selenium compound in preventative clinical trials involving prostate, lung, and colon carcinoma . We found that methioninase-activated MSC potentiates 7-ethyl-10-hydroxycamptothecin (SN-38)-induced cell lethality in vitro in the p53-defective human head and neck carcinoma A253 cells . Activated MSC increases chk2 phosphorylation at threonine-68 induced by SN-38, with no significant effect on chk1 phosphorylation . Cell cycle arrest induced by SN-38, however, was not abrogated or potentiated by MSC . These results suggest that the enhanced cellular lethality of SN-38 by MSC was not associated with cell cycle regulation pathways . Because chk2, in addition to its role in cell cycle arrest, can induce apoptosis by phosphorylation/activation, we examined whether increased chk2 phosphorylation could induce preapoptotic DNA fragmentation . DNA damage analysis showed that megabase DNA fragmentation is decreased, accompanied by the increased 30 to 300 kilobase pairs of DNA fragmentation after exposure to SN-38 with MSC, compared with SN-38 alone . No significant changes in the amount of DNA fragments were observed in cells treated with SN-38 or MSC alone . Moreover, proteolytic destruction of DNA replication-associated proteins cdc6, MCM2, and cdc25A may induce a DNA damage checkpoint response . The observed down-regulation of DNA replication proteins cdc6, MCM2, and cdc25A after exposure to SN-38 with MSC further indicates a relationship between drug response and DNA damage . Exposure to SN-38 with MSC resulted in a significant increase of poly(ADP-ribose) polymerasecleavage and caspase 3 activation . All together, the data support the hypothesis that enhanced lethality of this combination is associated with increased chk2 phosphorylation at Thr68 and down-regulation of specific DNA replication-associated proteins, which result in poly(ADP-ribose) polymerase cleavage, caspase 3 activation, and the induction of 30 to 300 kilobase pairs of DNA fragmentation.

J Biochem (Tokyo), 2004 Jun, 135(6), 695 - 700
Human histone deacetylase SIRT2 interacts with the homeobox transcription factor HOXA10; Bae NS et al.; Histone deacetylases are required for transcriptional repression in eukaryotes . Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution . Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2 . Here we show for the first time that SIRT2 interacts with the homeobox transcription factor, HOXA10, which was identified in a two-hybrid screen . Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts . Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development.

J Biol Chem, 2004 Aug 27, 279(35), 36462 - 9 Epub 2004 Jun 22.
DNA repair defects channel interstrand DNA cross-links into alternate recombinational and error-prone repair pathways; Saffran WA et al.; The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR) . In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR . An alternate error-prone repair pathway generates mutations at cross-link sites . We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations . Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells . Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast . Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any cross-link-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast . Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent error-prone pathways, one NER-dependent and one NER-independent.

Proteins, 2004 Aug 1, 56(2), 354 - 66
Predictive reconstruction of the mitochondrial iron-sulfur cluster assembly metabolism: I . The role of the protein pair ferredoxin-ferredoxin reductase (Yah1-Arh1); Alves R et al.; Adrenodoxin reductase homologue (Arh1) and yeast adrenodoxin homologue (Yah1) are essential Saccharomyces cerevisiae mitochondrial proteins involved in heme A biosynthesis and in iron-sulfur cluster (FeSC) assembly . Although the role of Arh1 and Yah1 in heme A biosynthesis is fairly well established, their systemic role on FeSC synthesis is not well understood . Also, while it is thought that the reductase Arh1 provides electrons for the ferredoxin Yah1, two hybrid experiments do not show interaction between the two proteins . In the first part of this article, we use structural bioinformatics methods to evaluate the possibility of interaction between Arh1 and Yah1 . Using protein model building and docking algorithms, we predict a complex between Arh1 and Yah1 that is similar to that of their bovine homologues (adrenodoxin reductase-adrenodoxin), suggesting that Arh1 can indeed reduce Yah1 . The predicted complex allows us to suggest point mutations to either molecule that could hinder Arh1-Yah1 interaction and test the role of Arh1 as the reductase for Yah1 . In the second part of this article, we investigate the physiological role of Arh1-Yah1 on FeSC assembly by deriving alternative mathematical models of the process, based on published information . Comparing the dynamical behavior of each model with that observed in reported experiments emphasizes the importance of Arh1-Yah1 providing electrons for in situ FeSC repair . Only when this mode of action of either of the two proteins in FeSC synthesis is considered can previously reported results be reproduced .

Proteins, 2004 Aug 1, 56(2), 188 - 200
Automatic target selection for structural genomics on eukaryotes; Liu J et al.; A central goal of structural genomics is to experimentally determine representative structures for all protein families . At least 14 structural genomics pilot projects are currently investigating the feasibility of high-throughput structure determination; the National Institutes of Health funded nine of these in the United States . Initiatives differ in the particular subset of "all families" on which they focus . At the NorthEast Structural Genomics consortium (NESG), we target eukaryotic protein domain families . The automatic target selection procedure has three aims: 1) identify all protein domain families from currently five entirely sequenced eukaryotic target organisms based on their sequence homology, 2) discard those families that can be modeled on the basis of structural information already present in the PDB, and 3) target representatives of the remaining families for structure determination . To guarantee that all members of one family share a common foldlike region, we had to begin by dissecting proteins into structural domain-like regions before clustering . Our hierarchical approach, CHOP, utilizing homology to PrISM, Pfam-A, and SWISS-PROT chopped the 103,796 eukaryotic proteins/ORFs into 247,222 fragments . Of these fragments, 122,999 appeared suitable targets that were grouped into >27,000 singletons and >18,000 multifragment clusters . Thus, our results suggested that it might be necessary to determine >40,000 structures to minimally cover the subset of five eukaryotic proteomes .

J Biol Chem, 2004 Sep 3, 279(36), 37751 - 62 Epub 2004 Jun 21.
The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions; Wendler P et al.; 26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins . As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus . 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes . We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway . Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta . Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed . Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs . The Rpt2 NLS deletion yielded wild type localization . However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function . Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta.

Cell, 2004 Jun 25, 117(7), 887 - 98
Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5; de Bruin RA et al.; G1-specific transcriptional activation by Cln3/CDK initiates the budding yeast cell cycle . To identify targets of Cln3/CDK, we analyzed the SBF and MBF transcription factor complexes by multidimensional protein interaction technology (MudPIT) . Whi5 was identified as a stably bound component of SBF but not MBF . Inactivation of Whi5 leads to premature expression of G1-specific genes and budding, whereas overexpression retards those processes . Whi5 inactivation bypasses the requirement for Cln3 both for transcriptional activation and cell cycle initiation . Whi5 associates with G1-specific promoters via SBF during early G1 phase, then dissociates coincident with transcriptional activation . Dissociation of Whi5 is promoted by Cln3 in vivo . Cln/CDK phosphorylation of Whi5 in vitro promotes its dissociation from SBF complexes . Mutation of putative CDK phosphorylation sites, at least five of which are phosphorylated in vivo, strongly reduces SBF-dependent transcription and delays cell cycle initiation . Like mammalian Rb, Whi5 is a G1-specific transcriptional repressor antagonized by CDK.

Cell, 2004 Jun 25, 117(7), 849 - 50
RB from a bud's eye view; Schaefer JB et al.; RB and related proteins block transcriptional activation of genes critical to initiation of the cell cycle and suppress unwanted cell division . The circuitry controlling this response is generally conserved from humans to yeast, but no negative regulator like RB has been found in yeast . In this issue of Cell, two studies reveal that Whi5 appears to play the role of RB in preventing precocious cell cycle entry in budding yeast.

Nat Cell Biol, 2004 Jul, 6(7), 634 - 41 Epub 2004 Jun 20.
Proteolysis-independent regulation of the transcription factor Met4 by a single Lys 48-linked ubiquitin chain; Flick K et al.; The ubiquitin ligase SCF(Met30) is required for cell cycle progression in budding yeast . The critical function of SCF(Met30) is inactivation of the transcriptional activator Met4 . Here we show that a single ubiquitin chain is attached to Met4 through lysine at position 163 . Inhibition of Met4 ubiquitination by mutating lysine to arginine at this position constitutively activates, but does not stabilize, Met4 . This supports a proteolysis-independent role of Cdc34-SCF(Met30)-catalysed Met4 ubiquitination . Surprisingly, the ubiquitin chain attached to Met4 is linked through Lys 48 in ubiquitin, a ubiquitin chain structure that is usually required for substrate targeting to the 26S proteasome . These results suggest that Lys 48-linked ubiquitin chains can have a regulatory role independent of proteolysis.

Ann N Y Acad Sci, 2004 May, 1020, 212 - 26
Bayesian decomposition: analyzing microarray data within a biological context; Ochs MF et al.; The detection and correct identification of cancer, especially at an early stage, are vitally important for patient survival and quality of life . Since signaling pathways play critical roles in cancer development and metastasis, methods that reliably assess the activity of these pathways are critical to understand cancer and the response to therapy . Bayesian Decomposition (BD) identifies signatures of expression that can be linked directly to signaling pathway activity, allowing the changes in mRNA levels to be used as downstream indicators of pathway activity . Here, we demonstrate this ability by identifying the downstream expression signal associated with the mating response in Saccharomyces cerevisiae and showing that this signal disappears in deletion mutants of genes critical to the MAPK signaling cascade used to trigger the response . We also show the use of BD in the context of supervised learning, by analyzing the Mus musculus tissue-specific data set provided by Project Normal . The algorithm correctly removes routine metabolic processes, allowing tissue-specific signatures of expression to be identified . Gene ontology is used to interpret these signatures . Since a number of modern therapeutics specifically target signaling proteins, it is important to be able to identify changes in signaling pathways in order to use microarray data to interpret cancer response . By removing routine metabolic signatures and linking specific signatures to signaling pathway activity, BD makes it possible to link changes in microarray results to signaling pathways.

Biochem Biophys Res Commun, 2004 Jul 16, 320(1), 197 - 204
In silico characterization of the INO80 subfamily of SWI2/SNF2 chromatin remodeling proteins; Bakshi R et al.; Proteins belonging to SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression . The yeast Ino80, the catalytic ATPase subunit of the INO80 complex, is the most recently described member of the SNF2 family . Outside the conserved ATPase domain, it has very little similarity with other well-characterized SNF2 proteins hence it is believed to represent a new subfamily . We have identified new members of this subfamily in different organisms and have detected characteristic features of this subfamily . Using various data mining tools we have identified a new, previously undetected domain in all members of this subfamily . This domain designated DBINO is characteristic of the INO80 subfamily and is predicted to have DNA-binding function . The presence of this domain in all the INO80 subfamily proteins from different organisms suggests its conserved function in evolution.

Curr Biol, 2004 Jun 22, 14(12), R483 - 5
Membrane targeting: getting Arl to the Golgi; Graham TR; Post-translational modification with myristoyl or prenyl groups is essential for membrane association of many small GTPases in the Ras superfamily . Two recent papers show that, rather than myristoylation, amino-terminal acetylation of the Arf-like protein Arl3p is required for Golgi targeting via an interaction with an integral membrane protein called Sys1.

Neurochem Res, 2004 Jul, 29(7), 1405 - 9
Syntaxin 1A and receptor for activated C kinase interact with the N-terminal region of human dopamine transporter; Lee KH et al.; The dopamine transporter (DAT) regulates the extent and duration of dopamine receptor activation through sodium-dependant reuptake of dopamine into presynaptic neurons, resulting in termination of dopaminergic neurotransmission . Using the yeast two-hybrid system, we have identified novel interactions between DAT, the SNARE protein syntaxin 1A, and the receptor for activated C kinases (RACK1) . This association involves the intracellular N-terminal domain of human DAT (hDAT) . Our data suggest that hDAT may exist as dimers or oligomers and that its protein-protein interactions with syntaxin 1A and RACK1 form functional regulatory complexes that may mediate DAT trafficking through modulation of hDAT phosphorylation by PKC.

Nature, 2004 Jun 17, 429(6993), 724 - 30
Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex; Bowman GD et al.; Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases . Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA) . Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring . Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix . This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.

Toxicol Sci, 2004 Sep, 81(1), 133 - 8 Epub 2004 Jun 16.
TCDD affects DNA double strand-break repair; Chan CY et al.; 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant, elicits a spectrum of deleterious biological responses including carcinogenesis . We hypothesize that TCDD exposure exerts its carcinogenicity, in part, by affecting the repair of DNA double strand breaks (DSBs) through homologous recombination (HR), mediated by the AhR signaling pathway . To investigate this hypothesis we used a Chinese hamster ovary (CHO) cell line (CHO 33) containing a neo direct repeat recombination reporter substrate to determine whether TCDD affects DNA DSB repair . The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI was used to induce a site specific DSB within the upstream neo recombination substrate in the CHO 33 cells . The cells were then exposed to 500 pM of TCDD in the presence or absence of the AhR antagonist alpha-naphthoflavone (0.1 microM) for 24 h . Two weeks later HR frequencies were determined by counting the number of functional neo expressing, G418-resistant colonies per live cells plated . TCDD significantly increased HR frequency, demonstrating that it does in fact modulate the repair of DNA DSBs . Southern blot analysis of G418-resistant colonies using a cDNA neo probe determined that both gene conversion and gene deletion HR events occurred as a result of DNA DSB repair and TCDD exposure . Exposure of cells to alpha-naphthoflavone resulted in a significant decrease in TCDD-induced HR frequency . These results demonstrate that TCDD, potentially acting via the AhR, can modulate HR repair of DNA DSBs in CHO 33 cells.

Proc Natl Acad Sci U S A, 2004 Jun 22, 101(25), 9315 - 20 Epub 2004 Jun 15.
Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1; Sugawara N et al.; Recombination between moderately divergent DNA sequences is impaired compared with identical sequences . In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences . A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences . This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant . In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type . Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta mlh3 Delta triple mutant resembled mlh1 Delta . However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6 . An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded . This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.

Mol Cell Biol, 2004 Jul, 24(13), 6029 - 39
Molecular requirements for gene expression mediated by targeted histone acetyltransferases; Jacobson S et al.; Histone acetyltransferases (HATs) play fundamental roles in regulating gene expression . HAT complexes with distinct subunit composition and substrate specificity act on chromatin-embedded genes with different promoter architecture and chromosomal locations . Because requirements for HAT complexes vary, a central question in transcriptional regulation is how different HAT complexes function in different chromosomal contexts . Here, we have tested the ability of targeted yeast HATs to regulate gene expression of an epigenetically silenced locus . Of a panel of HAT fusion proteins targeted to a telomeric reporter gene, Sas3p and Gcn5p selectively increased expression of the silenced gene . Reporter gene expression was not solely dependent on acetyltransferase activity of the targeted HAT . Further analysis of Gcn5p-mediated gene expression revealed collateral requirements for HAT complex subunits Spt8p and Spt3p, which interact with TATA-binding protein, and for a gene-specific transcription factor . These data demonstrate plasticity of gene expression mediated by HATs upon encountering novel promoter architecture and chromatin context . The telomeric location of the reporter gene used in these studies also provides insight into the molecular requirements for heterochromatin boundary formation and for overcoming transcriptional silencing.

Mol Cell Biol, 2004 Jul, 24(13), 5639 - 49
Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression; Yokoyama A et al.; MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias . Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression . Here we report the biochemical purification of MLL and demonstrate that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein . Two other members of the novel MLL complex identified here are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties . Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex . Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression . Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components . These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis.

Extremophiles, 2004 Jun, 8(3), 253 - 8 Epub 2004 Apr 09.
Identification of replication origins in the genome of the methanogenic archaeon, Methanocaldococcus jannaschii; Zhang R et al.; Methanocaldococcus jannaschii has been notorious as an archaeon in which the replication origins are difficult to identify . Although extensive efforts have been exerted on this issue, the locations of replication origins still remain elusive 7 years after the publication of its complete genome sequence in 1996 . Ambiguous results were obtained in identifying the replication origins of M . jannaschii based on all theoretical and experimental approaches . In the genome of M . jannaschii, we found that an ORF (MJ0774), annotated as a hypothetical protein, is a homologue of the Cdc6 protein . The position of the gene is at a global minimum of the x component of the Z curve, i.e., RY disparity curve, which has been used to identify replication origins in other Archaea . In addition, an intergenic region (694,540-695,226 bp) that is between the cdc6 gene and an adjacent ORF shows almost all the characteristics of known replication origins, i.e., it is highly rich in AT composition (80%) and contains multiple copies of repeat elements and AT stretches . Therefore, these lines of evidence strongly suggest that the identified region is a replication origin, which is designated as oriC1 . The analysis of the y component of the Z curve, i.e., MK disparity curve, suggests the presence of another replication origin corresponding to one of the peaks in the MK disparity curve at around 1,388 kb of the genome .

Planta, 2004 Sep, 219(5), 856 - 66 Epub 2004 Jun 10.
Localization to the rhizoid tip implicates a Fucus distichus Rho family GTPase in a conserved cell polarity pathway; Fowler JE et al.; Generation and expression of cell polarity in brown algal zygotes of the Fucales involve regulation of the actin cytoskeleton and localized secretion . We used degenerate PCR to isolate cDNAs that encode two small GTPases, FdRac1 and FdRab8, from zygotes of Fucus distichus (L.) Powell . Sequence analysis placed FdRac1 in the Rho family, which regulates actin, and FdRab8 in the Rab family, which regulates vesicle transport . As expected, bacterially expressed forms of both proteins bound GTP in vitro . When expressed in budding yeast, FdRac1 showed some functional overlap with CDC42, the Saccharomyces cerevisiae Rho family gene required for yeast cell polarity . Immunolocalization revealed an asymmetric distribution of FdRac1 in polarized zygotes and embryos, with FdRac1 concentrated at or near the growing tip of the algal rhizoid . Our data support the hypothesis that FdRac1 regulates algal cell polarity, possibly via the actin cytoskeleton . Because brown algae belong to the heterokont group, which diverged from other groups early in eukaryotic evolution, we argue that the Rho family function of regulating cell polarity is ancient and may extend throughout the eukaryotes.

Cell Mol Life Sci, 2004 Jun, 61(12), 1485 - 97
Molecular characterization of Arabidopsis PHO80-like proteins, a novel class of CDKA;1-interacting cyclins; Torres Acosta JA et al.; Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle . In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T) . A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs) . They all share a conserved central region of 100 amino acids ("cyclin box") displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T . brucei . The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast . The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments . P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues . The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.

Genome Res, 2004 Jul, 14(7), 1232 - 47 Epub 2004 Jun 14.
Local definition of Ty1 target preference by long terminal repeats and clustered tRNA genes; Bachman N et al.; LTR-containing retrotransposons reverse transcribe their RNA genomes, and the resulting cDNAs are integrated into the genome by the element-encoded integrase protein . The yeast LTR retrotransposon Ty1 preferentially integrates into a target window upstream of tDNAs (tRNA genes) in the yeast genome . We investigated the nature of these insertions and the target window on a genomic scale by analyzing several hundred de novo insertions upstream of tDNAs in two different multicopy gene families . The pattern of insertion upstream of tDNAs was nonrandom and periodic, with peaks separated by approximately 80 bp . Insertions were not distributed equally throughout the genome, as certain tDNAs within a given family received higher frequencies of upstream Ty1 insertions than others . We showed that the presence and relative position of additional tDNAs and LTRs surrounding the target tDNA dramatically influenced the frequency of insertion events upstream of that target .

Curr Opin Genet Dev, 2004 Apr, 14(2), 218 - 26
RNA polymerase II structure: from core to functional complexes; Cramer P; New structural studies of RNA polymerase II (Pol II) complexes mark the beginning of a detailed mechanistic analysis of the eukaryotic mRNA transcription cycle . Crystallographic models of the complete Pol II, together with new biochemical and electron microscopic data, give insights into transcription initiation . The first X-ray analysis of a Pol II complex with a transcription factor, the elongation factor TFIIS, supports the idea that the polymerase has a 'tunable' active site that switches between mRNA synthesis and cleavage . The new studies also show that domains of transcription factors can enter polymerase openings, to modulate function during transcription.

Curr Opin Genet Dev, 2004 Apr, 14(2), 147 - 54
The highly conserved and multifunctional NuA4 HAT complex; Doyon Y et al.; Histone acetyltransferase complexes have been shown to be key regulators of gene expression . Among these, the NuA4 complex, first characterized in yeast, stands out as it controls multiple key nuclear functions in eukaryotic cells . Many subunits of this protein assembly have been directly linked to global and targeted acetylation of histone H4 tails in vivo, regulation of transcription, cell-cycle progression as well as to the process of DNA repair . Recent studies presented here have established its remarkable structural conservation from yeast to human cells and contributed to the understanding of its diverse functions.

Curr Opin Genet Dev, 2004 Apr, 14(2), 139 - 46
Facts about FACT and transcript elongation through chromatin; Belotserkovskaya R et al.; The regulation of transcription elongation within the context of chromatin is a topic of great interest . Even though chromatin presents a barrier to transcription by the PolII machinery in vitro, this process is rather efficient in vivo . Importantly, the chromatin structure of the actively transcribed genes is altered as part of this process . A large number of factors implicated in the control of transcript elongation have been identified through genetics, biochemistry and targeted proteomics approaches . However the precise roles and mechanisms of action of these factors remain obscure . A significant advance came about this past year with the elucidation of the roles of FACT and Spt6 in transcription elongation . These factors facilitate PolII passage through chromatin by destabilizing the nucleosome structure as well as reassemble nucleosomes traversed by PolII.

J Biol Chem, 2004 Sep 3, 279(36), 37741 - 50 Epub 2004 Jun 11.
Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway; Meiling-Wesse K et al.; Atg21 and Atg18 are homologue yeast proteins . Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway . Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles . Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate . This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy . The intracellular localization of Atg21 is unique among the Atg proteins . In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions . Cells with a single vacuole show most of the protein at few perivacuolar punctae . This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex . In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure . Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded . Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors . However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.

Cancer Cell, 2004 Jun, 5(6), 519 - 23
Lost in translation: dysregulation of cap-dependent translation and cancer; Bjornsti MA et al.; Activation of the phosphatidylinositol 3' kinase-Akt pathway has long been associated with malignant transformation and antiapoptotic signaling . Mutations downstream of Akt that activate the TOR kinase are found in tumor-prone syndromes, while overexpression of translation initiation complex components, such as eIF4E, occurs frequently in human cancer . However, direct roles for TOR signaling or eIF4E overexpression, in the genesis of cancer, have been lacking . Recent papers, including one by in this issue of Cancer Cell, clearly establish that dysregulation of cap-dependent translation confers malignant characteristics and induces cancer by suppressing apoptosis, underscoring the potential of therapeutics that selectively target the Akt-TOR-eIF4E pathway.

Science, 2004 Jun 11, 304(5677), 1644 - 7
Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid; Loewen CJ et al.; Cells regulate the biophysical properties of their membranes by coordinated synthesis of different classes of lipids . Here, we identified a highly dynamic feedback mechanism by which the budding yeast Saccharomyces cerevisiae can regulate phospholipid biosynthesis . Phosphatidic acid on the endoplasmic reticulum directly bound to the soluble transcriptional repressor Opi1p to maintain it as inactive outside the nucleus . After the addition of the lipid precursor inositol, this phosphatidic acid was rapidly consumed, releasing Opi1p from the endoplasmic reticulum and allowing its nuclear translocation and repression of target genes . Thus, phosphatidic acid appears to be both an essential ubiquitous metabolic intermediate and a signaling lipid.

Boll Chim Farm, 2004 Mar, 143(2), 62 - 4
Pharmacokinetics of selenium following oral administration selenium preparation in rabbits; Szulc-Musiol B et al.; The aim of study was to investigate the bioavailability of selenium after oral administration of selenium yeast . As a reference preparation was used sodium selenite . The preparations were investigated in rabbits, according to a randomized two way crossover design in the fasted state . Each animal was given selenium preparation in the form of the single oral dose 0.5 mg Se/kg body weight . A washout period of one week separated both treatment periods . The selenium concentration was determined in serum spectrofluorometry . The divalent equation of one-compartment model was the simplest formula describing the course of selenium changes in serum of rabbits and giving the pharmacokinetic parameters . Pharmacokinetic variables (mean maximum plasma concentration, mean time to reach maximum plasma concentration, and the mean area under the plasma concentration-time curve) were not statistically different for the two preparations . It can be concluded that the two selenium preparations are likely to be bioequivalent.

J Biol Chem, 2004 Jul 30, 279(31), 32401 - 6 Epub 2004 Jun 09.
Inhibition of TATA binding protein dimerization by RNA polymerase III transcription initiation factor Brf1; Alexander DE et al.; The Brf1 subunit of TFIIIB plays an important role in recruiting the TATA-binding protein (TBP) to the up-stream region of genes transcribed by RNA polymerase III . When TBP is not bound to promoters, it sequesters its DNA binding domain through dimerization . Promoter assembly factors therefore might be required to dissociate TBP into productively binding monomers . Here we show that Saccharomyces cerevisiae Brf1 induces TBP dimers to dissociate . The high affinity TBP binding domain of Brf1 is not sufficient to promote TBP dimer dissociation but in addition requires the TFIIB homology domain of Brf1 . A model is proposed to explain how two distinct functional domains of Brf1 work in concert to dissociate TBP into monomers.

Eukaryot Cell, 2004 Jun, 3(3), 598 - 609
The signal from the initiation of meiotic recombination to the first division of meiosis; Malone RE et al.; Two of the unique events that occur in meiosis are high levels of genetic recombination and the reductional division . Our previous work demonstrated that the REC102, REC104, REC114, and RAD50 genes, required to initiate meiotic recombination in Saccharomyces cerevisiae, are needed for the proper timing of the first meiotic (MI) division . If these genes are absent, the MI division actually begins at an earlier time . This paper demonstrates that the meiotic recombination genes MER2/REC107, SPO11, and MRE2 and the synaptonemal complex genes HOP1 and RED1 are also required for the normal delay of the MI division . A rec103/ski8 mutant starts the MI division at the same time as in wild-type cells . Our data indicate no obvious correlation between the timing of premeiotic S phase and the timing of the first division in Rec- mutants . Cells with rec102 or rec104 mutations form MI spindles before wild-type cells, suggesting that the initiation signal acts prior to spindle formation . Neither RAD9 nor RAD24 is needed to transduce the signal, which delays the first division . The timing of the MI division in RAD24 mutants indicates that the pachytene checkpoint is not active in Rec+ cells and suggests that the coordination between recombination and the MI division in wild-type cells may occur primarily due to the initiation signal . Finally, at least one of the targets of the recombination initiation signal is the NDT80 gene, a transcriptional regulator of middle meiotic gene expression required for the first division .

Annu Rev Biochem, 2004, 73, 177 - 208
Regulation of telomerase by telomeric proteins; Smogorzewska A et al.; Telomeres are essential for genome stability in all eukaryotes . Changes in telomere functions and the associated chromosomal abnormalities have been implicated in human aging and cancer . Telomeres are composed of repetitive sequences that can be maintained by telomerase, a complex containing a reverse transcriptase (hTERT in humans and Est2 in budding yeast), a template RNA (hTERC in humans and Tlc1 in yeast), and accessory factors (the Est1 proteins and dyskerin in humans and Est1, Est3, and Sm proteins in budding yeast) . Telomerase is regulated in cis by proteins that bind to telomeric DNA . This regulation can take place at the telomere terminus, involving single-stranded DNA-binding proteins (POT1 in humans and Cdc13 in budding yeast), which have been proposed to contribute to the recruitment of telomerase and may also regulate the extent or frequency of elongation . In addition, proteins that bind along the length of the telomere (TRF1/TIN2/tankyrase in humans and Rap1/Rif1/Rif2 in budding yeast) are part of a negative feedback loop that regulates telomere length . Here we discuss the details of telomerase and its regulation by the telomere.

Curr Protein Pept Sci, 2004 Jun, 5(3), 201 - 11
Deubiquitinating enzymes are IN/(trinsic to proteasome function); Guterman A et al.; Covalent conjugation of the ubiquitin tag to cellular proteins plays a central role in a number of processes, the most notable among them being degradation by the 26S proteasome . A fundamental property of this process is that ubiquitination, in contrast to subsequent degradation, is reversible due to a number of deubiquitinating enzymes that mediate the disassembly of ubiquitin-protein conjugates . The uniqueness of ubiquitin as a reversible tag necessitates mechanisms to guarantee its efficiency . Interestingly, some deubiquitinating enzymes are associated with the 26S proteasome itself . We include a brief overview of the key proteasome-associated deubiquitinating enzymes such as Rpn11/POH1, UCH37/Uch2, Ubp6/Usp14 and Doa4/Ubp4 . We go on to discuss how these enzymes may contribute to, or possibly counteract, proteolysis by the proteasome . For example, cumulative evidence points to a partitioning of proteasome action between proteolysis and deubiquitination . On the one hand, inhibition of proteolysis promotes deubiquitination, while on the other hand, inhibition of deubiquitination can promote proteolysis . The plethora of deubiquitinating enzymes may serve as proof reading devices altering the equilibrium between these two processes and allowing for reversal of fortune at various stages of the process . To promote degradation over deubiquitination, certain polyubiquitin conformations could be stabilized or protected from deubiquitinating enzymes in order that they can serve as efficient targeting signals leading to the proteasome . We hypothesize that polvubiquitin chains could also serve as "timers": by slowing down chain disassembly, longer chains allow ample time for unfolding and proteolysis of the substrate.

Proteomics, 2004 May, 4(5), 1433 - 8
Differential phosphoproteome profiling by affinity capture and tandem matrix-assisted laser desorption/ionization mass spectrometry; Metodiev MV et al.; Protein phosphorylation is a ubiquitous post-translational modification that affects a significant subset of the proteome and plays an especially important role in signal transduction and cell cycle control in eukaryotic organisms . Recently developed methods that couple multidimensional liquid chromatography to electrospray mass spectrometers can be used to analyze entire phosphoproteomes . However, they require considerable investments and technical skills that are only available in a few highly specialized laboratories . These methods also appear to be biased . Statistical analyses show that peptides from abundant proteins and multiply phosphorylated peptides are disproportionately identified . We describe an economic alternative that utilizes a phospho-affinity step to isolate the intact phosphoproteins . These are subsequently characterized by electrophoresis and identified by direct de novo sequencing using tandem mass spectrometry . We applied this technique to probe signal-induced changes in the phosphoproteome of human U937 cells, and found that the pools of two cancer-related phosphoproteins implicated in intracellular hormones signaling are dramatically altered in the course of monocyte to macrophage differentiation.

J Immunol, 2004 Jun 15, 172(12), 7548 - 55
Bap29/31 influences the intracellular traffic of MHC class I molecules; Paquet ME et al.; In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER) . We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with beta(2)-microglobulin and maintained until the class I molecule has left the ER . We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation . Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I . Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats . This observation is also associated with a delay in class I traffic from ER to Golgi . Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.

Cell, 2004 Jun 11, 117(6), 721 - 33
Mapping global histone acetylation patterns to gene expression; Kurdistani SK et al.; Histone acetyltransferases and deacetylases with specificities for different sites of acetylation affect common chromatin regions . This could generate unique patterns of acetylation that may specify downstream biological processes . To search for existence of these patterns and their relationship to gene activity, we analyzed the genome-wide acetylation profiles for eleven lysines in the four core histones of Saccharomyces cerevisiae . We find that both hyper- and hypoacetylation of individual lysines are associated with transcription, generating distinct patterns of acetylation that define groups of biologically related genes . The genes within these groups are significantly coexpressed, mediate similar physiological processes, share unique cis-regulatory DNA motifs, and are enriched for binding of specific transcription factors . Our data also indicate that the in vivo binding of the transcription factor Bdf1 is associated with acetylation on most lysines but relative deacetylation on H4 lysine 16 . Thus, certain acetylation patterns may be used as surfaces for specific protein-histone interactions, providing one mechanism for coordinate regulation of chromatin processes that are biologically related.

Curr Biol, 2004 May 25, 14(10), R397 - 9
Peroxisome membrane biogenesis: the stage is set; Schliebs W et al.; Pex3p and Pex19p are key players in the post-translational import of peroxisomal membrane proteins . New data suggest that these peroxins act in tandem, Pex19p as a cytosolic chaperone and import receptor for peroxisomal membrane proteins, and Pex3p as docking factor at the peroxisomal membrane.

Nat Biotechnol, 2004 Jul, 22(7), 871 - 6 Epub 2004 Jun 06.
Temperature-sensitive control of protein activity by conditionally splicing inteins; Zeidler MP et al.; Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function . Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems . However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use . Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins . We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80 . We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster . Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.

Free Radic Biol Med, 2004 Jul 1, 37(1), 23 - 35
Genome-wide transcriptional responses to a lipid hydroperoxide: adaptation occurs without induction of oxidant defenses; Alic N et al.; Free radicals can initiate the oxidation of polyunsaturated fatty acids in cells through the process of lipid peroxidation . The genome-wide transcriptional changes in Saccharomyces cerevisiae after treatment with the toxic lipid peroxidation product linoleic acid hydroperoxide (LoaOOH) were identified . High-dose treatment led to a switch in transcription from biosynthetic to protective functions . This response encompassed a set of genes stimulated predominantly by LoaOOH, and not by other oxidants or heat shock, which contained components of the pleiotropic drug resistance system . The dose dependence of the transcriptional response revealed that large and widespread changes occur only in response to higher doses . Pretreatment of cells with sublethal doses of LoaOOH induces resistance to an otherwise lethal dose through the process of adaptation . Adaptive doses elicited a more subtle transcriptional response affecting metabolic functions, including an increase in the capacity for detoxification and downregulation of the rate of protein synthesis . Surprisingly, the cellular response to adaptive doses did not include induction of oxidative-stress defense enzymes nor of transcripts involved in general cellular defense systems .

Biol Cell, 2004 Apr, 96(3), 193 - 9
An overview of the KIN1/PAR-1/MARK kinase family; Tassan JP et al.; Members of the KIN1/PAR-1/MARK kinase family are conserved from yeast to humans and share a similar primary structural organization . Several kinases of this family appear to be at the crossroads of various biological functions including cell polarity, cell cycle control, intracellular signalisation, microtubules stability and protein stability . Here we present an overview of known roles of KIN1/PAR-1/MARK kinases including pEg3 a newly identified member which is regulated during the cell cycle and is a potential regulator of the cell cycle progression . Some common modes of action can be deciphered for this protein kinase family.

Curr Biol, 2004 Jun 8, 14(11), 1002 - 6
A barrier to lateral diffusion in the cleavage furrow of dividing mammalian cells; Schmidt K et al.; Barriers to diffusion of proteins and lipids play an important role in generating functionally specialized regions of the plasma membrane . Such barriers have been reported at the base of axons, at the bud neck in Saccharomyces cerevisiae, as well as at the tight junctions of epithelia . How diffusion barriers are formed and how they effect behavior of both inner and outer leaflets of the bilayer are not fully understood . Here, we provide evidence for a cortical barrier to diffusion within the cleavage furrow of mammalian cells . Photobleaching-based assays were used to measure diffusion of three membrane proteins with differing topologies and putative lipid raft association, as well as the lipid analog dialkylindocarbocyanine (DiI C18, ), across the cleavage furrow . There was a block in diffusion of proteins with a cytosolic domain, but not of proteins anchored in the outer leaflet of the PM or of DiI . Diffusion of lipid raft proteins in the inner and outer leaflets of the membrane was not directly coupled . The distribution of Septin proteins, as opposed to cortical actin, was consistent with a functional role in limiting diffusion .

Plant Physiol, 2004 Jun, 135(2), 1027 - 39 Epub 2004 Jun 04.
A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis; Song WY et al.; Cadmium (Cd) is a widespread pollutant that is toxic to plant growth . However, only a few genes that contribute to Cd resistance in plants have been identified . To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1 . This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs) . Database searches revealed that there are nine close homologs in Arabidopsis . Homologs were also found in other plants . Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1 . AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis . Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II) . AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd . Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants.

Bioinformatics, 2004 Nov 22, 20(17), 3013 - 20 Epub 2004 Nov 22.
Protein complex prediction via cost-based clustering; King AD et al.; MOTIVATION: Understanding principles of cellular organization and function can be enhanced if we detect known and predict still undiscovered protein complexes within the cell's protein-protein interaction (PPI) network . Such predictions may be used as an inexpensive tool to direct biological experiments . The increasing amount of available PPI data necessitates an accurate and scalable approach to protein complex identification . RESULTS: We have developed the Restricted Neighborhood Search Clustering Algorithm (RNSC) to efficiently partition networks into clusters using a cost function . We applied this cost-based clustering algorithm to PPI networks of Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans to identify and predict protein complexes . We have determined functional and graph-theoretic properties of true protein complexes from the MIPS database . Based on these properties, we defined filters to distinguish between identified network clusters and true protein complexes . Conclusions: Our application of the cost-based clustering algorithm provides an accurate and scalable method of detecting and predicting protein complexes within a PPI network.

Curr Protein Pept Sci, 2004 Jun, 5(3), 177 - 84
Cullin-based ubiquitin ligase and its control by NEDD8-conjugating system; Chiba T et al.; Several studies have examined the importance of ubiquitin-like posttranslational modifiers (which consist of an unexpectedly large family) . Of these, NEDD8 (also called Rub1, related to ubiquitin 1) with a high homology to ubiquitin is covalently linked to all members of cullin (Cul)-family proteins through an enzymatic cascade analogous to ubiquitylation . Cul-family proteins are scaffold proteins for a wide series of ubiquitin-protein ligase complexes, such as SCFs (Skp1, Cul-1, Roc1, and F-box proteins), which regulate the degradation of broad range of cellular proteins . Unlike ubiquitin, which mostly acts as a degradation signal for the target proteins, NEDD8 acts as an activation signal for Cul-family proteins; i.e., Cul-based ubiquitin-protein ligases . Accordingly, the NEDD8 conjugation pathway regulating Cul-protein function is responsible for a diverse array of biologically important processes, such as the cell cycle progression, signalling cascades and developmental programs . Furthermore, recent studies have revealed that the COP9/Signalosome complex interacts physically and genetically with Cul-family proteins, and catalyzes deconjugation of NEDD8 ligated to Cul-family proteins . This review summarizes recent advances in biochemical and genetic studies on how the NEDD8-modifying system regulates Cul-family proteins and their physiology.

Mol Cells, 2004 Apr 30, 17(2), 223 - 9
Interaction of tomato mosaic virus movement protein with tobacco RIO kinase; Yoshioka K et al.; Tomato mosaic virus (ToMV) has a regulatory gene encoding a movement protein (MP) that is involved in the cell-to-cell movement of viral RNA through plasmodesmata . To identify the host cell factors interacting with ToMV MP, we used a recombinant MP probe to isolate cDNA clones from a phage expression library of Nicotiana tabacum by a far-Western screening method . One of the cDNA clones encoded an MP-interacting protein, MIP-T7, homologous to the yeast novel protein kinase, Rio1p . We isolated a full-length cDNA by RT-PCR . The putative gene product was designated NtRIO, and shared 33 and 73% amino acid identity with yeast and Arabidopsis RIO kinases, respectively . In vitro analyses using recombinant proteins showed that NtRIO also interacted with a different MP derived from Cucumber mosaic virus . NtRIO had autophosphorylation activity and phosphorylated ToMV MP . Addition of recombinant tobacco casein kinase 2 resulted in a marked increase in the phosphorylation of NtRIO . The interaction between NtRIO and ToMV MP was inhibited by phosphorylation of NtRIO.

Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 8987 - 92 Epub 2004 Jun 03.
Spatiotemporal retinoid-X receptor activation detected in live vertebrate embryos; Luria A et al.; Most studies on the nuclear retinoid-X receptor (RXR) have focused on its role as a heterodimeric partner but less about its own activation pattern during development and the distribution of potential endogenous ligands . The aim of this study is to visualize the distribution of activated RXRalpha in live transgenic Xenopus laevis embryos across a wide range of developmental stages . We adopted a nuclear receptor-Gal4 fusion/upstream activation sequence-based reporter system for our assay . Strong activation of the RXRalpha ligand-binding domain was observed in a segment of the spinal cord just posterior to the hindbrain . This activation is first detected in neurula stage embryos and persists up to swimming tadpole stages, after which activation strongly declines . Addition of exogenous ligands, such as 9-cis retinoic acid or all-trans retinoic acid, expands the activation of RXR throughout the spinal cord but not in the brain, whereas the RXR-specific ligand LG268 expanded the Gal4-RXR activation into the brain and olfactory epithelia . Treatment with the RAR-specific ligand 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid or thyroid hormone had no effect on Gal4-RXR activation, whereas these compounds significantly increased their corresponding Gal4/receptor fusion proteins under similar conditions . Embryos expressing a Gal4-RXR fusion protein with a deletion in the ligand-dependent activation domain (AF2) show no reporter gene activation . The results shown in this paper reveal a specific activation pattern for Gal4-RXRalpha specifically in the developing spinal cord and suggest the existence of RXR ligand "hot-spots" in this region.

J Biol Chem, 2004 Aug 6, 279(32), 33456 - 62 Epub 2004 Jun 03.
Isohumulones, bitter acids derived from hops, activate both peroxisome proliferator-activated receptor alpha and gamma and reduce insulin resistance; Yajima H et al.; The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism . The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPARalpha and -gamma, respectively . In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPARalpha and -gamma in transient co-transfection studies . Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPARalpha and -gamma . Diabetic KK-Ay mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone . Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group) . C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance . Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes . A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0) . These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes.

J Biol Chem, 2004 Jul 30, 279(31), 32373 - 84 Epub 2004 Jun 03.
PINA is essential for growth and positively influences NIMA function in Aspergillus nidulans; Joseph JD et al.; The phospho-Ser/Thr-directed prolyl-isomerase Pin1 was originally identified in vertebrate systems as a negative regulator of NIMA, a Ser/Thr protein kinase that regulates the G(2)/M transition in Aspergillus nidulans . Here we explore the physiological roles of the Pin1 orthologue, PINA, in A . nidulans and evaluate the relevance of the interaction of PINA with NIMA in this fungus . We find pinA to be an essential gene in A . nidulans . In addition, when PINA levels are reduced 50-fold the cells grow at a reduced rate . Upon germination under conditions that repress PINA expression, the cells are delayed in the interphase activation of NIMX(cdc2), whereas they traverse the other phases of the cell cycle at a similar rate to controls . These results indicate that a marked reduction of PINA results in a lengthening of G(1) . Additionally, PINA repression increases the rate at which the cells enter mitosis following release from a hydroxyurea arrest without altering the sensitivity of the fungus to agents that activate the replication or DNA damage checkpoints . In contrast to predictions based on Pin1, the physical interaction between PINA and NIMA is primarily dependent upon the prolylisomerase domain of PINA and the C-terminal 303 amino acids of NIMA . Finally, reduction of PINA levels exacerbates the nimA5 temperature-sensitive mutant, whereas overexpression of PINA decreases the severity of this mutation, results that are consistent with a positive genetic interaction between PINA and NIMA . Thus, although PINA is essential and positively regulates NIMA function, A . nidulans is most sensitive to a reduction in PINA concentration in G(1) rather than in G(2)/M.

Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 456 - 63
Dibasic amino acid residues at the carboxy-terminal end of kinase homology domain participate in the plasma membrane localization and function of phosphatidylinositol 5-kinase gamma; Arioka M et al.; Type I phosphatidylinositol 4-phosphate (PI(4)P) 5-kinases (PIP5Ks) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic . The protein localizes to the plasma membrane where its activity has been shown to be regulated by small GTPase ARFs and/or phosphatidic acid . Deletion analysis of amino- or carboxy-terminal sequences of PIP5Kgamma fused with EGFP demonstrated that the presence of central kinase homology domain (KHD), a 380 amino acid-long region highly conserved among PIP5K family, was necessary and sufficient for the plasma membrane localization of PIP5Kgamma . Particularly, the dibasic Arg-Lys sequence located at the carboxy-terminal end of KHD was shown to be crucial for the plasma membrane targeting of PIP5Kgamma, since the deletion or charge-reversal mutation of this dibasic sequence resulted in the mislocalization of the protein to the cytoplasm . Mislocalized mutants also failed to complement the temperature-sensitive growth of Saccharomyces cerevisiae mss4-1 mutant defective in PIP5K function . The presence of dibasic residues at the C-terminal end of KHD was conserved among mammalian as well as invertebrate PIP5K family members, but not in the type II PIPKs that are not targeted to the plasma membrane, suggesting that the conserved dibasic motif provides a mechanism essential for the proper localization and cellular function of PIP5Ks.

Carbohydr Res, 2004 Jun 1, 339(8), 1453 - 7
Synthesis, (1-->3)-beta-D-glucanase-binding ability, and phytoalexin-elicitor activity of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides; Huang GL et al.; We describe a approach for the synthesis of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides . The particular (1-->3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method, and it was characterized by FTIR spectroscopy . The acid-solubilized (1-->3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan particles, which were qualitatively analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) . The peracetylated 3-butenyl (1-->3)-beta-D-oligoglucosides were synthesized by treating peracetylated (1-->3)-beta-D-oligoglucosides with the 3-butenyl alcohols and a Lewis acid (SnCl4) catalyst . Epoxidation of the peracetylated 3-butenyl oligoglucosides took place with m-chloroperoxybenzoic acid (m-CPBA) . NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture in an overall yield of 21% . The sample was analyzed by positive-ion electrospray ionization mass spectrometry (ESIMS) . In a 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside-binding (1-->3)-beta-D-glucanase assay, we found that the (1-->3)-beta-D-glucanase was obviously inactivated by the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides . At the same time, we found the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture was more active as compared to the underivatized oligoglucoside mixture in eliciting phytoalexin accumulation in tobacco cotyledon tissue . Furthermore, it could be kept for a longer time than a (1-->3)-beta-D-oligoglucoside mixture, which indicated it is much more stable than (1-->3)-beta-D-oligoglucosides .

FEBS Lett, 2004 Jun 4, 567(2-3), 302 - 6
Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins; Ketelaar T et al.; Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells . Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of yeast Atg8 (Apg8/Aut7) and Atg4 (Apg4/Aut2) partially complement the yeast deletion strains . The yeast double mutant, a deletion strain with respect to both Atg8 and Atg4, could not be complemented by Arabidopsis Atg8, indicating that Arabidopsis Atg8 requires Atg4 for its function . Moreover, Arabidopsis Atg8 and Arabidopsis Atg4 interact directly in a two-hybrid assay . Interestingly, Atg8 shows significant homology with the microtubule binding light chain 3 of MAP1A and B, and here we show that Arabidopsis Atg8 binds microtubules . Our results demonstrate that a principle component of the autophagic pathway in plants is similar to that in yeast and we suggest that microtubule binding plays a role in this process.

FEBS Lett, 2004 Jun 4, 567(2-3), 259 - 64
Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome; London MK et al.; Analysis of several Saccharomyces cerevisiae ump mutants with defects in ubiquitin (Ub)-mediated proteolysis yielded insights into the regulation of the polyubiquitin gene UBI4 and of proteasome genes . High-molecular weight Ub-protein conjugates accumulated in ump mutants with impaired proteasome function with a concomitant decrease in the amount of free Ub . In these mutants, transcriptional induction of UBI4 was depending in part on the transcription factor Rpn4 . Deletion of UBI4 partially suppressed the growth defects of ump1 mutants, indicating that accumulation of polyubiquitylated proteins is deleterious to cell growth . Transcription of proteasome subunit genes was induced in ump mutants affecting the proteasome, as well as under conditions that mediate DNA damage or the formation of abnormal proteins . This induction required the transcriptional activator Rpn4 . Elevated Rpn4 levels in proteasome-deficient mutants or as a response to abnormal proteins were due to increased metabolic stability . Up-regulation of proteasome genes in response to DNA damage, in contrast, is shown to operate via induction of RPN4 transcription.

Gene, 2004 May 26, 333, 135 - 41
Representing GC variation along eukaryotic chromosomes; Paces J et al.; Genome sequencing now permits direct visual representation, at any scale, of GC heterogeneity along the chromosomes of several higher eukaryotes . Plots can be easily obtained from the chromosomal sequences, yet sequence releases of mammalian or plant chromosomes still tend to use small scales or window sizes that obscure important large-scale compositional features . To faithfully reveal, at one glance, the compositional variation at a given scale, we have devised a simple scheme that combines line plots with color-coded shading of the regions underneath the plots . The scheme can be applied to different eukaryotic genomes to facilitate their comparison, as illustrated here for a sample of chromosomes chosen from seven selected species . As a complement to a previously published compact view of isochores in the human genome sequence, we include here an analogous map for the recently sequenced mouse genome, and discuss the contribution of repetitive DNA to the GC variation along the plots . Supplementary information, including a database of color-coded GC profiles for all recently sequenced eukaryotes and the program draw_chromosomes_gc.pl used to obtain them, are available at.

DNA Repair (Amst), 2004 Jul 2, 3(7), 769 - 76
The Hog1 MAP kinase pathway and the Mec1 DNA damage checkpoint pathway independently control the cellular responses to hydrogen peroxide; Haghnazari E et al.; The DNA damage checkpoint pathway and the MAP kinase pathway respond to various forms of environmental as well as endogenous stresses through signal transduction mechanisms involving protein kinases . Both pathways are intertwined in mammalian cells, but potential crosstalk between these two pathways in budding yeast has not been examined yet . We show that the Rad53 checkpoint kinase and the Hog1 MAP kinase of Saccharomyces cerevisiae become phosphorylated upon exposure to hydrogen peroxide, indicative of activation of the DNA damage checkpoint and MAP kinase pathways in response to oxidative stress . Rad53 kinase is equally activated in wild type and in hog1-Delta cells . Likewise, the activation of Hog1 MAP kinase is not dependent on Mec1 kinase, the central checkpoint kinase in budding yeast . Mutants in either pathway are sensitive to hydrogen peroxide and the double mutants exhibit a near perfectly additive phenotype . These data demonstrate that the DNA damage checkpoint pathway and the MAP kinase pathway respond to oxidative stress independently of each other and suggest that these two stress signaling pathways are activated by different types of insults induced by hydrogen peroxide.

DNA Repair (Amst), 2004 Jul 2, 3(7), 729 - 42
Mutations in the nucleotide-binding domain of MutS homologs uncouple cell death from cell survival; Drotschmann K et al.; After genotoxic insult, the decision to repair or undergo cell death is pivotal for undamaged cell survival, and requires a highly controlled coordination of both pathways . Disruption of this regulation results in tumorigenesis and failure of cancer therapy . Mismatch repair (MMR) proteins have a unique role by contributing to both pathways, though direct evidence for their function in the DNA damage response is ambiguous . We report separation of function mutants in the ATPase domains of yeast MutS homologous (MSH) proteins that uncouple MMR-dependent DNA repair from damage response to cisplatin . While mutations in the ATPase domain have devastating effects on the mutation rate of the cell, ATPase processing is mostly dispensable for the cell death phenotype; only limited processing by the MSH6 subunit is required in DNA damage response . Different DNA binding patterns and nucleotide sensitivity of Msh2/Msh6-DNA adduct and protein-mismatch complexes, respectively, suggest that the presence of different DNA lesions influences the requirement for ATP . Limited proteolysis of purified protein gives first indications for differences in nucleotide-induced conformational changes in the presence of platinated DNA . Structural modeling of bacterial MutS proteins reinforces nucleotide-dependent differences in structures that contribute to the distinction between DNA damage response and repair . Our results demonstrate the uncoupling of MMR-dependent damage response from repair and present first indications for the involvement of distinct conformational changes in MSH proteins in this process . These data present evidence for a mechanism of MMR-dependent damage response that differs from MMR; these results have strong implications for the chemotherapeutic treatment of MMR-defective tumors.

Dev Cell, 2004 Jun, 6(6), 865 - 73
Lumen morphogenesis in C . elegans requires the membrane-cytoskeleton linker erm-1; Gobel V et al.; Epithelial tubes are basic building blocks of complex organs, but their architectural requirements are not well understood . Here we show that erm-1 is a unique C . elegans ortholog of the ERM family of cytoskeleton-membrane linkers, with an essential role in lumen morphogenesis . ERM-1 localizes to the luminal membranes of those tubular organ epithelia which lack stabilization by cuticle . RNA interference (RNAi), a germline deletion, and overexpression of erm-1 cause cystic luminal phenotypes in these epithelia . Confocal and ultrastructural analyses indicate that erm-1 functions directly in apical membrane morphogenesis, rather than in epithelial polarity and junction assembly as has been previously proposed for ERMs . We also show that act-5/cytoplasmic actin and sma-1/beta-H-spectrin are required for lumen formation and functionally interact with erm-1 . Our findings suggest that there are common structural constraints on the architecture of diverse organ lumina.

Dev Cell, 2004 Jun, 6(6), 831 - 43
Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules; Busch KE et al.; The positioning of growth sites in fission yeast cells is mediated by spatially controlled microtubule dynamics brought about by tip1p, a CLIP-170-like protein, which is localized at the microtubule tips and guides them to the cell ends . The kinesin tea2p is also located at microtubule tips and affects microtubule dynamics . Here we show that tea2p interacts with tip1p and that the two proteins move with high velocity along the microtubules toward their growing tips . There, tea2p and tip1p accumulate in larger particles . Particle formation requires the EB1 homolog, mal3p . Our results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips.

Dev Cell, 2004 Jun, 6(6), 815 - 29
Cell cycle control of kinesin-mediated transport of Bik1 (CLIP-170) regulates microtubule stability and dynein activation; Carvalho P et al.; CLIPs are microtubule plus end-associated proteins that mediate interactions required for cell polarity and cell division . Here we demonstrate that budding yeast Bik1, unlike its human ortholog CLIP-170, is targeted to the microtubule plus end by a kinesin-dependent transport mechanism . Bik1 forms a complex with the kinesin Kip2 . Fluorescently labeled Bik1 and Kip2 comigrate along individual microtubules . Bik1 exists in distinct intracellular pools: a stable pool at the spindle pole body that is depleted during cell cycle progression, a soluble pool from which Bik1 can be recruited during microtubule initiation, and a dynamic plus end pool maintained by Kip2 . Kip2 stabilizes microtubules by targeting Bik1 to the plus end and Kip2 levels are controlled during the cell cycle . As with Bik1, the targeting of dynein to the microtubule plus end requires Kip2 . These findings reveal a central role for Kip2-dependent transport in the cell cycle control of microtubule dynamics and dynein-dependent motility.

Dev Cell, 2004 Jun, 6(6), 746 - 8
CLIP-170 family members: a motor-driven ride to microtubule plus ends; Maekawa H et al.; CLIP-170 family proteins regulate microtubule plus end dynamics . Two reports published in this issue of Developmental Cell show that Bik1 and tip1p, the CLIP-170-like proteins of budding and fission yeast, are carried to microtubule plus ends by kinesin motor proteins . These findings indicate a complex interplay between microtubule-associated proteins and suggest a novel mechanism by which kinesin proteins stabilize microtubules.

Mol Cell, 2004 Jun 4, 14(5), 685 - 91
A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology; Sato S et al.; The Mediator is a multiprotein transcriptional coactivator that is expressed ubiquitously in eukaryotes from yeast to mammals and is required for induction of RNA polymerase II (pol II) transcription by DNA binding transcription factors . In the work described here, we exploit multidimensional protein identification technology (MudPIT) to carry out a proteomic analysis of the subunit composition of the mammalian Mediator complex . By comparing MudPIT data sets obtained from six independent Mediator preparations immunoaffinity purified through their Nut2 (MED10), Med25 (MED9), Intersex (MED29), LCMR1 (MED19), AK007855 (MED28), or CRSP70 (MED26) subunits, we identify a set of consensus mammalian Mediator subunits . In addition, we identify as Mediator-associated proteins the CDK8-like cyclin-dependent kinase CDK11 and the TRAP240-like KIAA1025 protein (MED13L), which is mutated in patients with the congenital heart defect transposition of the great arteries (TGA).

Mol Cell, 2004 Jun 4, 14(5), 667 - 73
Removal of promoter nucleosomes by disassembly rather than sliding in vivo; Boeger H et al.; Previous work demonstrated the removal of nucleosomes from the PHO5 promoter upon transcriptional activation in yeast . Removal could occur by nucleosome disassembly or by sliding of nucleosomes away from the promoter . We have now activated the PHO5 promoter on chromatin circles following excision from the chromosomal locus . Whereas sliding would conserve the number of nucleosomes on the circle, we found that the number was diminished, demonstrating chromatin remodeling by nucleosome disassembly.

Proteomics, 2004 Jun, 4(6), 1581 - 90
Predotar: A tool for rapidly screening proteomes for N-terminal targeting sequences; Small I et al.; Probably more than 25% of the proteins encoded by the nuclear genomes of multicellular eukaryotes are targeted to membrane-bound compartments by N-terminal targeting signals . The major signals are those for the endoplasmic reticulum, the mitochondria, and in plants, plastids . The most abundant of these targeted proteins are well-known and well-studied, but a large proportion remain unknown, including most of those involved in regulation of organellar gene expression or regulation of biochemical pathways . The discovery and characterization of these proteins by biochemical means will be long and difficult . An alternative method is to identify candidate organellar proteins via their characteristic N-terminal targeting sequences . We have developed a neural network-based approach (Predotar--Prediction of Organelle Targeting sequences) for identifying genes encoding these proteins amongst eukaryotic genome sequences . The power of this approach for identifying and annotating novel gene families has been illustrated by the discovery of the pentatricopeptide repeat family.

Methods Mol Biol, 2004, 284, 147 - 62
Characterization of protein-DNA association in vivo by chromatin immunoprecipitation; Kuras L; Chromatin immunoprecipitation (ChIP) is one of the most powerful methods to identify and characterize the association of proteins with specific genomic regions in the context of intact cells . In this method, cells are first treated with formaldehyde to crosslink protein-protein and protein-DNA complexes in situ . Next, the crosslinked chromatin is sheared by sonication to generate small chromatin fragments, and the fragments associated with the protein of interest are immunoprecipitated using antibodies to the protein . Finally, protein-DNA crosslinks are reversed and the DNA is examined for the presence of particular sequences by quantitative polymerase chain reaction (PCR) . Enrichment of specific sequences in the precipitate indicates that the sequences are associated with the protein of interest in vivo . The ChIP method described here is intended for studying protein-DNA association in the budding yeast Saccharomyces cerevisiae, but it can be easily implemented in other cell types, including fly, mammalian, and plant cells.

Methods Mol Biol, 2004, 284, 79 - 90
Assaying protein kinase activity; Brabek J et al.; Protein kinases, encoded by approx 2% of eukaryotic genes, represent one of the major classes of cell-regulatory molecules . Assessment of the catalytic activity of a specific protein kinase can be an important step in elucidating signal-transduction pathways that affect cell behavior . As an example of approaches taken to measure protein kinase activity, this chapter presents methods useful for determination of the activity of the oncogenic protein-tyrosine kinase v-Src . Included are protocols for heterologous expression of the kinase in yeast Saccharomyces cerevisiae, immunoaffinity purification from yeast cell lysates, kinase reactions using incorporation of 32P into peptide substrates, and quantifying protein kinase activity . The Notes section discusses alternative approaches for assaying the activity of Src recovered from vertebrate cells and it gives recommendations for assaying the activity of the other protein kinases with respect to the substrate specifity and the composition of kinase reaction buffer.

Methods Mol Biol, 2004, 284, 1 - 14
Making protein immunoprecipitates; Elion EA et al.; A wide variety of methods used in the study of signal transduction in eukaryotes rely on the ability to precipitate proteins from whole cell extracts . Immunoprecipitation and related methods of affinity purification are routinely used to assess binding partner interactions and enzyme activity in addition to the size of a protein, rates of protein synthesis and turnover, and protein abundance, thus making it a mainstay of a wide variety of protocols . This chapter will provide starting-point methods for immunoprecipitation of proteins under denaturing and nondenaturing conditions and the detection of protein-protein interactions by co-precipitation . The Notes section gives recommendations on how to troubleshoot potential problems that can arise while doing these methodologies.

Nucleic Acids Res, 2004 Jun 01, 32(10), 3005 - 16 Print 2004.
The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit; Sanchez Garcia J et al.; DNA polymerase delta (Pol delta) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination . In fission yeast, Pol delta is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1 . Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex . Here it is shown that removal of the tandem zinc finger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the C-terminal zinc finger module ZnF2 is both necessary and sufficient for binding to the B-subunit in vivo and in vitro . Extensive mutagenesis of the ZnF2 module identifies important residues for B-subunit binding . In particular, disruption of the ZnF2 module by substitution of the putative metal-coordinating cysteines with alanine abolishes B-subunit binding and in vivo function . Finally, evidence is presented suggesting that the ZnF region is post-translationally modified in fission yeast cells.

J Cell Sci, 2004 Jun 15, 117(Pt 14), 2997 - 3009 Epub 2004 Jun 01.
Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B; Fujita H et al.; SKD1 belongs to the AAA-ATPase family and is one of the mammalian class E Vps (vacuolar protein sorting) proteins . Previously we have reported that the overexpression of an ATPase activity-deficient form of SKD1 (suppressor of potassium transport growth defect), SKD1(E235Q), leads the perturbation of membrane transport through endosomes and lysosomes, however, the molecular mechanism behind the action of SKD1 is poorly understood . We have identified two SKD1-binding proteins, SBP1 and mVps2, by yeast two-hybrid screening and we assign them as mammalian class E Vps proteins . The primary sequence of SBP1 indicates 22.5% identity with that of Vta1p from Saccharomyces cerevisiae, which was recently identified as a novel class E Vps protein binding to Vps4p . In fact, SBP1 binds directly to SKD1 through its C-terminal region (198-309) . Endogenous SBP1 is exclusively localized to cytosol, however it is redirected to an aberrant endosomal structure, the E235Q compartment, in the cells expressing SKD1(E235Q) . The ATPase activity of SKD1 regulates both the membrane association of, and assembly of, a large hetero-oligomer protein complex, containing SBP1, which is potentially involved in membrane transport through endosomes and lysosomes . The N-terminal half (1-157) of human SBP1 is identical to lyst-interacting protein 5 and intriguingly, SKD1 ATPase activity significantly influences the membrane association of lyst protein . The SKD1-SBP1 complex, together with lyst protein, may function in endosomal membrane transport . A primary sequence of mVps2, a mouse homologue of human CHMP2A/BC-2, indicates 44.4% identity with Vps2p/Did4p/Chm2p from Saccharomyces cerevisiae . mVps2 also interacts with SKD1 and is localized to the E235Q compartment . Intriguingly, the N-terminal coiled-coil region of mVps2 is required for the formation of the E235Q compartment but not for binding to SKD1 . We propose that both SBP1 and mVps2 regulate SKD1 function in mammalian cells.

Genome Res, 2004 Jun, 14(6), 1107 - 18
Annotation transfer between genomes: protein-protein interologs and protein-DNA regulogs; Yu H et al.; Proteins function mainly through interactions, especially with DNA and other proteins . While some large-scale interaction networks are now available for a number of model organisms, their experimental generation remains difficult . Consequently, interolog mapping--the transfer of interaction annotation from one organism to another using comparative genomics--is of significant value . Here we quantitatively assess the degree to which interologs can be reliably transferred between species as a function of the sequence similarity of the corresponding interacting proteins . Using interaction information from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Helicobacter pylori, we find that protein-protein interactions can be transferred when a pair of proteins has a joint sequence identity >80% or a joint E-value <10(-70) . (These "joint" quantities are the geometric means of the identities or E-values for the two pairs of interacting proteins.) We generalize our interolog analysis to protein-DNA binding, finding such interactions are conserved at specific thresholds between 30% and 60% sequence identity depending on the protein family . Furthermore, we introduce the concept of a "regulog"--a conserved regulatory relationship between proteins across different species . We map interologs and regulogs from yeast to a number of genomes with limited experimental annotation (e.g., Arabidopsis thaliana) and make these available through an online database at Specifically, we are able to transfer approximately 90,000 potential protein-protein interactions to the worm . We test a number of these in two-hybrid experiments and are able to verify 45 overlaps, which we show to be statistically significant .

Cancer Res, 2004 Jun 1, 64(11), 3940 - 8
Genome-wide identification of genes conferring resistance to the anticancer agents cisplatin, oxaliplatin, and mitomycin C; Wu HI et al.; Cisplatin is a crucial agent in the treatment of many solid tumors, yet many tumors have either acquired or intrinsic resistance to the drug . We have used the homozygous diploid deletion pool of Saccharomyces cerevisiae, containing 4728 strains with individual deletion of all nonessential genes, to systematically identify genes that when deleted confer sensitivity to the anticancer agents cisplatin, oxaliplatin, and mitomycin C . We found that deletions of genes involved in nucleotide excision repair, recombinational repair, postreplication repair including translesional synthesis, and DNA interstrand cross-link repair resulted in sensitivity to all three of the agents, although with some differences between the platinum drugs and mitomycin C in the spectrum of required translesional polymerases . Putative defective repair of oxidative damage (imp2'Delta strain) also resulted in sensitivity to platinum and oxaliplatin, but not to mitomycin C . Surprisingly in light of their different profiles of clinical activity, cisplatin and oxaliplatin have very similar sensitivity profiles . Finally, we identified three novel genes (PSY1-3, "platinum sensitivity") that, when deleted, demonstrate sensitivity to cisplatin and oxaliplatin, but not to mitomycin C . Our results emphasize the importance of multiple DNA repair pathways responsible for normal cellular resistance to all three of the agents . Also, the similarity of the sensitivity profiles of the platinum agents with that of the known DNA interstrand cross-linking agent mitomycin C, and the importance of the gene PSO2 known to be involved in DNA interstrand cross-link repair strongly suggests that interstrand cross-links are important toxic lesions for cisplatin and oxaliplatin, at least in yeast.

Novartis Found Symp, 2004, 259, 63 - 73; discussion 73-7, 163-9
H2B ubiquitylation and de-ubiquitylation in gene activation; Wyce A et al.; Previous models for the role of histone modifications suggest that adding and removing modifications, such as acetylation/deacetylation in gene regulation, are functionally antagonistic . We have investigated a transcriptional role of H2B C-terminal ubiquitylation and de-ubiquitylation in Saccharomyces cerevisiae . H2B ubiquitylation is required for optimal transcription of SUC2 and GAL1 genes . The ubiquitin hydrolase Ubp8 is a stable component of SAGA but not ADA complexes, and is not required for overall integrity of SAGA . Biochemical and genetic evidence indicates that Ubp8 targets H2B for deubiquitylation . The dynamic balance of H2B ubiquitylation/deubiquitylation is important for GAL1 transcription since either substitution of the ubiquitylation site in H2B (Lys123), or loss of Ubp8, lowers GAL1 expression . Further, this balance of ubiquitylation appears to set the balance of histone H3 methylation at Lys4 relative to Lys36 . Thus, unlike acetylation/deacetylation whose functions are mutually opposing, both ubiquitylation and de-ubiquitylation are required for gene activation . These results suggest that ubiquitylation of histones has a unique role among histone modifications, possibly to orchestrate an ordered pathway of chromatin alterations.

Bioessays, 2004 Jun, 26(6), 629 - 38
hnRNP K: one protein multiple processes; Bomsztyk K et al.; Since its original identification as a component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex, K protein has been found not only in the nucleus but also in the cytoplasm and mitochondria and is implicated in chromatin remodeling, transcription, splicing and translation processes . K protein contains multiple modules that, on one hand, bind kinases while, on the other hand, recruit chromatin, transcription, splicing and translation factors . Moreover, the K- protein-mediated interactions are regulated by signaling cascades . These observations are consistent with K protein acting as a docking platform to integrate signaling cascades by facilitating cross-talk between kinases and factors that mediate nucleic-acid-directed processes . Comparison of K across species reveals that it is an essential factor in metazoans, but not in yeast . Although some of the K protein interactions and functions are conserved in eukaryotes from yeast to man, the mammalian protein seems to play a wider role . The greater diversity of mammalian K protein interactions and function may reflect gain of novel docking sites and expansion evolutionary of gene expression networks .

EMBO Rep, 2004 Jun, 5(6), 567 - 71
The J-protein family: modulating protein assembly, disassembly and translocation; Walsh P et al.; DnaJ is a molecular chaperone and the prototypical member of the J-protein family . J proteins are defined by the presence of a J domain that can regulate the activity of 70-kDa heat-shock proteins . Sequence analysis on the genome of Saccharomyces cerevisiae has revealed 22 proteins that establish four distinguishing structural features of the J domain: predicted helicity in segments I-IV, precisely placed interhelical contact residues, a lysine-rich surface on helix II and placement of the diagnostic sequence HPD between the predicted helices II and III . We suggest that this definition of the J-protein family could be used for other genome-wide studies . In addition, three J-like proteins were identified in yeast that contain regions closely resembling a J domain, but in which the HPD motif is non-conservatively replaced . We suggest that J-like proteins might function to regulate the activity of bona fide J proteins during protein translocation, assembly and disassembly.

Nat Cell Biol, 2004 Jun, 6(6), 473 - 5
Cdc14 phosphatase resolves the rDNA segregation delay; Pereira G et al.; Sister chromatid segregation in anaphase of mitosis is initiated through cleavage of cohesin by the protease separase . Two studies now show that this view is valid for most chromosomal DNA, but not for the highly repetitive ribosomal DNA (rDNA) and telomeres . The disjunction of these regions of the chromosome occurs in mid-anaphase, long after cohesin cleavage, and is regulated by the conserved phosphatase Cdc14.

Biochemistry, 2004 Jun 8, 43(22), 7111 - 20
An encephalitozoon cuniculi ortholog of the RNA polymerase II carboxyl-terminal domain (CTD) serine phosphatase Fcp1; Hausmann S et al.; Fcp1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7) . The CTD of the microsporidian parasite Encephalitozoon cuniculi consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast . Here we show that E . cuniculi encodes a minimized 411-aa Fcp1-like protein (EcFcp1), which consists of a DxDx(T/V) phosphatase domain and a BRCA1 carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp1 enzymes . Nonetheless, EcFcp1 can function in lieu of Saccharomyces cerevisiae Fcp1 to sustain yeast cell growth . Recombinant EcFcp1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-PO(4)) substrates . EcFcp1 dephosphorylates CTD positions Ser2 and Ser5 with similar efficacy in vitro . We exploit synthetic CTD Ser2-PO(4) and Ser5-PO(4) peptides to define minimized substrates for EcFcp1 and to illuminate the importance of CTD primary structure in Ser2 and Ser5 phosphatase activity.

Biochemistry, 2004 Jun 8, 43(22), 7028 - 37
Mutation and evolution of the magnesium-binding site of a class II aminoacyl-tRNA synthetase; Ador L et al.; Aminoacyl-tRNA synthetases contain one or three Mg(2+) ions in their catalytic sites . In addition to their role in ATP binding, these ions are presumed to play a role in catalysis by increasing the electropositivity of the alpha-phosphate and stabilizing the pentavalent transition state . In the class II aaRS, two highly conserved carboxylate residues have been shown to participate with Mg(2+) ions in binding and coordination . It is shown here that these carboxylate residues are absolutely required for the activity of Saccharomyces cerevisiae aspartyl-tRNA synthetase . Mutants of these residues exhibit pleiotropic effects on the kinetic parameters suggesting an effect at an early stage of the aminoacylation reaction, such as the binding of ATP, Mg(2+), aspartic acid, or the amino acid activation . Despite genetic selections in an APS-knockout yeast strain, we were unable to select a single active mutant of these carboxylate residues . Nevertheless, we isolated an intragenic suppressor from a combinatorial library . The active mutant showed a second substitution close to the first one, and exhibited a significant increase of the tRNA aminoacylation rate . Structural analysis suggests that the acceptor stem of the tRNA might be repositioned to give a more productive enzyme:tRNA complex . Thus, the initial defect of the activation reaction was compensated by a significant increase of the aminoacylation rate that led to cellular complementation.

Mol Cell Biol, 2004 Jun, 24(12), 5534 - 47
Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors; Grigull J et al.; Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II) . Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1 . A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription . Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts . We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress . This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress . Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins . Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability.

Mol Cell Biol, 2004 Jun, 24(12), 5521 - 33
Positive and negative regulation of poly(A) nuclease; Mangus DA et al.; PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails . The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p) . We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions . Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease . Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN . Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p . However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct . Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus . The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

Mol Cell Biol, 2004 Jun, 24(12), 5485 - 95
The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for early embryonic development in mice; Roest HP et al.; The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions . In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins . Here, we describe the phenotype of cells and mice deficient for the mHR6A gene . Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight . The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable . In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile . However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation . The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H3 at this early stage of mouse embryonic development . These observations support redundant but dose-dependent roles for HR6A and HR6B in somatic cell types and germ line cells in mammals.

Mol Cell Biol, 2004 Jun, 24(12), 5249 - 56
Genome-wide analysis of the biology of stress responses through heat shock transcription factor; Hahn JS et al.; Heat shock transcription factor (HSF) and the promoter heat shock element (HSE) are among the most highly conserved transcriptional regulatory elements in nature . HSF mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses . While HSF is essential for cell viability in Saccharomyces cerevisiae, oogenesis and early development in Drosophila melanogaster, extended life span in Caenorhabditis elegans, and extraembryonic development and stress resistance in mammals, little is known about its full range of biological target genes . We used whole-genome analyses to identify virtually all of the direct transcriptional targets of yeast HSF, representing nearly 3% of the genomic loci . The majority of the identified loci are heat-inducibly bound by yeast HSF, and the target genes encode proteins that have a broad range of biological functions including protein folding and degradation, energy generation, protein trafficking, maintenance of cell integrity, small molecule transport, cell signaling, and transcription . This genome-wide identification of HSF target genes provides novel insights into the role of HSF in growth, development, disease, and aging and in the complex metabolic reprogramming that occurs in all cells in response to stress.

J Cell Sci, 2004 Jun 1, 117(Pt 13), 2805 - 12
LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation; Kabeya Y et al.; Rat LC3, a homologue of yeast Atg8 (Aut7/Apg8), localizes to autophagosomal membranes after post-translational modifications . The C-terminal fragment of LC3 is cleaved immediately following synthesis to yield a cytosolic form called LC3-I . A subpopulation of LC3-I is further converted to an autophagosome-associating form, LC3-II . Because yeast Atg8 is conjugated with phosphatidylethanolamine (PE) by a ubiquitin-like system, it has been hypothesized that LC3 is modified in a similar manner . Here, we show that {(14)C}-ethanolamine was preferentially incorporated into LC3-II, suggesting that LC3-II is a PE-conjugated form . LC3-II can be a substrate of mammalian Atg4B, a homologue of yeast Atg8-PE deconjugase, supporting the idea that LC3-II is LC3-PE . Moreover, two other mammalian homologues of yeast Atg8, gamma-aminobutyric-acid-type-A-receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE16) also generate form II, which are recovered in membrane fractions . Generation of the form II correlates with autophagosome association of GABARAP and GATE16 . These results suggest that all mammalian Atg8 homologues receive a common modification to associate with autophagosomal membrane as the form II.

J Biol Chem, 2004 Jul 16, 279(29), 29875 - 8 Epub 2004 May 27.
DNA damage-induced Def1-RNA polymerase II interaction and Def1 requirement for polymerase ubiquitylation in vitro; Reid J et al.; UV-induced DNA damage results in ubiquitylation and degradation of RNA polymerase II (RNAPII) . In yeast, this requires the DEF1 gene, the product of which forms a complex with the transcription-coupling repair factor, Rad26 . However, whether Def1 is directly involved in RNAPII ubiquitylation has remained unclear . Here we report the establishment of a reconstituted system for studying UV-induced RNAPII ubiquitylation, which mimics the known requirements for this process in vitro . Using this system, we show that Def1 is indeed directly required for RNAPII ubiquitylation . Moreover, Def1 interacts with RNAPII in a damage-dependent manner . These results support a model in which Def1 interacts with RNAPII in response to DNA damage, recruiting the ubiquitylation machinery to enable its modification and subsequent degradation.

J Biol Chem, 2004 Jul 30, 279(31), 32055 - 62 Epub 2004 May 27.
Superoxide inhibits 4Fe-4S cluster enzymes involved in amino acid biosynthesis . Cross-compartment protection by CuZn-superoxide dismutase; Wallace MA et al.; Among the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy; in the current work we show an additional leaky auxotrophy for leucine . The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively . We present evidence that direct aerobic inactivation of these enzymes in sod1 Delta yeast results in the auxotrophies . Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p . Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix . The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1 Delta mutants . We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial "free" (EPR-detectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities . Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.

Genetics, 2004 May, 167(1), 217 - 31
A large-scale screen for mutagen-sensitive loci in Drosophila; Laurencon A et al.; In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C . Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2) . Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes . The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219) . Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes . The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327) . We have initiated efforts to identify these genes at the molecular level and report here the first two identified . The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene . We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination . At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.

Bioinformatics, 2004 Jul 22, 20(11), 1766 - 71 Epub 2004 May 27.
Correcting the loss of cell-cycle synchrony in clustering analysis of microarray data using weights; Duan F et al.; MOTIVATION: Due to the existence of the loss of synchrony in cell-cycle data sets, standard clustering methods (e.g . k-means), which group open reading frames (ORFs) based on similar expression levels, are deficient unless the temporal pattern of the expression levels of the ORFs is taken into account . METHODS: We propose to improve the performance of the k-means method by assigning a decreasing weight on its variable level and evaluating the 'weighted k-means' on a yeast cell-cycle data set . Protein complexes from a public website are used as biological benchmarks . To compare the k-means clusters with the structures of the protein complexes, we measure the agreement between these two ways of clustering via the adjusted Rand index . RESULTS: Our results show the time-decreasing weight function--exp{-(1/2)(t(2)/C(2))}--which we assign to the variable level of k-means, generally increases the agreement between protein complexes and k-means clusters when C is near the length of two cell cycles.

Mol Microbiol, 2004 Jun, 52(5), 1271 - 80
Identification of novel dominant INO2c mutants with an Opi- phenotype; Gardenour KR et al.; The INO2 gene of Saccharomyces cerevisiae is required for derepression of the phospholipid biosynthetic genes in response to inositol depletion . Conversely, the OPI1 gene is required for repression in response to inositol supplementation . Results of an in vitro assay have led to a model in which Opi1p interacts with Ino2p . However, there is no in vivo evidence to support this model . Additionally, most of the previously isolated ino2 mutants offer little insight into this model . Here, we report the isolation of a new class of dominant mutations in the INO2 gene, which yield constitutive expression of a target gene (i.e . an Opi(-) mutant phenotype) . Two mutations reside in a region of the Ino2p required for interaction with Opi1p in vitro . Three other mutations are at the amino-terminus in a transcriptional activation domain.

J Nat Prod, 2004 May, 67(5), 908 - 10
Further bioactive piperidine alkaloids from the flowers and green fruits of Cassia spectabilis; Viegas C Jr et al.; The flowers of Cassia spectabilis yielded three new piperidine alkaloids, (-)-3-O-acetylspectaline (1), (-)-7-hydroxyspectaline (2), and iso-6-spectaline (3), together with the known (-)-spectaline (4) . The green fruits of this plant were also investigated, resulting in the isolation of 1 and 4 . Their structures were elucidated using a combination of multidimensional NMR and MS techniques, and relative stereochemistries were established by NOESY correlations and analysis of coupling constants . The DNA-damaging activity of these compounds was evaluated using a mutant yeast, Saccharomyces cerevisiae, assay.

Cell, 2004 May 28, 117(5), 601 - 10
Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell; Gross E et al.; The flavoenzyme Ero1p produces disulfide bonds for oxidative protein folding in the endoplasmic reticulum . Disulfides generated de novo within Ero1p are transferred to protein disulfide isomerase and then to substrate proteins by dithiol-disulfide exchange reactions . Despite this key role of Ero1p, little is known about the mechanism by which this enzyme catalyzes thiol oxidation . Here, we present the X-ray crystallographic structure of Ero1p, which reveals the molecular details of the catalytic center, the role of a CXXCXXC motif, and the spatial relationship between functionally significant cysteines and the bound cofactor . Remarkably, the Ero1p active site closely resembles that of the versatile thiol oxidase module of Erv2p, a protein with no sequence homology to Ero1p . Furthermore, both Ero1p and Erv2p display essential dicysteine motifs on mobile polypeptide segments, suggesting that shuttling electrons to a rigid active site using a flexible strand is a fundamental feature of disulfide-generating flavoenzymes.

Cell, 2004 May 28, 117(5), 555 - 6
Checkpoint adaptation; molecular mechanisms uncovered; Lupardus PJ et al.; Adaptation to the DNA damage checkpoint is a phenomenon long thought to be confined to the unicellular world . A new report in this issue of Cell by suggests the presence of a checkpoint adaptation pathway in Xenopus egg extracts that displays interesting molecular parallels to adaptation in yeast.

Cytogenet Genome Res, 2004, 104(1-4), 65 - 71
Progress towards understanding the nature of chromatid breakage; Bryant PE et al.; The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility . The mechanisms of chromatid breakage are not yet fully understood . However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB) . Three models of chromatid breakage are considered . The classical breakage-first and the Revell "exchange" models do not accord with current evidence . Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains . Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site . However, these interchromatid rearrangements comprise less than 20% of the total breaks . The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain . Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites . It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB . Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks . Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected . The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived . Techniques to study and improve such systems are under way in different cell lines . Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions .

Cytogenet Genome Res, 2004, 104(1-4), 21 - 7
The role of homologous recombination repair in the formation of chromosome aberrations; Griffin CS et al.; The repair of DNA double strand breaks by homologous recombination can occur by at least two pathways: a Rad51-dependent pathway that is predominantly error free, and a Rad51-independent pathway (single strand annealing, SSA) that is error prone . In theory, chromosome exchanges can result from (mis)repair by either pathway . Both repair pathways will involve a search for homologous sequence, leading to co-localization of chromatin . Genes involved in homologous recombination repair (HRR) have now been successfully knocked out in mice and the role of HRR in the formation of chromosome exchanges, particularly after ionising radiation, is discussed in the light of new evidence .

Nucleic Acids Res, 2004 May 25, 32(9), 2947 - 56 Print 2004.
Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors; Fingerman IM et al.; The Ndt80 protein of the yeast Saccharomyces cerevisiae is the founding member of a new sub-family of proteins in the Ig-fold superfamily of transcription factors . The crystal structure of Ndt80 bound to DNA shows that it makes contacts through several loops on one side of the protein that connect beta-strands which form the beta-sandwich fold common to proteins in this superfamily . However, the DNA-binding domain of Ndt80 is considerably larger than many other members of the Ig-fold superfamily and it appears to make a larger number of contacts with the DNA than these proteins . To determine the contribution of each of these contacts and to examine if the mechanism of Ndt80 DNA binding was similar to other members of the Ig-fold superfamily, amino acid substitutions were introduced at each residue that contacts the DNA and assayed for their effect on Ndt80 activity . Many of the mutations caused significant decreases in DNA-binding affinity and transcriptional activation . Several of these are in residues that are not found in other sub-families of Ig-fold proteins . These additional contacts are likely responsible for Ndt80's ability to bind DNA as a monomer while most other members require additional domains or cofactors to recognize their sites.

J Biol Chem, 2004 Aug 6, 279(32), 33875 - 81 Epub 2004 May 25.
Modulation of FcgammaRI (CD64) ligand binding by blocking peptides of periplakin; Beekman JM et al.; FcgammaRI requires both the intracellular domain of the alpha-chain and associated leukocyte Fc receptor (FcR) gamma-chains for its biological function . We recently found the C terminus of periplakin to selectively interact with the cytoplasmic domain of the FcgammaRI alpha-chain . It thereby enhances the capacity of FcgammaRI to bind, internalize, and present antigens on MHC class II . Here, we characterized the domains involved in FcgammaRI-periplakin interaction using truncated and alanine-substituted FcgammaRI mutants and randomly mutagenized periplakin . This allowed us to design TAT peptides that selectively interfered with endogenous FcgammaRI-periplakin interactions . The addition of these peptides to FcgammaRI-expressing cells modulated FcgammaRI ligand binding, as assessed by erythrocyte-antibody-rosetting . These data support a dominant-negative role of C-terminal periplakin for FcgammaRI biological activity and implicate periplakin as a novel regulator of FcgammaRI in immune cells.

Plant Mol Biol, 2004 Jan, 54(2), 233 - 43
Arabidopsis Acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats; Li HY et al.; Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools . Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats . The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified . Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane . In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A . thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats . This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats . When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane . Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.

Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 103 - 9
Mutagenesis of the three conserved valine residues: consequence on the foldability of insulin; Guo ZY et al.; Natural polypeptide chain usually can spontaneously fold into tightly compact native structure . This capability is the so-called foldability . However, how the foldability is encoded in the polypeptide chain is still poorly understood . The structure of insulin has been well solved and extensively investigated . Therefore, insulin provides a good model for investigating the role of individual residue to the sequence foldability . In insulins from different species there are three highly conserved Val residues (A3Val, B12Val, and B18Val), but their contribution to the insulin foldability is still unknown . Here, a single-chain insulin (PIP) was used to investigate the contribution of the three conserved valine residues to the foldability . Five PIP mutants, {A3S}PIP, {A3T}PIP, {B12A}PIP, {B18T}PIP, and {B18L}PIP, were used in the studies, and their structural changes, secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency were analyzed . The effects of the mutations on the PIP foldability are multifold: as a whole, mutation of A3Val has only moderate effect; while mutation of B12Val has significant detriment; hydrophobic replacement of B18Val is more tolerant than hydrophilic substitution as foldability is concerned . Therefore, the three highly conserved valine residues have different contributions to the insulin foldability, and their contribution might be ranked as B12Val>B18Val>A3Val.

Biochem Biophys Res Commun, 2004 Jun 18, 319(1), 240 - 6
A molecular beacon assay for measuring base excision repair activities; Maksimenko A et al.; The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage . Current methods to measure BER activities are indirect and cumbersome . Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons . We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide . Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time . Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins . We have further developed the method to analyze BER in cultured cells . As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways . Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.

Biochem Biophys Res Commun, 2004 Jun 18, 319(1), 193 - 9
Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii; Vohra PK et al.; The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV . Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type . We have cloned and characterized PCSTE3 from P . carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3 . We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein . Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor . We isolated P . carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms . PCSTE3 receptor expression was not found on cysts.

Biochem Biophys Res Commun, 2004 Jun 18, 319(1), 78 - 82
Interaction of D-lactate dehydrogenase protein 2 (Dld2p) with F-actin: implication for an alternative function of Dld2p; Hachiya NS et al.; D-Lactate dehydrogenase protein 2 {Yeast 15 (1999) 1377; Biochem . Biophys . Res . Commun . 295 (2002) 910} was initially identified as the actin interacting protein 2 (Aip2p) using a two-hybrid screen to search for proteins that interact with actin {Nat . Struct . Biol . 2 (1995) 28}, but no other evidence indicating an interaction between Aip2p and actin cytoskeleton has been reported so far . During our search for the protein conformation modifying activity, we serendipitously identified Aip2p isolated from Saccharomyces cerevisiae as exhibiting an interaction with F-actin both in vitro and in vivo . Incubation with Aip2p facilitated the formation of the circular form of F-actin in vitro, which exhibited an aberrant trypsin susceptibility . Overexpression of Aip2p induced multi-buds in yeast cells, whereas reduced expression interfered with the formation of the cleavage furrow for the cell division, which was rescued by the introduction of wild-type Aip2p . While Aip2p-treated F-actin in the circular form was negligibly stained by rhodamine-labeled phalloidin (rhodamine-phalloidin) in vitro, rhodamine-phalloidin staining profiles in actin interacting protein 2 gene (AIP2)-modified cells suggested a correlation between the conformation of F-actin and the expression of Aip2p in vivo . AIP2-deleted cells became sensitive to osmotic conditions, a hallmark of actin dysfunction . Finally, immunoprecipitation of yeast cells using anti-Aip2p antibody demonstrated that Aip2p associates with actin . These properties suggest that Aip2p may interact with F-actin in vivo and play an important role in the yeast cell morphology.

FEMS Microbiol Lett, 2004 Jun 1, 235(1), 153 - 6
Biochemical requirements for PCBCK1 kinase activity, the Pneumocystis carinii MEKK involved in cell wall integrity; Vohra PK et al.; Fungal cell wall assembly is a complicated process involving multiple enzymes and coordinated signaling pathways . The cell wall integrity MAPK pathway acts to stabilize the fungal cell wall during conditions of elevated temperature by regulation of glucan synthesis . The upstream kinase, BCK1, is a critical component of this pathway . Pneumonia is a significant cause of death from the fungal opportunistic pathogen Pneumocystis in immunocompromised states, especially with HIV infection . We have previously shown that PCBCK1 functions in the cell wall integrity pathway in yeast as a functional protein kinase . Kinases have specific requirements for enzymatic function which have not been investigated in fungi . Here we examine the biochemical requirements for PCBCK1 kinase activity expressed in Saccharomyces cerevisiae bck1Delta yeast . PCBCK1 requires 10 mM MgCl(2), pH 6, temperature 30 degrees C, and 10 microM ATP for kinase activity . Interference of the Pneumocystis cell wall integrity pathway is an attractive target for drug development since glucan synthesis machinery is not present in humans.

Trends Biotechnol, 2004 Jun, 22(6), 261 - 5
The regulatory software of cellular metabolism; Segre D; Understanding the regulation of metabolic pathways in the cell is like unraveling the 'software' that is running on the 'hardware' of the metabolic network . Transcriptional regulation of enzymes is an important component of this software . A recent systematic analysis of metabolic gene-expression data in Saccharomyces cerevisiae reveals a complex modular organization of co-expressed genes, which could increase our ability to understand and engineer cellular metabolic functions.

Comput Methods Programs Biomed, 2004 Jul, 75(1), 1 - 9
Analysis of gene expression data using functional principal components; Barra V; The large amount of data involved in DNA microarrays implies the development of efficient computer algorithms to analyze the gene expressions, and thus to study the transcriptome . Numerous techniques already exist and we propose a new method based on the key idea that gene profiles may be considered as continuous curves . The analysis of the set of curves stemming from the DNA microarray may be then performed using a functional analysis which can exhibit the main modes of variations in this set, gather genes with similar variations and extract characteristic parameters of gene profiles . We aim here at introducing this method, called the Functional Principal Component Analysis . A prospective study has been performed on two available datasets, concerning on the one hand the sporulation data of the Saccharomyces cerevisiae, and on the other hand data of tumor cell lines . Results are very promising: the method is able to extract characteristic parameters from the datasets, to extract significant modes of variations in the set of gene profiles, and to link these variations to biological processes already studied in literature.

Chem Biol, 2004 May, 11(5), 681 - 9
Transforming a (beta/alpha)8--barrel enzyme into a split-protein sensor through directed evolution; Tafelmeyer P et al.; Split-protein sensors have become an important tool for the analysis of protein-protein interactions in living cells . We present here a combinatorial method for the generation of new split-protein sensors and demonstrate its application toward the (beta/alpha)(8)-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae . The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan . This powerful selection complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening.

Biochemistry, 2004 Jun 1, 43(21), 6596 - 605
Key elements for protein foldability revealed by a combinatorial approach among similarly folded but distantly related proteins; Morimoto S et al.; We have determined the key regions for protein foldability by creating multiple crossover libraries from two proteins that share similar fold but have low sequence identity and differ significantly in stability . One protein is the propeptide of a serine protease, subtilisin BPN', and the other is Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) . The propeptide has a compact structure when complexed with subtilisin but is unstructured when isolated, whereas POIA1 takes a stable structure . We selected four of the conserved amino acid residues for the boundaries of crossover sites and utilized these residues to make same cohesive-ends to assemble synthetic DNA fragments . Each segment has one or two secondary structure units, and the interchange of these structural elements produces 32 (= 2(5)) combinations, including the propeptide and POIA1 . The stability of these mutants was first screened by formation of turbid zones on skim milk plates containing subtilisin BPN' . It was shown that six variants were foldable and structural units necessary for folding were identified . Further fragmentation and recombination of these mutants (the "multisection" method) revealed that two interactions between secondary structures are important; one is interaction between the loop-alpha1 and beta2-turn-beta3, and the other is hydrophobic interaction between the adjoining beta1 and beta4 strands . We were also able to specify the significant amino acid combinations for tolerance to proteolysis . These combinatorial methods not only elucidate how domains can be interchanged to make the whole protein foldable but also extract essential regions for the function, which is correlated with the instability of the molecule.

Science, 2004 Jun 18, 304(5678), 1793 - 7 Epub 2004 May 20.
Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers; Shorter J et al.; The protein-remodeling factor Hsp104 governs inheritance of {PSI+}, a yeast prion formed by self-perpetuating amyloid conformers of the translation termination factor Sup35 . Perplexingly, either excess or insufficient Hsp104 eliminates {PSI+} . In vitro, at low concentrations, Hsp104 catalyzed the formation of oligomeric intermediates that proved critical for the nucleation of Sup 35 fibrillization de novo and displayed a conformation common among amyloidogenic polypeptides . At higher Hsp104 concentrations, amyloidogenic oligomerization and contingent fibrillization were abolished . Hsp104 also disassembled mature fibers in a manner that initially exposed new surfaces for conformational replication but eventually exterminated prion conformers . These Hsp104 activities differed in their reaction mechanism and can explain {PSI+} inheritance patterns.

Plant Cell, 2004 Jun, 16(6), 1392 - 405 Epub 2004 May 21.
The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation; Dill A et al.; The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants . In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA) . SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA . These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome . In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response . By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain . The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation . The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation . However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins . We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants . This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins .

Nucleic Acids Res, 2004 May 20, 32(9), 2889 - 900 Print 2004.
Identification of sparsely distributed clusters of cis-regulatory elements in sets of co-expressed genes; Kreiman G; Sequence information and high-throughput methods to measure gene expression levels open the door to explore transcriptional regulation using computational tools . Combinatorial regulation and sparseness of regulatory elements throughout the genome allow organisms to control the spatial and temporal patterns of gene expression . Here we study the organization of cis-regulatory elements in sets of co-regulated genes . We build an algorithm to search for combinations of transcription factor binding sites that are enriched in a set of potentially co-regulated genes with respect to the whole genome . No knowledge is assumed about involvement of specific sets of transcription factors . Instead, the search is exhaustively conducted over combinations of up to four binding sites obtained from databases or motif search algorithms . We evaluate the performance on random sets of genes as a negative control and on three biologically validated sets of co-regulated genes in yeasts, flies and humans . We show that we can detect DNA regions that play a role in the control of transcription . These results shed light on the structure of transcription regulatory regions in eukaryotes and can be directly applied to clusters of co-expressed genes obtained in gene expression studies . Supplementary information is available at approximately kreiman/resources/cisregul/.

J Biol Chem, 2004 Aug 6, 279(32), 33899 - 908 Epub 2004 May 21.
Redesign of carnitine acetyltransferase specificity by protein engineering; Cordente AG et al.; In eukaryotes, L-carnitine is involved in energy metabolism by facilitating beta-oxidation of fatty acids . Carnitine acetyltransferases (CrAT) catalyze the reversible conversion of acetyl-CoA and carnitine to acetylcarnitine and free CoA . To redesign the specificity of rat CrAT toward its substrates, we mutated Met564 . The M564G mutated CrAT showed higher activity toward longer chain acyl-CoAs: activity toward myristoyl-CoA was 1250-fold higher than that of the wild-type CrAT, and lower activity toward its natural substrate, acetyl-CoA . Kinetic constants of the mutant CrAT showed modification in favor of longer acyl-CoAs as substrates . In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in carnitine octanoyltransferase (COT) decreased activity toward its natural substrates, medium- and long-chain acyl-CoAs, and increased activity toward short-chain acyl-CoAs . Another CrAT mutant, M564A, was prepared and tested in the same way, with similar results . We conclude that Met564 blocks the entry of medium- and long-chain acyl-CoAs to the catalytic site of CrAT . Three-dimensional models of wild-type and mutated CrAT and COT support this hypothesis . We show for the first time that a single amino acid is able to determine the substrate specificity of CrAT and COT.

Vopr Pitan, 2004, 73(2), 17 - 21
{Biologically active food additives for correction of the chronic fatigue syndrome}; Dotsenko VA et al.; The syndrome of chronic fatigue is a rather new pathology, included in ADS 10 . There is no specific treatment . Vitamins and microelements are very important for the prevention and treatment of CFS . Of special significance are BAA on the basis of yarsts, which are universal in their biological chemical composition and contain high amounts of vitamins B, K and essential aminoacids . The aim this work is clinical diagnostic of "Nagipol" use, made on the basis of beer yeasts, for the prevention and treatment of CFS . Clinical-dietologic study showed that "Nagipol" biologically active additive, useful in CFS, prodvecins clinical status improvement in CFS, positively influencing cognitive CNS functions, symptoms of psychoemtional instability, normalizing blood biochemical parameters, can be recommended as one of the addition element in diet-therapy of patients with excessive body mass and otesitg with the associated CFS . Recommended "Nagipol" as preventive-medical dietetic means for this pathology.

Methods Mol Biol, 2004, 270, 277 - 86
A PCR-based method for gene deletion and protein tagging in Trypanosoma brucei; Arhin GK et al.; Sequence information on the Trypanosoma brucei genome is rapidly accumulating . As a consequence, there is a need for techniques to analyze gene function systematically . Here, we describe a polymerase chain reaction (PCR)-based method for direct gene deletion and the generation of epitope-tagged fusion proteins . The approach is based on methodologies developed for Saccharomyces cerevisiae and involves PCR amplification of a reporter cassette using primers containing flanking sequences specific to the target gene . The PCR product is then transfected directly into procyclic T . brucei cells, and homologous recombinants that carry the deleted or tagged target gene are identified.

Eur J Biochem, 2004 Jun, 271(11), 2189 - 203
Characterization of mammalian eIF4E-family members; Joshi B et al.; The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells . Through its interaction with the 5' cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome . The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns . Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates . Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis . Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen . Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure . However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities . eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs . Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs . Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast . It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.

Proc Natl Acad Sci U S A, 2004 Jun 1, 101(22), 8307 - 12 Epub 2004 May 18.
Structural basis for recognition of the AGNN tetraloop RNA fold by the double-stranded RNA-binding domain of Rnt1p RNase III; Wu H et al.; Specific recognition of double-stranded RNA (dsRNA) by dsRNA-binding domains (dsRBDs) is involved in a large number of biological and regulatory processes . Although structures of dsRBDs in complex with dsRNA have revealed how they can bind to dsRNA in general, these do not explain how a dsRBD can recognize specific RNAs . Rnt1p, a member of the RNase III family of dsRNA endonucleases, is a key component of the Saccharomyces cerevisiae RNA-processing machinery . The Rnt1p dsRBD has been implicated in targeting this endonuclease to its RNA substrates, by recognizing hairpins closed by AGNN tetraloops . We report the solution structure of Rnt1p dsRBD complexed to the 5' terminal hairpin of one of its small nucleolar RNA substrates, the snR47 precursor . The conserved AGNN tetraloop fold is retained in the protein-RNA complex . The dsRBD contacts the RNA at successive minor, major, and tetraloop minor grooves on one face of the helix . Surprisingly, neither the universally conserved G nor the highly conserved A are recognized by specific hydrogen bonds to the bases . Rather, the N-terminal helix fits snugly into the minor groove of the RNA tetraloop and top of the stem, interacting in a non-sequence-specific manner with the sugar-phosphate backbone and the two nonconserved tetraloop bases . Mutational analysis of residues that contact the tetraloop region show that they are functionally important for RNA processing in the context of the entire protein in vivo . These results show how a single dsRBD can convey specificity for particular RNA targets, by structure specific recognition of a conserved tetraloop fold.

J Cell Sci, 2004 Jun 1, 117(Pt 13), 2769 - 75 Epub 2004 May 18.
Role of the cytoskeleton in signaling networks; Forgacs G et al.; Intracellular signal transduction occurs through cascades of reactions involving dozens of proteins that transmit signals from the cell surface, through a crowded cellular environment filled with organelles and a filamentous cytoskeleton, to specific targets . Numerous signaling molecules are immobilized or transiently bound to the cytoskeleton, yet most models for signaling pathways have no specific role for this mesh, which is often presumed to function primarily as a scaffold that determines cell mechanics but not information flow . We combined analytical tools with several recently established large-scale protein-protein interaction maps for Saccharomyces cerevisiae to quantitatively address the role of the cytoskeleton in intracellular signaling . The results demonstrate that the network of signaling proteins is intimately linked to the cytoskeleton, suggesting that this interconnected filamentous structure plays a crucial and distinct functional role in signal transduction.

Mol Cell, 2004 May 21, 14(4), 479 - 89
Cellular stress alters the transcriptional properties of promoter-bound Mot1-TBP complexes; Geisberg JV et al.; Mot1 associates with transcriptionally active promoters, and it directly affects transcriptional activity in a positive or negative manner, depending on the gene . As determined by sequential chromatin immunoprecipitation, Mot1 co-occupies promoters with TBP, but not with TFIIB, TFIIA, or Pol II when cells are grown in normal conditions . This strongly suggests that the Mot1-TBP complex is transcriptionally inactive, and hence is in dynamic equilibrium with transcriptionally active forms of TBP . Surprisingly, in response to heat shock and other forms of environmental stress, Mot1 co-occupies promoters with TFIIB and elongation-competent Pol II, but not with TFIIA . This suggests that functional preinitiation complexes can contain Mot1 instead of TFIIA in vivo . Thus, Mot1-TBP complexes can exist in active and inactive forms that are regulated by environmental stress.

Mol Cell, 2004 May 21, 14(4), 457 - 64
Elongator interactions with nascent mRNA revealed by RNA immunoprecipitation; Gilbert C et al.; The histone acetyltransferase Elongator was originally isolated as a component of the elongating form of RNA polymerase II (RNAPII) and a plethora of data has since supported a role for the factor in transcription . However, recent data has suggested that it is predominantly cytoplasmic and does not associate with the DNA of transcribed genes in vivo . Here, we report that Elongator binds to RNA both in vitro and in vivo . Using a modified chromatin immunoprecipitation procedure, RNA immunoprecipitation (RIP), we show that Elongator is indeed present at several actively transcribed genes and that it associates with the nascent RNA emanating from elongating RNAPII along the entire coding region of a gene . These results strongly support a role for Elongator in transcript elongation.

Mol Cell, 2004 May 21, 14(4), 447 - 56
The Paf1 complex has functions independent of actively transcribing RNA polymerase II; Mueller CL et al.; The yeast Paf1 complex, minimally composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, was originally isolated in association with RNA polymerase II (Pol II) . Paf1 complex components are abundant and colocalize with Pol II on chromatin at promoters and in the coding regions of actively transcribed genes . Loss of Paf1 results in severe phenotypes and reduced amounts of other Paf1 factors, with little effect on abundance or chromatin distribution of Pol II, proteins important for transcriptional elongation (Spt5, Spt16), or RNA processing (Sub2) . Loss of Paf1 factors causes a reduction of Pol II Ser2 phosphorylation and shortened poly(A) tails, suggesting that the complex facilitates linkage of transcriptional and posttranscriptional events . Surprisingly, loss of Rtf1 or Cdc73, with little phenotypic consequence, results in loss of Paf1 factors from chromatin and a significant reduction in Paf1/Pol II association . Therefore, the major functions of Paf1 can be independent of actively transcribing Pol II.

Mol Cell, 2004 May 21, 14(4), 418 - 20
Stay close to your sister; Reid RJ et al.; Yeast Sir2 protein regulates chromatin structure and suppresses recombination at the multiple tandem rDNA array . In the current issue of Cell, Kobayashi and colleagues reinvestigate rDNA dynamics in sir2 strains and find that Sir2 is necessary to recruit cohesins to the array, which influences the nature of rDNA recombination events.

J Exp Clin Cancer Res, 2004 Mar, 23(1), 83 - 91
Induction of a multifactorial resistance phenotype by high paclitaxel selective pressure in a human ovarian carcinoma cell line; Violini S et al.; Paclitaxel (PTX) is a potent anti-neoplastic agent that is highly effective in treating ovarian cancer . Nevertheless, the emergence of PTX resistance has limited the control of this disease . To gain insight into the molecular alterations accompanying drug resistance in ovarian cancer, we generated a new stable PTX-resistant ovarian carcinoma cell line . CABA I cells, which display an intrinsic PTX resistance (IC50 = 800 ng/ml), were subjected to continuous exposure to PTX . From the residual surviving cells, the highly PTX-resistant line CABA-PTX (IC50 = 256000 ng/ml) was generated and stably maintained in vitro . Analysis of beta-tubulin expression indicated that only the HM40 and Hbeta9 isotypes were expressed in both parental and resistant cells . No specific point mutations in the HM40 were detected in either cell line, but expression levels of this isotype were significantly reduced (40%) in CABA-PTX cells . Hbeta9 levels were unchanged . In those cells, PTX resistance was associated with cross-resistance to vinblastine but not to methotrexate or 5-fluorouracil . Verapamil treatment did not reverse the intrinsic drug resistance of parental cells, but partially modulated the sensitivity of CABA-PTX cells to PTX and induced total sensitivity to vinblastine . No changes in the cell surface expression of the drug efflux pumps MRP1, MRP2 and P-glycoprotein were observed . PTX influx, monitored using a fluorescent drug derivative, was significantly reduced and delayed in CABA-PTX cells as compared to the parental cells . Together, these findings suggest that more than one mechanism is involved in PTX resistance, making CABA-PTX cell line a potentially valuable in vitro tool to study multifactorial acquired drug resistance in ovarian cancer.

Am J Hum Genet, 2004 Jul, 75(1), 146 - 50 Epub 2004 May 17.
Identification and functional analysis of a defect in the human ALG9 gene: definition of congenital disorder of glycosylation type IL; Frank CG et al.; Defects of lipid-linked oligosaccharide assembly lead to alterations of N-linked glycosylation known as "type I congenital disorders of glycosylation" (CDG) . Dysfunctions along this stepwise assembly pathway are characterized by intracellular accumulation of intermediate lipid-linked oligosaccharides, the detection of which contributes to the identification of underlying enzymatic defects . Using this approach, we have found, in a patient with CDG, a deficiency of the ALG9 alpha 1,2 mannosyltransferase enzyme, which causes an accumulation of lipid-linked-GlcNAc(2)Man(6) and -GlcNAc(2)Man(8) structures, which was paralleled by the transfer of incomplete oligosaccharides precursors to protein . A homozygous point-mutation 1567G-->A (amino acid substitution E523K) was detected in the ALG9 gene . The functional homology between the human ALG9 and Saccharomyces cerevisiae ALG9, as well as the deleterious effect of the E523K mutation detected in the patient with CDG, were confirmed by a yeast complementation assay lacking the ALG9 gene . The ALG9 defect found in the patient with CDG--who presented with developmental delay, hypotonia, seizures, and hepatomegaly--shows that efficient lipid-linked oligosaccharide synthesis is required for proper human development and physiology . The ALG9 defect presented here defines a novel form of CDG named "CDG-IL."

Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 8078 - 83 Epub 2004 May 17.
Cti1/C1D interacts with condensin SMC hinge and supports the DNA repair function of condensin; Chen ES et al.; Condensin is a conserved five-subunit complex containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits and plays a major role in mitotic chromosome condensation . Condensin also acts in interphase and is required for DNA repair and replication checkpoint control . We attempted to study the function of the condensin in greater detail by means of the isolation of interacting proteins with the two-hybrid system . Using the hinge domain of Cut3/SMC4 as bait, we found one Cut three-interacting (Cti) 14-kDa nuclear protein, Cti1 . GST pull-down assay and immunoprecipitation supported physical interaction between Cti1 and condensin . Cti1 is similar to human C1D, which associates tightly with genomic DNA and functions to activate DNA protein kinase . SpC1D is essential for viability . The null mutant could germinate but arrest after replication, indicating that it is required for interphase growth . Importantly, an elevated dosage of spC1D suppressed the temperature, UV irradiation, and hydroxyurea sensitivity of the mutant of Cnd2, a non-SMC subunit of condensin . Upon exposure to hydroxyurea, spC1D accumulated on the nuclear chromatin, and the fraction of spC1D that was chromatin-bound increased . Cti1 is the first example of the protein that interacts with the hinge domain of SMC . Cti1 may have a supporting role for the DNA repair function of condensin.

FEBS Lett, 2004 May 21, 566(1-3), 229 - 33
The TRAF6 RING finger domain mediates physical interaction with Ubc13; Wooff J et al.; Tumor necrosis factor receptor associated factor 6 (TRAF6) is an important signaling molecule involved in a diverse array of physiological processes . It has been proposed that TRAF6, a RING finger-containing protein, acts as a ubiquitin ligase (E3) and a target for Lys-63 linked polyubiquitination mediated by Ubc13-Uev, a ubiquitin conjugating (E2) complex . However, the physical interaction between TRAF6 and this E2 complex has not been reported . We used the yeast two-hybrid assay to demonstrate that TRAF6 indeed interacts with the E2 complex through its direct binding to Ubc13 . Either a single Cys-to-Ser substitution within the TRAF6 RING finger domain or an amino acid substitution on the Ubc13 surface, that is predicted to interact with RING finger proteins, is able to abolish the interaction . In addition, we found that TRAF6 can interact with itself and this self-interaction domain is mapped to the N-terminus containing the RING finger motif . Based on this study and our previous Ubc13-Uev structural analysis, the interface of Ubc13-TRAF6 RING finger can be predicted.

FEBS Lett, 2004 May 21, 566(1-3), 30 - 4
Molecular analysis of the interaction between palladin and alpha-actinin; Ronty M et al.; Palladin is a novel component of stress fiber dense regions . Antisense and transient overexpression studies have indicated an important role for palladin in the regulation of actin cytoskeleton . Palladin colocalizes and coimmunoprecipitates with alpha-actinin, a dense region component, but the molecular details and functional significance of the interaction have not been studied . We show here a direct association between the two proteins and have mapped the binding site within a short sequence of palladin and in the carboxy-terminal calmodulin domain of alpha-actinin . Using transfection-based targeting assays, we show that palladin is involved in targeting of alpha-actinin to specific subcellular foci indicating a functional interplay between the two actin-associated proteins.

Toxicology, 2004 Jul 1, 199(2-3), 207 - 17
Isolation and characterisation of a cytotoxic polyunsaturated aldehyde from the marine phytoplankter Phaeocystis pouchetii (Hariot) Lagerheim; Hansen E et al.; Several investigators have documented that the marine phytoplankter Phaeocystis pouchetii produce and excrete some compound that has adverse effects on its surroundings, but the chemical composition and structure of the active agent has so far been unknown . In the present study we used mass spectrometry to investigate the structural properties of the putative toxin . Colonial cells of P . pouchetii were collected along the coast of northern Norway and cultivated in the lab for a limited period of time prior to harvesting by filtration . Harvested cells and culture filtrate were extracted separately with organic solvents, and a yeast cell bioassay was used to track the toxic fractions during extraction and purification with HPLC . We found the organic extract from the culture filtrate to be toxic, and after purification with RP-HPLC the cytotoxic activity was recovered as one fraction . When the toxic fractions were pooled and analysed by GC-MS we were able to identify 2-trans-4-trans-decadienal by comparing retention time and fragmentation pattern to a commercial standard . This is the first report of a polyunsaturated aldehyde produced by a marine alga belonging to the class Haptophyceae, and this implies that production and release of these reactive compounds are not limited to diatoms.

Biochemistry, 2004 May 25, 43(20), 6200 - 7
Homodimeric mitochondrial phosphate transport protein . Transient subunit/subunit contact site between the transport relevant transmembrane helices A; Phelps A et al.; The three Cys of the yeast (Saccharomyces cerevisiae) mitochondrial phosphate transport protein (PTP) subunit were replaced with Ser . The seven mutants (single, double, and complete Cys replacements) were expressed in yeast, and the homodimeric mutant PTPs were purified from the mitochondria and reconstituted . The pH gradient-dependent net phosphate (Pi) transport uptake rates (initial conditions: 1 mM {Pi}e, pHe 6.80; 0 mM {Pi}i, pHi 8.07) catalyzed by these reconstituted mutants are similar to those of the wild-type protein and range from 15 to 80 micromol Pi/min mg PTP protein . Aerobic media inhibit only the Pi uptake rates catalyzed by PTPs with the conserved (yeast and bovine) Cys28 . This inhibition in the proteoliposomes is 84-95% and can be completely reversed by dithiothreitol . Transport by the wild type as well as by all mutant proteins with Cys28 is more than 90% inhibited by mersalyl . Transport catalyzed by mutant proteins with only Cys300 or only Cys134 is less sensitive, and that catalyzed by the no Cys mutant shows 40% inhibition by mersalyl . When dithiothreitol is removed from purified single Cys mutant proteins, only the mutant protein with Cys28 appears as a homodimer in a nonreducing SDS polyacrylamide gel . Thus, the function relevant transmembrane helix A, with Cys 28 about equidistant from the two inner membrane surfaces, is in close contact with parts of transmembrane helix A of the other subunit in the functional homodimeric PTP . The results identify for the first time not only a transmembrane helix contact site between the two subunits of a homodimeric mitochondrial transport protein but also a contact site that if locked into position blocks transport . The results are related to two available secondary transporter structures (lactose permease, glycerol-3-phosphate transporter) as well as to a low resolution projection structure and a high resolution structure of monomers of inhibitor ADP/ATP carrier complexes.

Nat Genet, 2004 Jun, 36(6), 636 - 41 Epub 2004 May 16.
NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome; Tonkin ET et al.; Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects . We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs . We named its protein product delangin . Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D . melanogaster Nipped-B . We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS . Genome analyses typically identify individual delangin or Nipped-B-like orthologs in diploid animal and plant genomes . The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.

RNA, 2004 Jun, 10(6), 895 - 7; discussion 898
Reevaluation of the conclusion that IRES-activity reported within the 5' leader of the TIF4631 gene is due to promoter activity; Mauro VP et al.; We previously reported that the 5' leader of the mRNA-encoding initiation factor eIF4G in Saccharomyces cerevisiae can function as a translational enhancer and as an internal ribosome entry site (IRES) when tested in cells . However, Verge and colleagues recently suggested that this sequence does not facilitate translation initiation, but inhibits translation in vitro and has promoter activity when tested in cells . We disagree with these conclusions and respond by showing that the data are most consistent with an internal initiation mechanism.

Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7868 - 73 Epub 2004 May 14.
Identification of Ald6p as the target of a class of small-molecule suppressors of FK506 and their use in network dissection; Butcher RA et al.; FK506 inhibits the Ca2+/calmodulin-dependent protein phosphatase calcineurin, which plays a critical role in yeast subjected to salt stress . A chemical genetic screen for small molecules that suppress growth inhibition by high NaCl plus FK506 identified a structurally related class of suppressors of FK506 (SFKs) named SFKs 2-4 . To identify possible protein targets for these small molecules, a genome-wide screen of approximately 4,700 haploid yeast deletion strains was undertaken for strains showing resistance to high NaCl plus FK506 . This screen yielded a number of genes not previously implicated in salt stress, including ALD6, which encodes an NADP(+)-dependent aldehyde dehydrogenase, and UTR1, which encodes an NAD+ kinase . Transcriptional profiling of yeast treated with SFK2 indicated that the SFKs target the Ald6p pathway . In addition, screening of the deletion strains for hypersensitivity to SFK2 yielded ZWF1, encoding glucose-6-phosphate dehydrogenase, which has been shown to play an overlapping role with Ald6p in NADPH production . Furthermore, the SFKs inhibited the activity of Ald6p in vitro . Having established that the SFKs target Ald6p, they were used as tools to implicate systematically other gene products in the Ald6p pathway, including Utr1p, which may function by supplying Ald6p with its NADP+ cofactor . Furthermore, growth improvement by the SFKs on high NaCl plus FK506 was shown to require GPD1, which encodes an NADH-dependent glycerol-3-phosphate dehydrogenase that is important for the production of glycerol in response to osmotic stress.

J Biol Chem, 2004 Jul 16, 279(29), 30182 - 8 Epub 2004 May 15.
Chemical clamping allows for efficient phosphorylation of the RNA carrier protein Npl3; Aubol BE et al.; Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation . Although these distal contacts may provide excellent binding affinities (low Km values) through stabilization of the enzyme-substrate complex, these contacts could reduce catalytic turnover (decrease kcat) through slow phosphoprotein release . To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated . Sky1p bound and phosphorylated Npl3 with a Km that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants . Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding . Rather, Npl3 achieves a low Km through a rapid and favorable phosphoryl transfer step . This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release . The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.

J Biol Chem, 2004 Jul 23, 279(30), 31910 - 20 Epub 2004 May 15.
Efficient incorporation of eukaryotic initiation factor 1 into the multifactor complex is critical for formation of functional ribosomal preinitiation complexes in vivo; Singh CR et al.; Eukaryotic initiation factor 1 (eIF1) is a low molecular weight factor critical for stringent AUG selection in eukaryotic translation . It is recruited to the 43 S complex in the multifactor complex (MFC) with eIF2, eIF3, and eIF5 via multiple interactions with the MFC constituents . Here we show that FLAG epitope tagging of eIF1 at either terminus abolishes its in vitro interactions with eIF5 and eIF2beta but not that with eIF3c . Nevertheless, both forms of FLAG-eIF1 fail to bind eIF3 and are incorporated into the 43 S complex inefficiently in vivo . C-terminal FLAG tagging of eIF1 is lethal; overexpression of C-terminal FLAG-eIF1 severely impedes 43 S complex formation and derepresses GCN4 translation due to limiting of eIF2.GTP.Met-tRNA(i)(Met) ternary complex binding to the ribosome . Furthermore, N-terminal FLAG-eIF1 overexpression reduces eIF2 binding to the ribosome and moderately derepresses GCN4 translation . Our results provide the first in vivo evidence that eIF1 plays an important role in promoting 43 S complex formation as a core of factor interactions . We propose that the coordinated recruitment of eIF1 to the 40 S ribosome in the MFC is critical for the production of functional 40 S preinitiation complex.

J Biol Chem, 2004 Jul 23, 279(30), 31943 - 7 Epub 2004 May 15.
COX23, a homologue of COX17, is required for cytochrome oxidase assembly; Barros MH et al.; Deletion of reading frame YHR116W of the Saccharomyces cerevisiae nuclear genome elicits a respiratory deficiency . The encoded product, here named Cox23p, is shown to be required for the expression of cytochrome oxidase . Cox23p is homologous to Cox17p, a water-soluble copper protein previously implicated in the maturation of the Cu(A) center of cytochrome oxidase . The respiratory defect of a cox23 null mutant is rescued by high concentrations of copper in the medium but only when the mutant harbors COX17 on a high copy plasmid . Overexpression of Cox17p by itself is not a sufficient condition to rescue the mutant phenotype . Cox23p, like Cox17p, is detected in the intermembrane space of mitochondria and in the postmitochondrial supernatant fraction, the latter consisting predominantly of cytosolic proteins . Because Cox23p and Cox17p are not part of a complex, the requirement of both for cytochrome oxidase assembly suggests that they function in a common pathway with Cox17p acting downstream of Cox23p.

Ukr Biokhim Zh, 2003 Nov-Dec, 75(6), 106 - 10
{Interferonogenic activity of immobilized ribopolynucleotides in vitro}; Zholobak NM et al.; In vitro experiments the authors have studied a property of yeast RNA--tilorone hydrochloride complex covalently linked to spheron to induce the synthesis of interferons type I (alpha- and beta-interferons) in the culture of peripheral mononuclear human cells . Such a complex is shown to possess a marked interferonogenic activity . The data obtained appear to be a proof of the interferon induction to be realised by a mechanism needing at the first stage the contact between the inducer and the cell surface without its penetration into the cell.

Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7885 - 90 Epub 2004 May 13.
A model for Ure2p prion filaments and other amyloids: the parallel superpleated beta-structure; Kajava AV et al.; In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold . The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, "prion" domain . The prion domain is also capable of forming fibrils when alone or when fused to other proteins . We have developed a model for the fibril that we call a parallel superpleated beta-structure . In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement . Serpentines are stacked axially, in register, generating an array of parallel beta-sheets, with a small and potentially variable left-hand twist . The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded . The model is consistent with current biophysical, biochemical, and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.

Mol Cell Biol, 2004 Jun, 24(11), 4869 - 79
The nuclear pore complex and the DEAD box protein Rat8p/Dbp5p have nonessential features which appear to facilitate mRNA export following heat shock; Rollenhagen C et al.; Nuclear pore complexes (NPCs) play an essential role in RNA export . Nucleoporins required for mRNA export in Saccharomyces cerevisiae are found in the Nup84p and Nup82p subcomplexes of the NPC . The Nup82p subcomplex contains Nup82p, Rat7p/Nup159p, Nsp1p, Gle1p/Rss1p, and Rip1p/Nup42p and is found only on the cytoplasmic face of NPCs . Both Rat7p and Gle1p contain binding sites for Rat8p/Dbp5p, an essential DEAD box protein and putative RNA helicase . Rip1p interacts directly with Gle1p and is the only protein known to be essential for mRNA export after heat shock but not under normal growth conditions . We report that in cells lacking Rip1p, both Gle1p and Rat8p dissociate from NPCs following heat shock at 42 degrees C . Rat8p but not Gle1p was retained at NPCs if rip1Delta cells were first shifted to 37 degrees C and then to 42 degrees C, and this was correlated with preserving mRNA export in heat-shocked rip1Delta cells . Export following ethanol shock was less dependent on the presence of Rip1p . Exposure to 10% ethanol led to dissociation of Rat8p from NPCs in both wild-type and rip1Delta cells . Following this treatment, Rat8p was primarily nuclear in wild-type cells but primarily cytoplasmic in rip1Delta cells . We also determined that efficient export of heat shock mRNA after heat shock depends upon a novel 6-amino-acid element within Rat8p . This motif is not required under normal growth conditions or following ethanol shock . These studies suggest that the molecular mechanism responsible for the defect in export of heat shock mRNAs in heat-shocked rip1Delta cells is dissociation of Rat8p from NPCs . These studies also suggest that both nuclear pores and Rat8p have features not required for mRNA export in growing cells but which enhance the ability of mRNAs to be exported following heat shock.

Mol Cell Biol, 2004 Jun, 24(11), 4734 - 42
Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2; Sawa C et al.; Bromodomain factor 1 (Bdf1) associates with Saccharomyces cerevisiae TFIID and corresponds to the C-terminal half of higher eukaryotic TAF1 . It also associates with the SWR-C complex, which is important for Htz1 deposition . Bdf1 binds preferentially to acetylated histone H4 . Bdf1 is phosphorylated, but the mechanism and significance of this modification have been unclear . Two distinct regions within Bdf1 are phosphorylated; one is just C terminal to the bromodomains and the other is near the C terminus . Mutational analysis shows that phosphorylation is necessary for Bdf1 function in vivo . Endogenous protein kinase CK2 purifies with Bdf1 and phosphorylates both domains . A similar mechanism may be responsible for phosphorylation of the C-terminal region of mammalian TAF1 . These findings suggest that CK2 phosphorylation of Bdf1 may regulate RNA polymerase II transcription and/or chromatin structure.

Mol Plant Microbe Interact, 2004 May, 17(5), 547 - 56
Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea; Li L et al.; In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth . PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways . In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M . grisea . Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection . In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly . Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds . The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology . Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced . On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology . However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis . These data suggest that MST20 is dispensable for plant infection in M . grisea, but CHM1 plays a critical role in appressorium formation and penetration . Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth . It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth . However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M . grisea.

EMBO J, 2004 Jun 2, 23(11), 2281 - 92 Epub 2004 May 13.
The unfolded protein response represses differentiation through the RPD3-SIN3 histone deacetylase; Schroder M et al.; In Saccharomyces cerevisiae, splicing of HAC1 mRNA is initiated in response to the accumulation of unfolded proteins in the endoplasmic reticulum by the transmembrane kinase-endoribonuclease Ire1p . Spliced Hac1p (Hac1(i)p) is a negative regulator of differentiation responses to nitrogen starvation, pseudohyphal growth, and meiosis . Here we show that the RPD3-SIN3 histone deacetylase complex (HDAC), its catalytic activity, recruitment of the HDAC to the promoters of early meiotic genes (EMGs) by Ume6p, and the Ume6p DNA-binding site URS1 in the promoters of EMGs are required for nitrogen-mediated negative regulation of EMGs and meiosis by Hac1(i)p . Co-immunoprecipitation experiments demonstrated that Hac1(i)p can interact with the HDAC in vivo . Systematic analysis of double deletion strains revealed that HAC1 is a peripheral component of the HDAC . In summary, nitrogen-induced synthesis of Hac1(i)p and association of Hac1(i)p with the HDAC are physiological events in the regulation of EMGs by nutrients . These data also define for the first time a gene class that is under negative control by the UPR, and provide the framework for a novel mechanism through which bZIP proteins repress transcription.

Sci Aging Knowledge Environ . 2004 May 12;2004(19):pe19.
Vital connections; Pletcher SD; Scientists are currently exploring the structure and behavior of complex biological systems, which consist of networks of interacting entities . A recent paper in Proceedings of the Royal Society of London Series B Biological Sciences describes the distinctive characteristics of aging-related proteins in the yeast protein-protein interaction network . In this Perspective, I discuss the implications of these findings for longevity research.

J Virol, 2004 Jun, 78(11), 5900 - 12
Processing of a pestivirus protein by a cellular protease specific for light chain 3 of microtubule-associated proteins; Fricke J et al.; The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p . The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p . Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with the Saccharomyces cerevisiae enzyme . The search for a system for functional testing of these putative LC3-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts . Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeast AUT2 deletion mutant . In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3 . Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2 . However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.

J Biol Chem, 2004 Aug 27, 279(35), 36219 - 27 Epub 2004 May 12.
Furin inhibition by compounds of copper and zinc; Podsiadlo P et al.; Furin, a human subtilisin-related proprotein convertase (SPC), is emerging as an important pharmaceutical target because it processes vital proteins of many aggressive pathogens . Furin inhibitors reported as yet are peptide derivatives and proteins, with the exception of andrographolides, which are natural compounds . Here we report that the small and highly stable compounds M(chelate)Cl(2) (M is copper or zinc) inhibit furin and Kex2, with Cu(TTP)Cl(2) and Zn(TTP)Cl(2) as the most efficient inhibitors . (TTP is 4'-{p-tolyl}-2,2 ':6',2"-terpyridine.) Inhibition is irreversible, competitive with substrate, and affected by substituents on the chelate . The free chelates are not inhibitors . Solvated Zn(2+) is less potent than its complexes . This is true also for copper and Kex2 . However, solvated Cu(2+) (k(on) of 25,000 +/- 2,500 s(-1)) is more potent than Cu(TTP)Cl(2) (k(on) = 140 +/- 13 s(-1) and allows recovery of furin activity prior to a second inhibition phase . A mechanism that involves coordination to the catalytic histidine is proposed for all inhibitors . Target specificity is indicated by the fact that these metal chelate inhibitors are much less potent toward Kex2, the yeast homologue of furin . For example, k(on) with Zn(TTP)Cl(2) is 120 +/- 20 s(-1) for furin, but only 1.2 +/- 0.1 s(-1) for Kex2.

Physiol Genomics, 2004 Jul 08, 18(2), 196 - 205
A combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression; Conour JE et al.; The intracellular reduction-oxidation (redox) environment influences cell cycle progression; however, underlying mechanisms are poorly understood . To examine potential mechanisms, the intracellular redox environment was characterized per cell cycle phase in Chinese hamster ovary fibroblasts via flow cytometry by measuring reduced glutathione (GSH), reactive oxygen species (ROS), and DNA content with monochlorobimane, 2',7'-dichlorohydrofluorescein diacetate (H2DCFDA), and DRAQ5, respectively . GSH content was significantly greater in G2/M compared with G1 phase cells, whereas GSH was intermediate in S phase cells . ROS content was similar among phases . Together, these data demonstrate that G2/M cells are more reduced than G1 cells . Conventional approaches to define regulatory mechanisms are subjective in nature and focus on single proteins/pathways . Proteome databases provide a means to overcome these inherent limitations . Therefore, a novel bioinformatic approach was developed to exhaustively identify putative redox-regulated cell cycle proteins containing redox-sensitive protein motifs . Using the InterPro database, we categorized 536 redox-sensitive motifs as: 1) active/functional-site cysteines, 2) electron transport, 3) heme, 4) iron binding, 5) zinc binding, 6) metal binding (non-Fe/Zn), and 7) disulfides . Comparing this list with 1,634 cell cycle-associated proteins from Swiss-Prot and SpTrEMBL revealed 92 candidate proteins . Three-fourths (69 of 92) of the candidate proteins function in the central cell cycle processes of transcription, nucleotide metabolism, (de)phosphorylation, and (de)ubiquitinylation . The majority of oxidant-sensitive candidate proteins (68.9%) function during G2/M phase . As the G2/M phase is more reduced than the G1 phase, oxidant-sensitive proteins may be temporally regulated by oscillation of the intracellular redox environment . Combined with evidence of intracellular redox compartmentalization, we propose a spatiotemporal mechanism that functionally links an oscillating intracellular redox environment with cell cycle progression.

J Biol Chem, 2004 Aug 6, 279(32), 33816 - 28 Epub 2004 May 11.
The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large; Ralston KJ et al.; Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response . But how clustering is achieved and sustained is not known . Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G . Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large . We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G . T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G . We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

J Biol Chem, 2004 Jul 16, 279(29), 30150 - 7 Epub 2004 May 11.
Mechanisms and functional properties of two peptide transporters, AtPTR2 and fPTR2; Chiang CS et al.; The Arabidopsis AtPTR2 and fungal fPTR2 genes, which encode H+/dipeptide cotransporters, belong to two different subgroups of the peptide transporter (PTR) (NRT1) family . In this study, the kinetics, substrate specificity, stoichiometry, and voltage dependence of these two transporters expressed in Xenopus oocytes were investigated using the two-microelectrode voltage-clamp method . The results showed that: 1) although AtPTR2 belongs to the same PTR family subgroup as certain H+/nitrate cotransporters, neither AtPTR2 nor fPTR2 exhibited any nitrate transporting activity; 2) AtPTR2 and fPTR2 transported a wide spectrum of dipeptides with apparent affinity constants in the range of 30 microM to 3 mM, the affinity being dependent on the side chain structure of both the N- and C-terminal amino acids; 3) larger maximal currents (Imax) were evoked by positively charged dipeptides in AtPTR2- or fPTR2-injected oocytes; 4) a major difference between AtPTR2 and fPTR2 was that, whereas fPTR2 exhibited low Ala-Asp- transporting activity, AtPTR2 transported Ala-Asp- as efficiently as some of the positively charged dipeptides; 5) kinetic analysis suggested that both fPTR2 and AtPTR2 transported by a random binding, simultaneous transport mechanism . The results also showed that AtPTR2 and fPTR2 were quite distinct from PepT1 and PepT2, two well characterized animal PTR transporters in terms of order of binding of substrate and proton(s), pH sensitivity, and voltage dependence.

J Biol Chem, 2004 Jul 16, 279(29), 29889 - 94 Epub 2004 May 11.
Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway; Shintani T et al.; Selective incorporation of cargo proteins into the forming vesicle is an important aspect of protein targeting via vesicular trafficking . Based on the current paradigm of cargo selection in vesicular transport, proteins to be sorted to other organelles are condensed at the vesicle budding site in the donor organelle, a process that is mediated by the interaction between cargo and coat proteins, which constitute part of the vesicle forming machinery . The cytoplasm to vacuole targeting (Cvt) pathway is an unconventional vesicular trafficking pathway in yeast, which is topologically and mechanistically related to autophagy . Aminopeptidase I (Ape1) is the major cargo protein of the Cvt pathway . Unlike the situation in conventional vesicular transport, precursor Ape1, along with its receptor Atg19/Cvt19, is packed into a huge complex, termed a Cvt complex, independent of the vesicle formation machinery . The Cvt complex is subsequently incorporated into the forming Cvt vesicle . The deletion of APE1 or ATG19 compromised the organization of the pre-autophagosomal structure (PAS), a site that is thought to play a critical role in Cvt vesicle/autophagosome formation . The proper organization of the PAS also required Atg11/Cvt9, a protein that localizes the cargo complex at the PAS . Accordingly, the deletion of APE1, ATG19, or ATG11 affected the formation of Cvt vesicles . These observations suggest a unique concept; in the case of the Cvt pathway, the cargo proteins facilitate receptor recruitment and vesicle formation rather than the situation with most vesicular transport, in which the forming vesicle concentrates the cargo proteins.

J Biol Chem, 2004 Jul 16, 279(29), 30307 - 15 Epub 2004 May 11.
The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf-Hirschhorn syndrome; Nowikovsky K et al.; The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa . Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype . We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane . Deletion of the yeast YOL027 gene (yol027Delta mutation) results in mitochondrial dysfunction . This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man . Mutant yol027Delta mitochondria have increased cation contents, particularly K+ and low-membrane-potential Deltapsi . They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K+/H+ exchange activity . Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K+ homeostasis and organelle volume control.

Cell, 2004 May 14, 117(4), 495 - 502
Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans; Bieganowski P et al.; NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems . Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s . The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast . Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin . Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.

Cell, 2004 May 14, 117(4), 455 - 69
Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA; D'Amours D et al.; Chromosome segregation is triggered by the cleavage of cohesins by separase . Here we show that in budding yeast separation of the ribosomal DNA (rDNA) and telomeres also requires Cdc14, a protein phosphatase known for its role in mitotic exit . Cdc14 shares this role with the FEAR network, which activates Cdc14 during early anaphase, but not the mitotic exit network, which promotes Cdc14 activity during late anaphase . We further show that CDC14 is necessary and sufficient to promote condensin enrichment at the rDNA locus and to trigger rDNA segregation in a condensin-dependent manner . We propose that Cdc14 released by the FEAR network mediates the partitioning of rDNA by facilitating the localization of condensin thereto . This dual role of the FEAR network in initiating mitotic exit and promoting chromosome segregation ensures that exit from mitosis is coupled to the completion of chromosome segregation.

Cell, 2004 May 14, 117(4), 427 - 39
Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization; Casolari JM et al.; The association of genes with the nuclear pore complex (NPC) and nuclear transport factors has been implicated in transcriptional regulation . We therefore examined the association of components of the nuclear transport machinery including karyopherins, nucleoporins, and the Ran guanine-nucleotide exchange factor (RanGEF) with the Saccharomyces cerevisiae genome . We find that most nucleoporins and karyopherins preferentially associate with a subset of highly transcribed genes and with genes that possess Rap1 binding sites whereas the RanGEF preferentially associates with transcriptionally inactive genes . Consistent with coupling of transcription to the nuclear pore, we show that transcriptional activation of the GAL genes results in their association with nuclear pore proteins, relocation to the nuclear periphery, and loss of RanGEF association . Taken together, these results indicate that the organization of the genome is coupled via transcriptional state to the nuclear transport machinery.

J Biol Chem, 2004 Jul 16, 279(29), 30047 - 52 Epub 2004 May 10.
Two conserved cysteine triads in human Ero1alpha cooperate for efficient disulfide bond formation in the endoplasmic reticulum; Bertoli G et al.; Human Ero1alpha is an endoplasmic reticulum (ER)-resident protein responsible for protein disulfide isomerase (PDI) oxidation . To clarify the molecular mechanisms underlying its function, we generated a panel of cysteine replacement mutants and analyzed their capability of: 1) complementing a temperature-sensitive yeast Ero1 mutant, 2) favoring oxidative folding in mammalian cells, 3) forming mixed disulfides with PDI and ERp44, and 4) adopting characteristic redox-dependent conformations . Our results reveal that two essential cysteine triads (Cys85-Cys94-Cys99 and Cys391-Cys394-Cys397) cooperate in electron transfer, with Cys94 likely forming mixed disulfides with PDI . Dominant negative phenotypes arise when critical residues within the triads are mutated (Cys394, Cys397, and to a lesser extent Cys99) . Replacing the first cysteine in either triad (Cys85 or Cys391) generates mutants with weaker activity . In addition, mutating either Cys85 or Cys391, but not Cys397, reverts the dominant negative phenotype of the C394A mutant . These findings suggest that interactions between the two triads, dependent on Cys85 and Cys391, are important for Ero1alpha function, possibly stabilizing a platform for efficient PDI oxidation.

J Biol Chem, 2004 Jul 16, 279(29), 30791 - 9 Epub 2004 May 10.
Probing the function of STAS domains of the Arabidopsis sulfate transporters; Shibagaki N et al.; Sulfate transporters in plants and animals are structurally conserved and have an amino-terminal domain that functions in transport and a carboxyl-terminal region that has been designated the STAS domain . The STAS domain in sulfate transporters has significant similarity to bacterial anti-sigma factor antagonists . To determine if the STAS domain has a role in controlling the activity of sulfate transporters, their stability, or their localization to the plasma membrane, we examined the effect of deleting or modifying the STAS domain of dominant sulfate transporters in roots of Arabidopsis thaliana . The A . thaliana Sultr1;2 and Sultr1;1 sulfate transporters rescue the methionine-dependent growth phenotype of the yeast sulfate transporter mutant strain CP154-7B . Constructs of Sultr1;2 in which the STAS domain was deleted (DeltaSTAS) resulted in synthesis of a truncated polypeptide that was unable to rescue the CP154-7B phenotype . The inability of these constructs to rescue the mutant phenotype probably reflected both low level cellular accumulation of the transporter and the inability of the truncated protein to localize to the plasma membrane . Fusing the STAS domain from other sulfate transporters to Sultr1;2 DeltaSTAS constructs restored elevated accumulation and plasma membrane localization, although the kinetics of sulfate uptake in the transformants were markedly altered with respect to transformants synthesizing wild-type Sultr1;2 protein . These results suggest that the STAS domain is essential, either directly or indirectly, for facilitating localization of the transporters to the plasma membrane, but it also appears to influence the kinetic properties of the catalytic domain of transporters.

J Mol Biol, 2004 May 28, 339(2), 259 - 64
The N-terminal prion domain of Ure2p converts from an unfolded to a thermally resistant conformation upon filament formation; Baxa U et al.; According to the "amyloid backbone" model of Ure2p prionogenesis, the N-terminal domain of Ure2p polymerizes to form an amyloid filament backbone surrounded by the C-terminal domains . The latter domains retain their native glutathione-S-transferase (GST)-like fold but are sterically inactivated from their regulatory role in nitrogen catabolism . We have tested this model by differential scanning calorimetry of soluble and filamentous Ure2p and of soluble C-terminal domains, combined with electron microscopy . As predicted, the C-terminal domains respond to thermal perturbation identically in all three states, exhibiting a single endotherm at 76 degrees C . In contrast, no thermal signal was associated with the N-terminal domains: in the soluble state of Ure2p, because they are unfolded; in the filamentous state, because their robust amyloid conformation resists heating to 100 degrees C.

FEMS Microbiol Lett, 2004 May 15, 234(2), 189 - 99
Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses; Dasgupta A et al.; A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation . This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA . Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery . In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation . We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation . The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements . These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins .

Immunol Lett, 2004 Apr 30, 93(1), 71 - 8
Microparticulate beta-glucan upregulates the expression of B7.1, B7.2, B7-H1, but not B7-DC on cultured murine peritoneal macrophages; Hunter KW Jr et al.; Beta-1,3-(D)-glucan from a variety of biological sources has been shown to enhance both humoral and cellular immune responses to a variety of antigens, infectious agents, and tumors . Nevertheless, its mode of action has not been fully defined . We sought to determine whether a 1-2 microm diameter microparticulate form of beta-glucan (MG) from the yeast Saccharomyces cerevisiae could regulate expression of B7 family glycoproteins on resident peritoneal macrophages from BALB/c mice . We discovered that MG uregulated B7.2 mRNA expression and enhanced the surface membrane expression of B7.2 glycoprotein . Although B7.1 mRNA was not upregulated above constitutive levels, MG treatment enhanced B7.1 glycoprotein expression on the macrophages, albeit to a lesser extent than B7.2 . At the same time, the gene and cell surface expression of B7-H1, a putative negative regulator of T cell activity, was also upregulated by MG . The expression of B7-DC, another B7 family molecule with negative regulatory activity, was not affected by incubation with MG . This study has demonstrated that a microparticulate form of beta-glucan can enhance B7 co-stimulatory molecule expression on macrophages, thereby enabling these antigen-presenting cells to deliver the second signal to T-lymphocytes that express CD28 . In addition, because MG also induces the expression of B7-H1, it may enable macrophages to provide a concomitant downregulatory signal to T-lymphocytes expressing PD-1 or related receptors.

Biochemistry, 2004 May 18, 43(19), 5637 - 50
Interaction of the retinal insulin receptor beta-subunit with the p85 subunit of phosphoinositide 3-kinase; Rajala RV et al.; Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit . In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta . Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322 . The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system . Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85 . We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins . Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates . On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina . These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.

Biochemistry, 2004 May 18, 43(19), 5592 - 9
Nucleotide excision repair defect influences lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent in the absence of base excision repair; Monti P et al.; Using a yeast shuttle vector system, we have previously reported on the toxicity and mutagenicity of Me-lex, {1-methyl-4-{1-methyl-4-{3-(methoxysulfonyl)propanamido}pyrrole-2-carboxamido}pyrrole-2-carboxamido}propane, a compound that selectively generates 3-methyladenine (3-MeA) . We observed that a mutant strain defective in Mag1, the glycosylase that excises 3-MeA in the initial step of base excision repair (BER) to generate an abasic site, is significantly more sensitive to the toxicity of Me-lex with respect to wild type but shows only a marginal increase in mutagenicity . A strain defective in AP endonuclease activity (Deltaapn1apn2), also required for functional BER, is equally sensitive to the toxicity as the Deltamag1 mutant but showed a significantly higher mutation frequency . In the present work, we have explored the role of nucleotide excision repair (NER) in Me-lex-induced toxicity and mutagenicity since it is known that NER acts on abasic sites in vivo in yeast and in vitro assays . To accomplish this, we have deleted one of the genes essential for NER in yeast, namely, RAD14, both in the context of an otherwise DNA repair-proficient strain (Deltarad14) and in a BER-defective isogenic derivative lacking the MAG1 gene (Deltamag1rad14) . Interestingly, no sensitivity to the treatment with Me-lex was conferred by the simple deletion of RAD14 . However, a significant enhancement in toxicity and mutagenicity was observed when cells lacked both Rad14 and Mag1 . The mutation spectrum induced by Me-lex in the Deltamag1rad14 strain is indistinguishable from that observed in the Deltaapn1/Deltaapn2 or in the Deltamag1 strains . The results indicate that in yeast NER can play a protective role against 3-MeA-mediated toxicity and mutagenicity; however, the role of NER is appreciable only in a BER-defective background.

Mol Biol Cell, 2004 Jul, 15(7), 3406 - 17 Epub 2004 May 07.
Peroxisomal membrane proteins contain common Pex19p-binding sites that are an integral part of their targeting signals; Rottensteiner H et al.; Targeting of peroxisomal membrane proteins (PMPs) is a multistep process that requires not only recognition of PMPs in the cytosol but also their insertion into the peroxisomal membrane . As a consequence, targeting signals of PMPs (mPTS) are rather complex . A candidate protein for the PMP recognition event is Pex19p, which interacts with most PMPs . However, the respective Pex19p-binding sites are ill-defined and it is currently disputed whether these sites are contained within mPTS . By using synthetic peptide scans and yeast two-hybrid analyses, we determined and characterized Pex19p-binding sites in Pex11p and Pex13p, two PMPs from Saccharomyces cerevisiae . The sites turned out to be composed of a short helical motif with a minimal length of 11 amino acids . With the acquired data, it proved possible to predict and experimentally verify Pex19p-binding sites in several other PMPs by applying a pattern search and a prediction matrix . A peroxisomally targeted Pex13p fragment became mislocalized to the endoplasmic reticulum in the absence of its Pex19p-binding site . By adding the heterologous binding site of Pex11p, peroxisomal targeting of the Pex13p fragment was restored . We conclude that Pex19p-binding sites are well-defined entities that represent an essential part of the mPTS.

Genes Dev, 2004 May 1, 18(9), 1022 - 34
Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA; Warfield L et al.; A surface that is required for rapid formation of preinitiation complexes (PICs) was identified on the N-terminal domain (NTD) of the RNA Pol II general transcription factor TFIIA . Site-specific photocross-linkers and tethered protein cleavage reagents positioned on the NTD of TFIIA and assembled in PICs identified the SAGA subunit Spt8 and the TFIID subunit Taf4 as located near this surface . In agreement with these findings, mutations in Spt8 and the TFIIA NTD interact genetically . Using purified proteins, it was found that TFIIA and Spt8 do not stably bind to each other, but rather both compete for binding to TBP . Consistent with this competition, Spt8 inhibits the binding of SAGA to PICs in the absence of activator . In the presence of activator, Spt8 enhances transcription in vitro, and the positive function of the TFIIA NTD is largely mediated through Spt8 . Our results suggest a mechanism for the previously observed positive and negative effects of Spt8 on transcription observed in vivo.

Genes Dev, 2004 May 1, 18(9), 992 - 1006
Pol12, the B subunit of DNA polymerase alpha, functions in both telomere capping and length regulation; Grossi S et al.; The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood . Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, as a new factor involved in this process . Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing . Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1 . Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping . These data reveal a novel function for Pol12 and a new connection between DNA polymerase alpha and Stn1 . We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.

Blood, 2004 Aug 15, 104(4), 1183 - 90 Epub 2004 May 06.
CD63 tetraspanin slows down cell migration and translocates to the endosomal-lysosomal-MIICs route after extracellular stimuli in human immature dendritic cells; Mantegazza AR et al.; We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration . We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs . Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles . Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated "outside-in" and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class II-enriched compartments (MIICs) in less than one hour . Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis . Moreover, an association of CD63 with the beta-glycan receptor dectin-1 was observed . Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP-5) and MIP-1alpha . Concomitantly, Mabs against CD63 and CD82 diminished the surface expression of CD29, CD11b, CD18, and alpha5 integrins . By immunoprecipitation experiments we found that CD63 associated with integrins CD11b and CD18 . These results suggest that CD9, CD63, CD81, and CD82 could play a role in modulating the interactions between immature DCs and their environment, slowing their migratory ability . However, only CD63 would intervene in the internalization of complex antigens.

Appl Bioinformatics, 2002, 1(4), 191 - 222
Artificial intelligence techniques for bioinformatics; Narayanan A et al.; This review provides an overview of the ways in which techniques from artificial intelligence (AI) can be usefully employed in bioinformatics, both for modelling biological data and for making new discoveries . The paper covers three techniques: symbolic machine learning approaches (nearest neighbour and identification tree techniques), artificial neural networks and genetic algorithms . Each technique is introduced and supported with examples taken from the bioinformatics literature . These examples include folding prediction, viral protease cleavage prediction, classification, multiple sequence alignment and microarray gene expression analysis.

Trends Cell Biol, 2004 May, 14(5), 222 - 5
Guardian at the gate: preventing unspliced pre-mRNA export; Casolari JM et al.; The production of a mature mRNA requires the assembly and cooperation of numerous complexes before nuclear export . The deleterious effects of intron-containing pre-mRNA leakage into the cytoplasm necessitate mechanisms to prevent premature export of partially processed or unprocessed messages . A new study demonstrates that the Saccharomyces cerevisiae protein Mlp1 specifically retains intron-containing pre-mRNAs in the nucleus.

Dev Cell, 2004 May, 6(5), 729 - 35
A role for the FEAR pathway in nuclear positioning during anaphase; Ross KE et al.; In budding yeast, cells lacking separase function exit mitosis with an undivided nucleus localized to the daughter cell . Here we show that the inability to separate sister chromatids per se is not sufficient to cause the daughter preference . Rather, separase affects nuclear positioning as part of the Cdc14 early anaphase release (FEAR) pathway . The role of the FEAR pathway in nuclear positioning is exerted during anaphase and is not shared by the mitotic exit network . We find that the nuclear segregation defect in FEAR mutants does not stem from nonfunctional spindle poles or the absence of cytoplasmic microtubules . Instead, the concomitant inactivation of sister chromatid separation and the FEAR pathway uncovered a mother-directed force in anaphase that was previously masked by the elongating spindle . We propose that at anaphase onset, the FEAR pathway activates cytoplasmic microtubule-associated forces that facilitate chromosome segregation to the mother cell.

Dev Cell, 2004 May, 6(5), 608 - 10
Mitosis: FEAR pulls them apart; Barral Y; The function of astral microtubules during mitosis is becoming the focus of increasing interest . A report published in this issue of Developmental Cell provides new insight into the complex and subtle regulation of these microtubules during anaphase.

Mar Biotechnol (NY) . 2004 May 6; {Epub ahead of print}
Zebrafish cDNA Encoding Multifunctional Fatty Acid Elongase Involved in Production of Eicosapentaenoic (20:5n-3) and Docosahexaenoic (22:6n-3) Acids; Agaba M et al.; Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids . The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina . A search of the GenBank database identified several expressed sequence tag sequences, including one obtained from zebrafish ( Danio rerio), with high similarity to gLELO . The full-length transcript ZfELO, encoding a polypeptide of 291 amino acid residues, was isolated from zebrafish liver cDNA . The predicted amino acid sequence of the open reading frame shared high similarity with the elongases of Caenorhabditis elegans and human . When expressed in Saccharomyces cerevisiae, the zebrafish open reading frame conferred the ability to lengthen the chain of a range of C(18), C(20), and C(22) polyunsaturated fatty acids, indicating not only that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is feasible, but also that one elongase enzyme can perform all three elongation steps required . The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously.

J Biol Chem, 2004 Jul 16, 279(29), 30459 - 68 Epub 2004 May 05.
Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14; Wang WQ et al.; The Cdc14 family of protein phosphatases is conserved within eukaryotes and antagonizes the action of cyclin-dependent kinases, thereby promoting mitotic exit and cytokinesis . We performed a detailed kinetic and mechanistic study of the Cdc14 phosphatases with both small molecule aryl phosphates and a physiological protein substrate hCdh1 . We found that Cdc14 displays a strong preference for two-ringed aryl phosphates over smaller one-ringed or larger, multi-ringed substrates, a finding that may have important implications for inhibitor design . Results from both leaving group and pH dependence of the Cdc14-catalyzed reaction are consistent with a general acid-independent mechanism for substrates with leaving group pKa < 7 and a general acid-dependent mechanism for substrates with leaving group pKa > 7 . The use of both low and high leaving group pKa substrates, in combination with steady-state and pre-steady-state kinetic techniques enabled the isolation and analysis of both the phosphoenzyme (E-P) formation and hydrolysis step . We established the requirement of general acid catalysis for E-P formation in reactions with high leaving group pKa substrates, and the presence of general base catalysis in E-P hydrolysis . Mutational study of invariant acidic residues in Cdc14 identified Asp253 as the general acid during E-P formation and the general base in E-P hydrolysis . We also identified several residues including Asp50, Asp129, Glu168, Glu171, and Asp177 in the Cdc14 active site cleft that are required for efficient dephosphorylation of hCdh1.

Development, 2004 May, 131(10), 2329 - 38
Patterning the forebrain: FoxA4a/Pintallavis and Xvent2 determine the posterior limit of Xanf1 expression in the neural plate; Martynova N et al.; During early development of the nervous system in vertebrates, expression of the homeobox gene Anf/Hesx1/Rpx is restricted to the anterior neural plate subdomain corresponding to the presumptive forebrain . This expression is essential for normal forebrain development and ectopic expression of Xenopus Anf, Xanf1 (also known as Xanf-1), results in severe forebrain abnormalities . By use of transgenic embryos and a novel bi-colour reporter technique, we have identified a cis-regulatory element responsible for transcriptional repression of Xanf1 that defines its posterior expression limit within the neural plate . Using this element as the target in a yeast one-hybrid system, we identified two transcription factors, FoxA4a/Pintallavis and Xvent2 (also known as Xvent-2), which are normally expressed posterior to Xanf1 . Overexpression of normal and dominant-negative versions of these factors, as well as inhibition of their mRNA translation by antisense morpholinos, show that they actually function as transcriptional repressors of Xanf1 just behind its posterior expression limit . The extremely high similarity of the identified Anf cis-regulatory sequences in Xenopus, chick and human, indicates that the mechanism restricting posterior expression of Anf in Xenopus is shared among vertebrates . Our findings support Nieuwkoop's activation-transformation model for neural patterning, according to which the entire neurectoderm is initially specified towards an anterior fate, which is later suppressed posteriorly as part of the trunk formation process.

Genome Biol . 2004;5(5):R35 . Epub 2004 Apr 16.
Prolinks: a database of protein functional linkages derived from coevolution; Bowers PM et al.; The advent of whole-genome sequencing has led to methods that infer protein function and linkages . We have combined four such algorithms (phylogenetic profile, Rosetta Stone, gene neighbor and gene cluster) in a single database--Prolinks--that spans 83 organisms and includes 10 million high-confidence links . The Proteome Navigator tool allows users to browse predicted linkage networks interactively, providing accompanying annotation from public databases . The Prolinks database and the Proteome Navigator tool are available for use online at http://dip.doe-mbi.ucla.edu/pronav.

J Cell Sci, 2004 May 1, 117(Pt 11), 2333 - 43
Cross-linking ATP synthase complexes in vivo eliminates mitochondrial cristae; Gavin PD et al.; We have used the tetrameric nature of the fluorescent protein DsRed to cross-link F(1)F(O)-ATPase complexes incorporating a subunit gamma-DsRed fusion protein in vivo . Cells expressing such a fusion protein have impaired growth relative to control cells . Strikingly, fluorescence microscopy of these cells revealed aberrant mitochondrial morphology . Electron microscopy of cell sections revealed the absence of cristae and multiple layers of unfolded inner mitochondrial membrane . Complexes recovered from detergent lysates of mitochondria were present largely as tetramers . Co-expression of 'free' DsRed targeted to the mitochondria reduced F(1)F(O)-ATPase oligomerisation and partially reversed the impaired growth and abnormal mitochondrial morphology . We conclude that the correct arrangement of F(1)F(O)-ATPase complexes within the mitochondrial inner membrane is crucial for the genesis and/or maintenance of mitochondrial cristae and morphology . Our findings further suggest that F(1)F(O)-ATPase can exist in oligomeric associations within the membrane during respiratory growth.

J Biol Chem, 2004 Jul 16, 279(29), 29952 - 62 Epub 2004 May 04.
YIH1 is an actin-binding protein that inhibits protein kinase GCN2 and impairs general amino acid control when overexpressed; Sattlegger E et al.; The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes . Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells . Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2 . The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression . YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction . However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity . We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response . This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells . We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.

J Biol Chem, 2004 Jul 9, 279(28), 29114 - 20 Epub 2004 May 04.
Mutual targeting of mediator and the TFIIH kinase Kin28; Guidi BW et al.; In Saccharomyces cerevisiae, Kin28 is a member of the cyclin-dependent kinase family . Kin28 is a subunit of the basal transcription factor holo-TFIIH and its trimeric sub-complex TFIIK . Kin28 is the primary kinase that phosphorylates the RNA polymerase II (RNA pol II) C-terminal domain (CTD) within a transcription initiation complex . Mediator, a global transcriptional co-activator, dramatically enhances the phosphorylation of the CTD of RNA pol II by holo-TFIIH in vitro . Using purified proteins we have determined that the subunits of TFIIK are sufficient for Mediator to enhance Kin28 CTD kinase activity and that Mediator enhances phosphorylation of a glutathione S-transferase-CTD fusion protein, despite the absence of multiple Mediator and/or TFIIH interactions with polymerase . Mediator does not stimulate the activity of several other CTD kinases, suggesting that the specific enhancement of TFIIH kinase activity results in Kin28 being the primary CTD kinase at initiation . In addition, we have found that Kin28 phosphorylates Mediator subunit Med4 in an assay, including purified holo-TFIIH, and either Mediator or recombinant Med4 alone . Furthermore, Kin28 appears to be, at least in part, responsible for the phosphorylation of Med4 in vivo . We have identified Thr-237 as the site of phosphorylation of Med4 by Kin28 in vitro . The mutation of Thr-237 to Ala has no effect on the growth of a yeast strain under normal conditions but confirms that Thr-237 is also the site of Med4 phosphorylation in vivo.

Cancer Res, 2004 May 1, 64(9), 2998 - 3001
Three classes of genes mutated in colorectal cancers with chromosomal instability; Wang Z et al.; Although most colorectal cancers are chromosomally unstable, the basis for this instability has not been defined . To determine whether genes shown to cause chromosomal instability in model systems were mutated in colorectal cancers, we identified their human homologues and determined their sequence in a panel of colorectal cancers . We found 19 somatic mutations in five genes representing three distinct instability pathways . Seven mutations were found in MRE11, whose product is involved in double-strand break repair . Four mutations were found among hZw10, hZwilch/FLJ10036, and hRod/KNTC, whose products bind to one another in a complex that localizes to kinetochores and controls chromosome segregation . Eight mutations were found in Ding, a previously uncharacterized gene with sequence similarity to the Saccharomyces cerevisiae Pds1, whose product is essential for proper chromosome disjunction . This analysis buttresses the evidence that chromosomal instability has a genetic basis and provides clues to the mechanistic basis of instability in cancers.

Ann N Y Acad Sci, 2004 Apr, 1011, 272 - 83
Genetic and functional analysis of mitochondrial DNA-encoded complex I genes; Bai Y et al.; Mammalian mitochondrial NADH dehydrogenase (complex I) is a multimeric complex consisting of at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA) . The function of these subunits is largely unknown . We have established an efficient method to isolate and characterize cells carrying mutations in various mtDNA-encoded complex I genes . With this method, 15 mouse cell lines with deficiencies in complex I-dependent respiration were obtained, and two near-homoplasmic mutations in mouse ND5 and ND6 genes were isolated . Furthermore, by generating a series of cell lines with the same nuclear background but different content of an mtDNA nonsense mutation, we analyzed the genetic and functional thresholds in mouse mitochondria . We found that in wild-type cells, about 40% of ND5 mRNA is in excess of that required to support a normal rate of ND5 subunit synthesis . However, there is no indication of compensatory upsurge in either transcription or translation with the increase in the proportion of mutant ND5 genes . Interestingly, the highest ND5 protein synthesis rate was just sufficient to support the maximum complex I-dependent respiration rate, suggesting a tight regulation at the translational level . In another line of research, we showed that the mitochondrial NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1), although consisting of a single subunit, can completely restore respiratory NADH dehydrogenase activity in mutant human cells that lack the essential mtDNA-encoded subunit ND4 . In particular, in these transfected cells, the yeast enzyme becomes integrated into the human respiratory chain and fully restores the capacity of the cells to grow in galactose medium.

Ann N Y Acad Sci, 2004 Apr, 1011, 61 - 8
Mitochondrial nucleoid and transcription factor A; Kanki T et al.; Nuclear DNA is tightly packed into nucleosomal structure . In contrast, human mitochondrial DNA (mtDNA) had long been believed to be rather naked because mitochondria lack histone . Mitochondrial transcription factor A (TFAM), a member of a high mobility group (HMG) protein family and a first-identified mitochondrial transcription factor, is essential for maintenance of mitochondrial DNA . Abf2, a yeast counterpart of human TFAM, is abundant enough to cover the whole region of mtDNA and to play a histone-like role in mitochondria . Human TFAM is indeed as abundant as Abf2, suggesting that TFAM also has a histone-like architectural role for maintenance of mtDNA . When human mitochondria are solubilized with non-ionic detergent Nonidet-P40 and then separated into soluble and particulate fractions, most TFAM is recovered from the particulate fraction together with mtDNA, suggesting that human mtDNA forms a nucleoid structure . TFAM is tightly associated with mtDNA as a main component of the nucleoid.

Biosystems, 2004 Apr-Jun, 74(1-3), 51 - 62
Gene duplication and hierarchical modularity in intracellular interaction networks; Hallinan J; Networks of interactions evolve in many different domains . They tend to have topological characteristics in common, possibly due to common factors in the way the networks grow and develop . It has been recently suggested that one such common characteristic is the presence of a hierarchically modular organization . In this paper, we describe a new algorithm for the detection and quantification of hierarchical modularity, and demonstrate that the yeast protein-protein interaction network does have a hierarchically modular organization . We further show that such organization is evident in artificial networks produced by computational evolution using a gene duplication operator, but not in those developing via preferential attachment of new nodes to highly connected existing nodes.

Mol Cell, 2004 May 7, 14(3), 387 - 94
Ssu72 Is an RNA polymerase II CTD phosphatase; Krishnamurthy S et al.; Phosphorylation of serine-2 (S2) and serine-5 (S5) of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates the transcription cycle and coordinates recruitment of RNA processing factors . The Fcp1 CTD phosphatase catalyzes dephosphorylation of S2-P . Here, we report that Ssu72, a component of the yeast cleavage/polyadenylation factor (CPF) complex, is a CTD phosphatase with specificity for S5-P . Ssu72 catalyzes CTD S5-P dephosphorylation in association with the Pta1 component of the CPF complex, although its essential role in 3' end processing is independent of catalytic activity . Depletion of Ssu72 impairs transcription in vitro, and this defect can be rescued by recombinant, catalytically active Ssu72 . We propose that Ssu72 has a dual role in transcription, one as a CTD S5-P phosphatase that regenerates the initiation-competent, hypophosphorylated form of RNAP II and the other as a factor necessary for cleavage of pre-mRNA and efficient transcription termination.

Mol Cell, 2004 May 7, 14(3), 343 - 54
Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants; Query CC et al.; Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site (BS) attacks the 5' splice site, and the second step, when the 5' exon attacks the 3' splice site, yielding mRNA and lariat-intron products . A genetic screen for suppressors of BS A-to-G mutants, which stall between the two steps, identified Prp8, the highly conserved spliceosomal factor . prp8 suppressors facilitate the second step for multiple intron mutants and interact functionally with first step suppressors, alleles of PRP16 and U6 snRNA . Genetic interactions among prp8, prp16, and U6 alleles suggest that these factors control a common stage in first-to-second step transition . We propose that mutant substrates are utilized by alteration of the equilibrium between first/second step conformations, resembling tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations . This mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity.

Biochem J, 2004 Aug 1, 381(Pt 3), 803 - 11
Copper pathways in Plasmodium falciparum infected erythrocytes indicate an efflux role for the copper P-ATPase; Rasoloson D et al.; Copper, like iron, is a transition metal that can generate oxygen radicals by the Fenton reaction . The Plasmodium parasite invades an erythrocyte host cell containing 20 microM copper, of which 70% is contained in the Cu/Zn SOD (cuprozinc superoxide dismutase) . In the present study, we follow the copper pathways in the Plasmodium-infected erythrocyte . Metal-determination analysis shows that the total copper content of Percoll-purified trophozoite-stage-infected erythrocytes is 66% that of uninfected erythrocytes . This decrease parallels the decrease seen in Cu/Zn SOD levels in parasite-infected erythrocytes . Neocuproine, an intracellular copper chelator, arrests parasites at the ring-to-trophozoite stage transition and also specifically decreases intraparasitic levels of Cu/Zn SOD and catalase . Up to 150 microM BCS (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulphonic acid), an extracellular copper chelator, has no effect on parasite growth . We characterized a single copy PfCuP-ATPase (Plasmodium falciparum copper P-ATPase) transporter, which, like the Crypto-sporidium parvum copper P-ATPase, has a single copper-binding domain: 'Met-Xaa-Cys-Xaa-Xaa-Cys' . Recombinant expression of the N-terminal metal-binding domain reveals that the protein specifically binds reduced copper . Transcription of the PfCuP-ATPase gene is the highest at late ring stage/early trophozoite, and is down-regulated in the presence of neocuproine . Immunofluorescence and electron microscopy indicate the transporter to be both in the parasite and on the erythrocyte membrane . Both the decrease in total copper and the location of the PfCuP-ATPase gene indicate a copper-efflux pathway from the infected erythrocyte.

Biochem J, 2004 Jul 1, 381(Pt 1), 19 - 23
A novel role of Mgm1p, a dynamin-related GTPase, in ATP synthase assembly and cristae formation/maintenance; Amutha B et al.; In Saccharomyces cerevisiae, two mitochondrial inner-membrane proteins play critical roles in organellar morphology . One is a dynamin-related GTPase, Mgm1p, which participates in mitochondrial fusion . Another is Tim11p, which is required for oligomeric assembly of F1Fo-ATP synthase, which generates ATP through oxidative phosphorylation . Our data bring these findings together and define a novel role for Mgm1p in the formation and maintenance of mitochondrial cristae . We show that Mgm1p serves as an upstream regulator of Tim11p protein stability, ATP synthase assembly, cristae morphology and cytochrome c storage within cristae.

J Biol Chem, 2004 Jul 2, 279(27), 28100 - 5 Epub 2004 Apr 28.
Dissecting the cell-killing mechanism of the topoisomerase II-targeting drug ICRF-193; Oestergaard VH et al.; Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs . One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis . Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure . Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear . In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA . We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast . Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments . Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.

J Biol Chem, 2004 Jul 2, 279(27), 28093 - 9 Epub 2004 Apr 28.
Hindering the strand passage reaction of human topoisomerase IIalpha without disturbing DNA cleavage, ATP hydrolysis, or the operation of the N-terminal clamp; Oestergaard VH et al.; DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed . While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication . We have characterized a human topoisomerase IIalpha enzyme with a two-amino acid insertion at position 351 in the transducer domain . The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport . Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage . The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage . Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis . However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIalpha enzyme . Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se . A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.

J Biol Chem, 2004 Jun 25, 279(26), 27225 - 32 Epub 2004 Apr 27.
Sec22p export from the endoplasmic reticulum is independent of SNARE pairing; Liu Y et al.; Molecularly distinct sets of SNARE proteins localize to specific intracellular compartments and catalyze membrane fusion events . Although their central role in membrane fusion is appreciated, little is known about the mechanisms by which individual SNARE proteins are targeted to specific organelles . Here we investigated functional domains in Sec22p that direct this SNARE protein to the endoplasmic reticulum (ER), to Golgi membranes, and into SNARE complexes with Bet1p, Bos1p, and Sed5p . A series of Sec22p deletion mutants were monitored in COPII budding assays, subcellular fractionation gradients, and SNARE complex immunoprecipitations . We found that the N-terminal "profilin-like" domain of Sec22p was required but not sufficient for COPII-dependent export of Sec22p from the ER . Interestingly, versions of Sec22p that lacked the N-terminal domain were assembled into ER/Golgi SNARE complexes . Analyses of Sec22p SNARE domain mutants revealed a second signal within the SNARE motif (between layers -4 and -1) that was required for efficient ER export . Other SNARE domain mutants that contained this signal were efficiently packaged into COPII vesicles but failed to assemble into SNARE complexes . Together these results indicated that SNARE complex formation is neither required nor sufficient for Sec22p packaging into COPII transport vesicles and subsequent targeting to the Golgi complex . We propose that the COPII budding machinery has a preference for unassembled ER/Golgi SNARE proteins.

J Biol Chem, 2004 Jul 9, 279(28), 29167 - 74 Epub 2004 Apr 30.
Sequence-specific interaction between mitochondrial Fe-S scaffold protein Isu and Hsp70 Ssq1 is essential for their in vivo function; Dutkiewicz R et al.; Isu, the scaffold for assembly of Fe-S clusters in the yeast mitochondrial matrix, is a substrate protein for the Hsp70 Ssq1 and the J-protein Jac1 in vitro . As expected for an Hsp70-substrate interaction, the formation of a stable complex between Isu and Ssq1 requires Jac1 in the presence of ATP . Here we report that a conserved tripeptide, PVK, of Isu is critical for interaction with Ssq1 because amino acid substitutions in this tripeptide inhibit both the formation of the Isu-Ssq1 complex and the ability of Isu to stimulate the ATPase activity of Ssq1 . These biochemical defects correlate well with the growth defects of cells expressing mutant Isu proteins . We conclude that the Ssq1-Isu substrate interaction is critical for Fe-S cluster biogenesis in vivo . The ability of Jac1 and mutant Isu proteins to cooperatively stimulate the ATPase activity of Ssq1 was also measured . Increasing the concentration of Jac1 and mutant Isu together but not individually partially overcame the effect of the reduced affinity of the Isu mutant proteins for Ssq1 . These results, along with the observation that overexpression of Jac1 was able to suppress the growth defect of an ISU mutant, support the hypothesis that Isu is "targeted" to Ssq1 by Jac1, with a preformed Jac1-Isu complex interacting with Ssq1.

J Mol Biol, 2004 May 21, 339(1), 185 - 97
Conformational properties of the iso-1-cytochrome C denatured state: dependence on guanidine hydrochloride concentration; Wandschneider E et al.; Production of seven single surface histidine variants of yeast iso-1-cytochrome c allowed measurement of the apparent pK(a), pK(a)(obs), for histidine-heme loop formation for loops of nine to 83 amino acid residues under varying denaturing conditions (2 M to 6 M guanidine hydrochloride, gdnHCl) . A linear correlation between pK(a)(obs) and the log of the loop size is expected for a random coil, pK(a)(obs) proportional to k log(n), where k is a scaling factor and n is the number of monomers in the loop . For small loops of nine, 16, and 22 monomers, no dependence of pK(a)(obs) on loop size was observed at any denaturant concentration indicating effects from chain stiffness . For larger loops of 37, 56, 72, and 83 monomers, the dependence of pK(a)(obs) on log(n) was linear and the slope of that dependence decreased with increasing concentration of denaturant . The scaling factor obtained at 5 M and 6 M gdnHCl for the larger loop sizes was approximately -2.0, close to the value of -2.2 expected for a random coil with excluded volume . However, scaling factors obtained under less harsh denaturing conditions (2 M to 4.5 M gdnHCl) deviated strongly from that expected for a random coil, being in the range -3 to -4 . The gdnHCl dependence of pK(a)(obs) at each loop size was also evaluated to obtain denaturant m-values . Short loops where chain stiffness dominates had similar m-values of approximately 0.25 kcal/mol M . For larger loops m-values decrease with increasing loop size indicating that less hydrophobic area is sequestered when larger loops form . It is known that the earliest events in protein folding involve the formation of simple loops . The data from these studies provide direct insight into the relative probability with which loops of different sizes will form, as well as the factors which affect loop formation.

J Mol Biol, 2004 May 21, 339(1), 131 - 44
Regulation of LIP level and ROS formation through interaction of H-ferritin with G-CSF receptor; Yuan X et al.; A variety of hematopoietic factors including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3) and thrombopoietin (TPO) induce a rapid increase of intracellular reactive oxygen species (ROS) . ROS induces the activation of many signaling molecules, including Shc, Lck, syk, PKC, MAPK, STAT3, through inhibition of protein phosphatase . Each growth factor has a specific cell-surface receptor, which activates both unique and shared signal transduction pathways . The processes of signal transduction linking cell-surface receptor to the formation of intracellular ROS have not been elucidated fully . Ferritins are composed of two subunit types, H and L, and made of 24 subunits that sequester up to 4500 atoms of iron . When the stored iron atoms are released from H-ferritin, through iron-catalyzed reaction, they have the capacity to promote the formation of ROS . Here, the interaction of G-CSFR and H-ferritin was confirmed by yeast two-hybrid screen, mammalian two-hybrid assays, glutathione-S-transferase (GST) pull-down experiments and immunoprecipitation studies in vitro and in vivo . Additional immunofluorescence assay showed that the two proteins colocalized along the plasma membrane and partly in the cytoplasm . The binding site for H-ferritin was demonstrated to locate to the box3 motif on the C-terminal region of granulocyte colony-stimulating factor receptor (G-CSFR) . Furthermore, we found the interaction of full-length G-CSFR with H-ferritin was dissociated at 30 minutes after G-CSF induction and then began to assemble at 45 minutes . The labile iron pool (LIP) is a pool of redox-active iron complexes, which is regulated tightly by the expression of H-ferritin . Experiments showed that the level of LIP increased significantly at 30 minutes after G-CSF stimulation and intracellular ROS formation changed in a pattern similar to LIP response to G-CSF in bone-marrow hematopoietic cells . G-CSF-induced changes in the level of LIP and ROS formation could be blocked by pretreatment with iron chelators that repressed the expression of H-ferritin . In addition, the phosphorylation of STAT3 induced by G-CSF was decreased in iron chelator-treated hematopoietic cells . These data suggested that LIP may be released from the dissociated H-ferritin, and then induce intracellular ROS formation in the bone-marrow hematopoietic cells . ROS, acting as a second messenger, might take part in G-CSF receptor signal transduction . So, here, a new G-CSFR-H-ferritin-LIP-ROS pathway is proposed for regulation of intracellular ROS formation in bone-marrow hematopoietic cells.

Chem Biol, 2004 Mar, 11(3), 379 - 87
Cytochrome p450 taxadiene 5alpha-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis; Jennewein S et al.; The first oxygenation step in the biosynthesis of the anticancer drug taxol in Taxus species is the cytochrome p450-mediated hydroxylation (with double bond migration) of the diterpene olefin precursor taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol . A homology-based cloning strategy, employing an induced Taxus cell library, yielded a cDNA encoding taxadiene 5alpha-hydroxylase, which was functionally expressed in yeast and insect cells . The recombinant enzyme was characterized and shown to efficiently utilize both taxa-4(5),11(12)-diene and taxa-4(20),11(12)-diene (as an adventitious substrate) to synthesize taxa-4(20),11(12)-dien-5alpha-ol . This hydroxylase resembles, in sequence and properties, other cytochrome p450 oxygenases of taxol biosynthesis . The utilization of both taxadiene isomers in the formation of taxa-4(20),11(12)-dien-5alpha-ol is novel, suggesting a reaction mechanism involving promiscuous radical abstraction with selective oxygen insertion rather than epoxidation of the C4,C5-alkene of the natural substrate and allylic rearrangement of the resulting taxa-11(12)-en-4,5epoxide.

Biochemistry, 2004 May 11, 43(18), 5119 - 25
Association of human neuroglobin with cystatin C, a cysteine proteinase inhibitor; Wakasugi K et al.; Neuroglobin (Ngb) is a newly discovered globin that is expressed in vertebrate brain . It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that Ngb protects neurons from hypoxia . However, the mechanism of this neuroprotection remains unclear . In the present study, we identified human cystatin C, a cysteine proteinase inhibitor, as an Ngb-binding protein by using a yeast two-hybrid system . Surface plasmon resonance experiments verified that Ngb binds to cystatin C dimers, not to the monomers . Because both intracellular cystatin C and the amyloidogenic variant of cystatin C form dimers, Ngb may modulate the intracellular transport (or secretion) of cystatin C to protect against neuronal death under conditions of oxidative stress and/or it may have a role in the development of neurodegenerative diseases.

Nat Genet, 2004 Jun, 36(6), 577 - 9 Epub 2004 May 02.
Duplicate genes increase gene expression diversity within and between species; Gu Z et al.; Using microarray gene expression data from several Drosophila species and strains, we show that duplicated genes, compared with single-copy genes, significantly increase gene expression diversity during development . We show further that duplicate genes tend to cause expression divergences between Drosophila species (or strains) to evolve faster than do single-copy genes . This conclusion is also supported by data from different yeast strains.

Plant Physiol, 2004 May, 135(1), 400 - 11 Epub 2004 Apr 30.
Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis; Wang Y et al.; PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis . The genome of Arabidopsis contains 11 members of the PHO1 gene family . The cDNAs of all PHO1 homologs have been cloned and sequenced . All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion . The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes . The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain . Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters . Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers . Analysis of the activity of the promoter of all PHO1 homologs using promoter-beta-glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers . beta-Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains . The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.

Nucleic Acids Res, 2004 Apr 30, 32(8), 2453 - 63 Print 2004.
Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases; Bujnicki JM et al.; Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA . The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods . On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes . Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site . The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical . The newly identified residues include a Lys residue in motif I and an Asp in motif IV . A conserved Gln found in motif X was found to be dispensable for MTase activity . Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374 . Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases . We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.

Mol Cell Biol, 2004 May, 24(10), 4196 - 206
Identification of factors regulating poly(A) tail synthesis and maturation; Mangus DA et al.; Posttranscriptional maturation of the 3' end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length . While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized . Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation . To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts . Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3'-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene . In vitro polyadenylation reactions utilizing extracts derived from fir1 Delta and pbp1 Delta cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA . Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis . We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).

Mol Cell Biol, 2004 May, 24(10), 4104 - 17
An array of coactivators is required for optimal recruitment of TATA binding protein and RNA polymerase II by promoter-bound Gcn4p; Qiu H et al.; Wild-type transcriptional activation by Gcn4p is dependent on multiple coactivators, including SAGA, SWI/SNF, Srb mediator, CCR4-NOT, and RSC, which are all recruited by Gcn4p to its target promoters in vivo . It was not known whether these coactivators are required for assembly of the preinitiation complex (PIC) or for subsequent steps in the initiation or elongation phase of transcription . We find that mutations in subunits of these coactivators reduce the recruitment of TATA binding protein (TBP) and RNA polymerase II (Pol II) by Gcn4p at ARG1, ARG4, and SNZ1, implicating all five coactivators in PIC assembly at Gcn4p target genes . Recruitment of Pol II at SNZ1 and ARG1 was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p . However, several mutations in SAGA subunits and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and Srb mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment . Our results reveal an unexpected complexity in the cofactor requirements for the enhancement of PIC assembly by a single activator protein.

Exp Cell Res, 2004 May 15, 296(1), 71 - 9
Mismatch repair proteins, meiosis, and mice: understanding the complexities of mammalian meiosis; Svetlanov A et al.; Mammalian meiosis differs from that seen in lower eukaryotes in several respects, not least of which is the added complexity of dealing with chromosomal interactions across a much larger genome (12 MB over 16 chromosome pairs in Saccharomyces cerevisiae compared to 2500 MB over 19 autosome pairs in Mus musculus) . Thus, the recombination machinery, while being highly conserved through eukaryotes, has evolved to accommodate such issues to preserve genome integrity and to ensure propagation of the species . One group of highly conserved meiotic regulators is the DNA mismatch repair protein family that, as their name implies, were first identified as proteins that act to repair DNA mismatches that arise primarily during DNA replication . Their function in ensuring chromosomal integrity has also translated into a critical role for this family in meiotic recombination in most sexually reproducing organisms . In mice, targeted deletion of certain family members results in severe consequences for meiotic progression and infertility . This review will focus on the studies involving these mutant mouse models, with occasional comparison to the function of these proteins in other organisms.

Curr Biol, 2004 May 4, 14(9), 752 - 62
Mnd1 is required for meiotic interhomolog repair; Zierhut C et al.; BACKGROUND: While double-strand break (DSB) repair is vital to the survival of cells during both meiosis and mitosis, the preferred mechanism of repair differs drastically between the two types of cell cycle . Thus, during meiosis, it is the homologous chromosome rather than the sister chromatid that is used as a repair template . RESULTS: Cells attempting to undergo meiosis in the absence of Mnd1 arrest in prophase I due to the activation of the Mec1 DNA-damage checkpoint accumulating hyperresected DSBs and aberrant synapsis . Sporulation of mnd1Delta strains can be restored by deleting RED1 or HOP1, which permits repair of DSBs by using the sister chromatid as a repair template . Mnd1 localizes to chromatin as foci independently of DSB formation, axial element (AE) formation, and synaptonemal complex (SC) formation and does not colocalize with Rad51 . Mnd1 does not preferentially associate with hotspots of recombination . CONCLUSIONS: Our results suggest that Mnd1 acts specifically to promote DSB repair by using the homologous chromosome as a repair template . The presence of Rec8, Red1, or Hop1 renders Mnd1 indispensable for DNA repair, presumably through the establishment of interhomolog (IH) bias . Localization studies suggest that Mnd1 carries out this function without being specifically recruited to the sites of DNA repair . We propose a model in which Mnd1 facilitates chromatin accessibility, which is required to allow strand invasion in meiotic chromatin.

Nucleic Acids Res, 2004 Apr 26, 32(8), 2298 - 305 Print 2004.
Alpha1-induced DNA bending is required for transcriptional activation by the Mcm1-alpha1 complex; Carr EA et al.; The yeast Mcm1 protein is a founding member of the MADS-box family of transcription factors that is involved in the regulation of diverse sets of genes through interactions with distinct cofactor proteins . Mcm1 interacts with the Matalpha1 protein to activate the expression of the alpha-cell type-specific genes . To understand the requirement of the cofactor alpha1 for Mcm1-alpha1-dependent transcriptional activation we analyzed the recruitment of Mcm1 to the promoters of alpha-specific genes in vivo and found that Mcm1 is able to bind to the promoters of alpha-specific genes in the absence of alpha1 . This suggests the function of alpha1 is more complex than simply recruiting Mcm1 . Several MADS-box transcription factors, including Mcm1, induce DNA bending and there is evidence the proper bend may be required for transcriptional activation . We analyzed Mcm1-dependent bending of a Mcm1-alpha1 binding site in the presence and absence of alpha1 and found that Mcm1 alone shows a reduced DNA-bend at this site compared with other Mcm1 binding sites . However, the addition of alpha1 markedly increases the DNA-bend and we present evidence this bend is required for full transcriptional activation . These results support a model in which proper DNA-bending by the Mcm1-alpha1 complex is required for transcriptional activation.

J Biol Chem, 2004 Jun 25, 279(26), 26817 - 22 Epub 2004 Apr 26.
Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome; Elsasser S et al.; The selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation . The receptors that mediate this step have yet to be clearly defined although specific candidates exist . Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor . Both binding events can be observed in purified biochemical systems . A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo . Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains . Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding . Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains.

J Clin Gastroenterol, 2004 May-Jun, 38(5 Suppl), S36 - 40
Pathology of indeterminate colitis; Odze RD; The term indeterminate colitis (IC) is an interim, or preliminary, descriptive term used by pathologists for cases of inflammatory bowel disease (IBD) in which a definite diagnosis of ulcerative colitis (UC) or Crohn's disease (CD) cannot be established based on the information available at the time of surgical sign-out . Most cases are due to fulminant ulcerative colitis, a condition in which the classic pathologic features of UC are often obscured and may overlap with CD . For instance, fulminant UC may show early superficial fissuring ulceration, transmural lymphoid aggregates and relative rectal sparing, simulating CD . Other common causes for establishing a diagnosis of IC include confusion of backwash ileitis in UC for terminal ileal involvement in CD, failure to accept hard criteria, such as granulomas, or segmental disease, as representative of CD, and failure to recognize unusual variants of UC that can cause CD-like patchiness of disease . Also, a diagnosis of IC should be avoided on biopsy studies and should not be established until all available clinical, endoscopic, radiologic and pathologic information are available for review . Some cases of IC may represent other forms of colitis as well, such as chronic ischemic or infectious colitis . The natural history of IC more closely resembles that of UC than CD . In fact, most cases of IC represent UC upon long-term clinical follow-up . Although, in some instances, serologic testing for ANCA or ASCA may be helpful in separating UC from CD in patients with IC, there is much overlap in the results of these assays for cases in which CD involves the colon in a UC-like pattern . Approximately 20% of IC patients develop severe pouch complications, which is intermediate in frequency between that seen in UC or CD . The risk of pouch complications, such as perianal fistulas or abscesses, and the risk of pouch breakdown is, overall, quite similar between IC and UC patients, supporting the notion that most patients with IC probably have UC and can safely undergo an ileal pouch-anal anastomosis procedure and have a reasonably good chance of having a good outcome.

Nucleic Acids Res, 2004 Apr 28, 32(8), 2362 - 71 Print 2004.
Transcription factor binding element detection using functional clustering of mutant expression data; Chen G et al.; As a powerful tool to reveal gene functions, gene mutation has been used extensively in molecular biology studies . With high throughput technologies, such as DNA microarray, genome-wide gene expression changes can be monitored in mutants . Here we present a simple approach to detect the transcription-factor-binding motif using microarray expression data from a mutant in which the relevant transcription factor is deleted . A core part of our approach is clustering of differentially expressed genes based on functional annotations, such as Gene Ontology (GO) . We tested our method with eight microarray data sets from the Rosetta Compendium and were able to detect canonical binding motifs for at least four transcription factors . With the support of chromatin IP chip data, we also predict a possible variant of the Swi4 binding motif and recover a core motif for Arg80 . Our approach should be readily applicable to microarray experiments using other types of molecular biology techniques, such as conditional knockout/overexpression or RNAi-mediated 'knockdown', to perturb the expression of a transcription factor . Functional clustering included in our approach may also provide new insights into the function of the relevant transcription factor.

Hum Mol Genet, 2004 Jul 1, 13(13), 1389 - 405 Epub 2004 Apr 28.
Progressive decrease in chaperone protein levels in a mouse model of Huntington's disease and induction of stress proteins as a therapeutic approach; Hay DG et al.; The manipulation of chaperone levels has been shown to inhibit aggregation and/or rescue cell death in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and cell culture models of Huntington's disease (HD) and other polyglutamine (polyQ) disorders . We show here that a progressive decrease in Hdj1, Hdj2, Hsp70, alphaSGT and betaSGT brain levels likely contributes to disease pathogenesis in the R6/2 mouse model of HD . Despite a predominantly extranuclear location, Hdj1, Hdj2, Hsc70, alphaSGT and betaSGT were found to co-localize with nuclear but not with extranuclear aggregates . Quantification of Hdj1 and alphaSGT mRNA levels showed that these do not change and therefore the decrease in protein levels may be a consequence of their sequestration to aggregates, or an increase in protein turnover, possibly as a consequence of their relocation to the nucleus . We have used genetic and pharmacological approaches to assess the therapeutic potential of chaperone manipulation . Ubiquitous overexpression of Hsp70 in the R6/2 mouse (as a result of crossing to Hsp70 transgenics) delays aggregate formation by 1 week, has no effect on the detergent solubility of aggregates and does not alter the course of the neurological phenotype . We used an organotypic slice culture assay to show that pharmacological induction of the heat shock response might be a more useful approach . Radicicol and geldanamycin could both maintain chaperone induction for at least 3 weeks and alter the detergent soluble properties of polyQ aggregates over this time course .

J Med Chem, 2004 May 6, 47(10), 2635 - 44
Inhibitors of Sir2: evaluation of splitomicin analogues; Posakony J et al.; Splitomicin (1) and 41 analogues were prepared and evaluated in cell-based Sir2 inhibition and toxicity assays and an in vitro Sir2 inhibition assay . Lactone ring or naphthalene (positions 7-9) substituents decrease activity, but other naphthalene substitutions (positions 5 and 6) are well-tolerated . The hydrolytically unstable aromatic lactone is important for activity . Lactone hydrolysis rates were used as a measure of reactivity; hydrolysis rates correlate with inhibitory activity . The most potent Sir2 inhibitors were structurally similar to and had hydrolysis rates similar to 1.

J Biochem (Tokyo), 2004 Mar, 135(3), 289 - 95
A novel mechanism of intragenic complementation between Phe to Ala calmodulin mutations; Okano H et al.; Calmodulin (CaM) performs essential functions in cell proliferation in Saccharomyces cerevisiae . Previously, we isolated fourteen temperature-sensitive Phe-to-Ala mutations of the CaM-encoding gene CMD1 . These mutations were classified into four intragenic complementation groups, suggesting that each group represents a loss of CaM interaction with its specific essential target protein . Nuf1p/Spc110p, one of the essential targets, is a spindle pole body component that is required for proper mitosis . We investigated which intragenic complementation group of CaM represents the malfunction of Nuf1p . Immunoprecipitation analysis showed that two cmd1 mutations belonging to two distinct intragenic complementation groups had the most severely impaired complex formation with Nuf1p at the restrictive temperature . The temperature-sensitive growth of these cmd1 mutants was suppressed by a CaM-independent dominant allele of NUF1 . Additionally, these mutants displayed characteristic mitotic defects: an increased ratio of artificial chromosome loss, which could be suppressed by the CaM-independent dominant allele of NUF1, and aberrant microtubule structures . These results indicate that these cmd1 mutants display the temperature-sensitive growth due to the compromised interaction with Nuf1p . However, the interaction was restored in a heterozygous diploid of the two cmd1 alleles, suggesting that intragenic complementation between these cmd1 alleles occurs by a novel mechanism, whereby co-presence of both mutant proteins rescues the interaction with Nuf1p.

Metab Eng, 2004 Apr, 6(2), 140 - 54
Non-linear reduction for kinetic models of metabolic reaction networks; Gerdtzen ZP et al.; Kinetic models of metabolic networks are essential for predicting and optimizing the transient behavior of cells in culture . However, such models are inherently high dimensional and stiff due to the large number of species and reactions involved and to kinetic rate constants of widely different orders of magnitude . In this paper we address the problem of deriving non-stiff, reduced-order non-linear models of the dominant dynamics of metabolic networks with fast and slow reactions . We present a method, based on singular perturbation analysis, which allows the systematic identification of quasi-steady-state conditions for the fast reactions, and the derivation of explicit non-linear models of the slow dynamics independent of the fast reaction rate expressions . The method is successfully applied to detailed models of metabolism in human erythrocytes and Saccharomyces cerevisiae.

J Agric Food Chem, 2004 May 5, 52(9), 2577 - 81
Influence of blending on the content of different compounds in the biological aging of sherry dry wines; Berlanga TM et al.; Principal components analysis to examine the effect of blending (viz . the mixing and transfer of wine between cask rows in a "criaderas and solera" system) on metabolic activity in flor yeasts during biological aging of sherry dry wines was carried out . The variables used in the analysis were the wine compounds most deeply involved in the flor yeast metabolism, namely ethanol, acetaldehyde, glycerol, acetic acid, and l-proline . The greatest blending effect was found to be on the third and second "criadera", which are the stages where the yeasts show a high metabolic activity . The stages holding the oldest wine (viz . the first criadera and the solera) exhibited no differences before and after blending; therefore, the yeasts have a decreased biological activity in them and physical-chemical aging processes seemingly prevail over it.

Tsitologiia, 2004, 46(1), 43 - 52
{Dynamics of some postreplication DNA repair proteins in carcinogen-damaged mammalian cells}; Nikiforov AA et al.; Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks . This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA . The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR) . In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA . In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known . Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA . We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells . In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif . We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci . Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine . Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.

Int J Parasitol, 2004 May, 34(6), 675 - 82
Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide; Harmsen MM et al.; Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections . We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica . Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide . Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR . The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F . hepatica and are, therefore, referred to as CL3 . The CL3 clones encoded asparagine at position P1 of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation . Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form . The propeptide of CL-like proteins was predicted to contain important B-cell epitopes . To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence . A subsequent challenge infection resulted in a significant (P < 0.05) reduction of fluke load compared to adjuvant controls . We conclude that the propeptide of CL3 plays an important role in inducing immunity against F . hepatica infection.

J Mol Biol, 2004 May 14, 338(5), 877 - 93
Distinct DNA elements contribute to Rap1p affinity for its binding sites; Del Vescovo V et al.; The essential Saccharomyces cerevisiae regulatory protein Rap1 contains two tandem Myb-like DNA binding sub-domains that interact with two defined DNA "hemisites", separated by a trinucleotide linker sequence . We have mapped the thermodynamically defined DNA-binding site of Rap1 by a primer extension method coupled with electrophoretic separation of bound and unbound DNAs . Relative to published consensus sequences, we detect binding interactions that extend 3 bp beyond the 5'-end of the putative DNA-binding site . This new site of interaction is located where the DNA minor groove faces the protein, and may account for the major DNA bending induced by Rap1p that previous studies have mapped to a site immediately upstream of the consensus binding site . In addition, we show that a minimal DNA-binding site made of one single consensus hemisite, preceded or followed by a spacer trinucleotide that interacts with the unstructured protein linker between the two Rap1p DNA binding domains, is able to bind the protein, although at lower affinity . These findings may explain the observed in vivo binding properties of Rap1p at many promoters that lack canonical binding sites.

Cell, 2004 Apr 30, 117(3), 323 - 35
Telomere length homeostasis is achieved via a switch between telomerase- extendible and -nonextendible states; Teixeira MT et al.; Telomerase counteracts telomere erosion that stems from incomplete chromosome end replication and nucleolytic processing . A precise understanding of telomere length homeostasis has been hampered by the lack of assays that delineate the nonuniform telomere extension events of single chromosome molecules . Here, we measure telomere elongation at nucleotide resolution in Saccharomyces cerevisiae . The number of nucleotides added to a telomere in a single cell cycle varies between a few to more than 100 nucleotides and is independent of telomere length . Telomerase does not act on every telomere in each cell cycle, however . Instead, it exhibits an increasing preference for telomeres as their lengths decline . Deletion of the telomeric proteins Rif1 or Rif2 gives rise to longer telomeres by increasing the frequency of elongation events . Thus, by taking a molecular snapshot of a single round of telomere replication, we demonstrate that telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states.

Cell, 2004 Apr 30, 117(3), 311 - 21
Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation; Paushkin SV et al.; tRNA splicing is a fundamental process required for cell growth and division . The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease . The enzyme responsible for intron removal in mammalian cells is unknown . We present the identification and characterization of the human tRNA splicing endonuclease . This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits . Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity . Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors . Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA . These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.

Cell, 2004 Apr 30, 117(3), 279 - 80
Telomerase regulation at the telomere: a binary switch; Loayza D et al.; Telomerase is known to preferentially elongate the shortest telomeres in a cell . Using an elegant yeast assay, Texeira et al . (2004 {this issue of Cell}) address what aspect of telomerase action is regulated by telomere length: the frequency or the extent of telomere elongation . They show that short telomeres are elongated more frequently than long telomeres, arguing that telomeres switch between two states, one that allows telomere extension and one that does not.

Curr Genet, 2004 Jun, 45(6), 339 - 49 Epub 2004 Apr 17.
Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth; Richman TJ et al.; The Cdc42p GTPase regulates multiple signal transduction pathways through its interactions with downstream effectors . Specific functional domains within Cdc42p are required for guanine-nucleotide binding, interactions with downstream effectors, and membrane localization . However, little is known about how Cdc42p is clustered at polarized growth sites or is extracted from membranes by Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) at specific times in the cell cycle . To address these points, localization studies were performed in Saccharomyces cerevisiae using green fluorescent protein (GFP)-tagged Cdc42p and the RhoGDI Rdi1p . GFP-Rdi1p localized to polarized growth sites at specific times of the cell cycle but not to other sites of Cdc42p localization . Overexpression of Rdi1p led to loss of GFP-Cdc42p from internal and plasma membranes . This effect was mediated through the Cdc42p Rho-insert domain, which was also implicated in interactions with the Bni1p scaffold protein . These data suggested that Rdi1p functions in cell cycle-specific Cdc42p membrane detachment . Additional genetic and time-lapse microscopy analyses implicated nucleotide binding in the clustering of Cdc42p . Taken together, these results provide insight into the complicated nature of the relationships between Cdc42p localization, nucleotide binding, and protein-protein interactions.

Nat Cell Biol, 2004 May, 6(5), 451 - 7 Epub 2004 Apr 25.
Amplitude control of cell-cycle waves by nuclear import; Becskei A et al.; Propagation of waves of biochemical activities through consecutive stages of the cell cycle is essential to execute the steps of cell division in a strict temporal order . Mechanisms that ensure the proper amplitude and timing of these waves are poorly understood . Using a synthetic gene circuit, we show that a transcriptional activator driven by yeast cell-cycle promoters propagates transcriptional oscillations with substantial damping . Although regulated nuclear translocation has been implicated in the timing of oscillatory events, mathematical analysis shows that increasing the rate of nuclear transport is an example of a general regulatory principle, which enhances the fidelity of wave propagation . Indeed, increasing the constitutive import rate of the activator counteracts the damping of waves and concurrently preserves the intensity of the signal . In contrast to the regulatory range of nuclear transport, the range of mRNA turnover considerably limits transcriptional wave propagation . This classification of cellular processes outlines potential regulatory mechanisms that can contribute to faithful transmission of oscillations at different stages of the cell cycle.

Nat Genet, 2004 May, 36(5), 462 - 70 Epub 2004 Apr 25.
The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression; Kim JC et al.; BBS4 is one of several proteins that cause Bardet-Biedl syndrome (BBS), a multisystemic disorder of genetic and clinical complexity . Here we show that BBS4 localizes to the centriolar satellites of centrosomes and basal bodies of primary cilia, where it functions as an adaptor of the p150(glued) subunit of the dynein transport machinery to recruit PCM1 (pericentriolar material 1 protein) and its associated cargo to the satellites . Silencing of BBS4 induces PCM1 mislocalization and concomitant deanchoring of centrosomal microtubules, arrest in cell division and apoptotic cell death . Expression of two truncated forms of BBS4 that are similar to those found in some individuals with BBS had a similar effect on PCM1 and microtubules . Our findings indicate that defective targeting or anchoring of pericentriolar proteins and microtubule disorganization contribute to the BBS phenotype and provide new insights into possible causes of familial obesity, diabetes and retinal degeneration.

Nat Genet, 2004 May, 36(5), 507 - 11 Epub 2004 Apr 11.
SUMO modification is required for in vivo Hox gene regulation by the Caenorhabditis elegans Polycomb group protein SOP-2; Zhang H et al.; Post-translational modification of proteins by the ubiquitin-like molecule SUMO (sumoylation) regulates their subcellular localization and affects their functional properties in vitro, but the physiological function of sumoylation in multicellular organisms is largely unknown . Here, we show that the C . elegans Polycomb group (PcG) protein SOP-2 interacts with the SUMO-conjugating enzyme UBC-9 through its evolutionarily conserved SAM domain . Sumoylation of SOP-2 is required for its localization to nuclear bodies in vivo and for its physiological repression of Hox genes . Global disruption of sumoylation phenocopies a sop-2 mutation by causing ectopic Hox gene expression and homeotic transformations . Chimeric constructs in which the SOP-2 SAM domain is replaced with that derived from fruit fly or mammalian PcG proteins, but not those in which the SOP-2 SAM domain is replaced with the SAM domains of non-PcG proteins, confer appropriate in vivo nuclear localization and Hox gene repression . These observations indicate that sumoylation of PcG proteins, modulated by their evolutionarily conserved SAM domain, is essential to their physiological repression of Hox genes.

Cell Cycle, 2004 May, 3(5), 601 - 3 Epub 2004 May 31.
Inactivating Cdc25, mitotic style; Wolfe BA et al.; Mitotic entry and exit require activation and inactivation of the Cdk1-cyclin B kinase complex, respectively . The Cdc25 protein phosphatase family activates Cdk1-cyclin B at the G2/M transition by removing inhibitory phosphate groups . Cdc25 family members, held inactive during interphase, are activated during mitotic progression in an amplification loop involving Cdk1-cyclin B . While Cdc25 activation at the G2/M transition is required for the timely initiation of mitosis, recent evidence suggests that the inactivation of Cdc25 in late mitosis may play a role in supporting Cdk1-cyclin B inactivation . Here, we discuss the mechanisms of Cdc25 regulation and how they pertain to both mitotic entry and exit.

J Vet Med Sci, 2004 Mar, 66(3), 243 - 50
A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage; Horiuchi H et al.; A new mouse monoclonal antibody (mAb), HUKT was raised against chicken peripheral blood thrombocytes . The mAb HUKT appeared to detect a specific marker on the surface of chicken thrombocytes . Flow cytometry (FCM) analysis revealed that it did not react with cells from the normal thymus, bursa of Fabricius, six kinds of chicken cell lines, chicken erythrocytes or human platelets . In addition, HUKT(+) cells in peripheral blood leukocytes (PBL) were CD45(low), Bu-1a(-) and CD3(-) cells . Immunoblotting analysis showed that the molecule recognized by HUKT is a monomer with an apparent molecular weight of 150 kDa under non-reducing and reducing conditions . Tissue distribution studies revealed that only cells of thrombocyte lineage in bone marrow and embryonic blood cells were stained by HUKT . The HUKT mAb presented here may be useful for both ontogenetic studies of thrombocyte lineage and immunological studies in the chicken.

Protein J, 2004 Feb, 23(2), 119 - 26
Estimation of the compaction of the denatured state by a protein variant involved in a reverse hydrophobic effect; Zhang MM et al.; Fluorescence resonance energy transfer methods have been used to evaluate changes in the dimension of the denatured state for position 73 variants of iso-1-cytochrome c that show a reverse hydrophobic effect {Herrmann et al . (1995)} . The experiments take advantage of the Trp 59/heme donor-acceptor pair in cytochrome c . Two large aliphatic variants, Ile 73 and Leu 73, were compared directly to the wild-type protein (lysine 73) . The Leu 73 was an outlier in the original work and serves as an internal control . The data show that the volume of the denatured state is contracted by a small but significant degree, 4-6%, for the Ile 73 variant whereas the Leu 73, which does not conform to the reverse hydrophobic effect, shows no significant compaction . Given that position 73 is beyond Trp 59 in the sequence, the denatured state compaction appears to be a global effect.

Trends Cell Biol, 2004 Feb, 14(2), 61 - 3
The exocyst meets the translocon: a regulatory circuit for secretion and protein synthesis?
Guo W, Novick P.
The translocon is responsible for the translocation of proteins across the membrane of the endoplasmic reticulum into its lumen, whereas the exocyst acts at the other end of the secretory pathway, tethering secretory vesicles to the sites of exocytosis . Here, we discuss three independent lines of evidence that indicate surprising genetic, physical and functional interactions between the two complexes . Although much of the existing evidence is rather preliminary in nature, these interactions might serve to coordinate the biosynthetic capacity of the cell with the function of the secretory machinery.

Genes Dev, 2004 May 1, 18(9), 981 - 91 Epub 2004 Apr 22.
Interaction of the S-phase cyclin Clb5 with an "RXL" docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch; Wilmes GM et al.; Cyclin-dependent kinases are critical regulators of eukaryotic DNA replication . We show that the S-phase cyclin Clb5 binds stably and directly to the origin recognition complex (ORC) . This interaction is mediated by an "RXL" target sequence, or "Cy" motif, in the Orc6 subunit that is recognized by the "hydrophobic patch" region on Clb5 . The Clb5-Orc6 interaction requires replication initiation, and is maintained throughout the remainder of S phase and into M phase . Eliminating the Clb5-Orc6 interaction has no effect on initiation of replication but instead sensitizes cells to lethal overreplication . We propose that Clb5 binding to ORC provides an origin-localized replication control switch that specifically prevents reinitiation at replicated origins.

Ann N Y Acad Sci, 2004 Mar, 1012, 299 - 305
Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis; Xu X et al.; In 1987, Miyajima et al . first characterized an autosomal recessive, adult-onset neurodegenerative disorder resembling Parkinson's disease associated with near-absent circulating serum ceruloplasmin levels . Coined "familial apoceruloplasmin deficiency", they described a patient with a presenting triad of diabetes mellitus, retinal degeneration, and neurodegeneration with blepharospasm . Neuropathological evaluation revealed abundant iron deposition in selected neurons of the basal ganglia and substantia nigra with associated neuronal dropout and spongioform degeneration without evidence of reactive gliosis . Subsequently, mutations in the ceruloplasmin gene have been determined to result in the excessive iron accumulation seen in the pancreas, retina, and brain . Elevated serum ferritin suggests a systemic iron overload syndrome, yet affected patients had low transferrin saturation and a mild anemia . This new disease, "aceruloplasminemia", reveals a role for ceruloplasmin as an essential ferroxidase critical for iron homeostasis . This multicopper oxidase promotes efficient iron efflux such that individuals lacking ceruloplasmin develop a presumed oxidative injury secondary to iron accumulation and significant neuronal damage . Aceruloplasminemic mice provide a valuable model to further study the mechanisms by which ceruloplasmin regulates iron trafficking and the role of iron in oxidative injury . Despite the dependence of ceruloplasmin on copper for its function, aceruloplasminemia represents an iron storage disease and not a defect in copper metabolism . However, recent evidence in Saccharomyces cerevisiae indicates that Fet3, the yeast homologue of ceruloplasmin, functions as an essential cuprous oxidase . Further investigation into the mechanisms by which ceruloplasmin regulates iron and copper homeostasis will provide valuable insight into the pathogenesis of metallo-mediated diseases and elucidate mechanisms for transition metal (copper, iron) neuropathology.

Antimicrob Agents Chemother, 2004 May, 48(5), 1515 - 9
The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insights on diamidine resistance in african trypanosomes; de Koning HP et al.; Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals . It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite . We describe here the mechanisms by which {(3)H}diminazene is transported by Trypanosoma brucei brucei bloodstream forms . Diminazene was rapidly accumulated through a single transporter, with a K(m) of 0.45 +/- 0.11 micro M, which was dose dependently inhibited by pentamidine and adenosine . The K(i) values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter . Yeast expressing TbAT1 acquired the ability to take up {(3)H}diminazene and {(3)H}pentamidine . TbAT1-null mutants had lost almost all capacity for {(3)H}diminazene transport . However, this cell line still displayed a small but detectable rate of {(3)H}diminazene accumulation, in a nonsaturable manner . We conclude that TbAT1 mediates {(3)H}diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

Biochem J, 2004 Aug 1, 381(Pt 3), 645 - 53
Modular organization of a Cdc6-like protein from the crenarchaeon Sulfolobus solfataricus; De Felice M et al.; In the present paper, we report that a Cdc6 (cell-division control)-like factor from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as SsoCdc6-2) has a modular organization of its biological functions . A reliable model of the SsoCdc6-2 three-dimensional structure was built up, based on the significant sequence identity with the Pyrobaculum aerophylum Cdc6 (PaeCdc6), whose crystallographic structure is known . This allowed us to design two truncated forms of SsoCdc6-2: the DeltaC (residues 1-297, molecular mass 35 kDa) and the DeltaN (residues 298-400, molecular mass 11 kDa) proteins . The DeltaC protein contains the nucleotide-binding Rossmann fold and the Sensor-2 motif (Domains I and II in the PaeCdc6 structure), and retains the ability to bind and hydrolyse ATP . On the other hand, the DeltaN protein contains the C-terminal WH (winged helix)-fold (Domain III), and is able to bind DNA molecules and to inhibit the DNA helicase activity of the SsoMCM (mini-chromosome maintenance) complex, although with lesser efficiency with respect to the full-sized SsoCdc6-2 . These results provide direct biochemical evidence that the Cdc6 WH-domain is responsible for DNA-binding and inhibition of MCM DNA helicase activity.

EMBO J, 2004 May 19, 23(10), 2105 - 15 Epub 2004 Apr 22.
The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins; Balk J et al.; The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases . A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus . However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins . Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron-sulphur (Fe/S) clusters . Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p . Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins . Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus . In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes.

EMBO J, 2004 May 5, 23(9), 1922 - 33 Epub 2004 Apr 22.
Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors; Dove SK et al.; Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies . To isolate PtdIns(3,5)P(2) effectors, we identified Saccharomyces cerevisiae mutants that display fab1Delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene . Expressed Svp1p displays PtdIns(3,5)P(2) binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1Delta cells fail to recycle a marker protein from the vacuole to the Golgi . Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P(2) . These observations identify Svp1p as a PtdIns(3,5)P(2) effector required for PtdIns(3,5)P(2)-dependent membrane recycling from the vacuole . Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P(2) similarly . Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P(2)-binding site is on the beta-propeller . It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P(2) effectors . Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P(2) to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P(2) effectors must exist.

J Biol Chem, 2004 Jul 9, 279(28), 29774 - 86 Epub 2004 Apr 21.
Structure of mouse Golgi alpha-mannosidase IA reveals the molecular basis for substrate specificity among class 1 (family 47 glycosylhydrolase) alpha1,2-mannosidases; Tempel W et al.; Three subfamilies of mammalian Class 1 processing alpha1,2-mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation . In an effort to define the structural basis for substrate recognition among Class 1 mannosidases, we have crystallized murine Golgi mannosidase IA (space group P2(1)2(1)2(1)), and the structure was solved to 1.5-A resolution by molecular replacement . The enzyme assumes an (alphaalpha)(7) barrel structure with a Ca(2+) ion coordinated at the base of the barrel similar to other Class 1 mannosidases . Critical residues within the barrel structure that coordinate the Ca(2+) ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved . A Man(6)GlcNAc(2) oligosaccharide attached to Asn(515) in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex . In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket . A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes.

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