S. cerevisiae

Protein Pept Lett, 2004 Feb, 11(1), 23 - 8
NMR studies of the prionogenic peptide derived from Sup35 protein; Chae YK et al.; The NMR studies of the prionogenic peptide derived from Sup35 are presented . The peptide molecules were dissolved in the half-aqueous solution to prevent severe aggregation, and were found to be in an extended conformation from the chemical shift and the coupling constant data . They could form higher order multimers by making intermolecular hydrogen bonds, judging from the observation that the NMR sample became a gel-like state at lower temperatures . This work reports the first structural information in the solution state about the prionogenic peptide mimicking the state of amyloid fibrils, and provides a solid foundation for further structural analysis of peptide molecules forming insoluble aggregates.

Curr Drug Metab, 2004 Feb, 5(1), 109 - 24
Intestinal drug transporters: in vivo function and clinical importance; Kunta JR et al.; The oral route of drug administration remains the most popular and convenient route of administration, despite its many shortcomings and challenges . Although the advantages associated with this route far outweigh any limitations, a prominent limitation relates to the interactions of drugs with intestinal membrane transporters . The complexities of these interactions and their impact on drug absorption and absorption variability are only now becoming recognized . The rapidly growing awareness of transporter-mediated secretion, saturable absorption, and even the concerted actions of transporters in intestinal drug absorption and secretion has attracted the attention of pharmaceutical scientists in academia, the pharmaceutical industry and the regulatory agencies . This is evidenced by the recent rapid accumulation of data in the literature, the routine conducting of transport studies in the discovery and development of drugs, and finally by the recognition of the importance of transporter (e.g . P-glycoprotein and OATP) mediated secretion of drugs by regulatory authorities such as the U.S . Food and Drug Administration . In this mini-review, we focus on the handful of absorptive and secretory transporters that have been relatively well studied and illustrate the impact of these intestinal transporters on oral drug absorption using published reports from preclinical and clinical studies.

Cell Cycle, 2004 Apr, 3(4), 439 - 42 Epub 2004 Apr 01.
A field guide to the Mps1 family of protein kinases; Fisk HA et al.; Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability . The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity . The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis . Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint . More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.

Science, 2004 Feb 13, 303(5660), 1014 - 6
Structural basis of transcription: separation of RNA from DNA by RNA polymerase II; Westover KD et al.; The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix . At the opposite end of the hybrid helix, the RNA separates from the template DNA . This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network . Formation of the network must occur in the transition from abortive initiation to promoter escape.

Science, 2004 Feb 13, 303(5660), 983 - 8
Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms; Bushnell DA et al.; The structure of the general transcription factor IIB (TFIIB) in a complex with RNA polymerase II reveals three features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the "dock" domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a "finger" domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein (TBP)-promoter DNA complex orients the DNA for unwinding and transcription . TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region . It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape . The trajectory of promoter DNA determined by the C-terminal domain of TFIIB traverses sites of interaction with TFIIE, TFIIF, and TFIIH, serving to define their roles in the transcription initiation process.

Genomics, 2004 Mar, 83(3), 473 - 81
Functional assessment of the carboxy-terminus of the Wilson disease copper-transporting ATPase, ATP7B; Hsi G et al.; The carboxy-terminus of ATP7B, the protein defective in the copper-transport disorder Wilson disease, was investigated with respect to its role in copper delivery to the ferroxidase ceruloplasmin . We use yeast as a model system to assess the functional capabilities of ATP7B variants . The yeast ferroxidase, Fet3p, acquires copper from Ccc2p and cannot function if Ccc2p is impaired; expression of wild-type ATP7B in ccc2 yeast complements the iron-deficient phenotype . Our results demonstrate that the C-terminus of ATP7B is necessary for protein stability, as removal of the nonmembranous terminus leads to reduced protein levels and cessation of growth in iron-limited medium . Growth is partially restored when an additional three amino acids are present and is near wild-type levels when only one-third of the C-terminus is present . Measurement of ferroxidase activity is a more sensitive indicator of copper transport function and allowed identification of impaired variants not detected with the growth assay.

Nature, 2004 Mar 4, 428(6978), 93 - 7 Epub 2004 Feb 11.
Tension between two kinetochores suffices for their bi-orientation on the mitotic spindle; Dewar H et al.; The movement of sister chromatids to opposite spindle poles during anaphase depends on the prior capture of sister kinetochores by microtubules with opposing orientations (amphitelic attachment or bi-orientation) . In addition to proteins necessary for the kinetochore-microtubule attachment, bi-orientation requires the Ipl1 (Aurora B in animal cells) protein kinase and tethering of sister chromatids by cohesin . Syntelic attachments, in which sister kinetochores attach to microtubules with the same orientation, must be either 'avoided' or 'corrected' . Avoidance might be facilitated by the juxtaposition of sister kinetochores such that they face in opposite directions; kinetochore geometry is therefore deemed important . Error correction, by contrast, is thought to stem from the stabilization of kinetochore-spindle pole connections by tension in microtubules, kinetochores, or the surrounding chromatin arising from amphitelic but not syntelic attachment . The tension model predicts that any type of connection between two kinetochores suffices for efficient bi-orientation . Here we show that the two kinetochores of engineered, unreplicated dicentric chromosomes in Saccharomyces cerevisiae bi-orient efficiently, implying that sister kinetochore geometry is dispensable for bi-orientation . We also show that Ipl1 facilitates bi-orientation by promoting the turnover of kinetochore-spindle pole connections in a tension-dependent manner.

Bioinformatics, 2004 Feb 12, 20(3), 381 - 8 Epub 2004 Jan 22.
A graph-theoretic modeling on GO space for biological interpretation of gene clusters; Lee SG et al.; MOTIVATION: With the advent of DNA microarray technologies, the parallel quantification of genome-wide transcriptions has been a great opportunity to systematically understand the complicated biological phenomena . Amidst the enthusiastic investigations into the intricate gene expression data, clustering methods have been the useful tools to uncover the meaningful patterns hidden in those data . The mathematical techniques, however, entirely based on the numerical expression data, do not show biologically relevant information on the clustering results . RESULTS: We present a novel methodology for biological interpretation of gene clusters . Our graph theoretic algorithm extracts common biological attributes of the genes within a cluster or a group of interest through the modified structure of gene ontology (GO) called GO tree . After genes are annotated with GO terms, the hierarchical nature of GO terms is used to find the representative biological meanings of the gene clusters . In addition, the biological significance of gene clusters can be assessed quantitatively by defining a distance function on the GO tree . Our approach has a complementary meaning to many statistical clustering techniques; we can see clustering problems from a different viewpoint by use of biological ontology . We applied this algorithm to the well-known data set and successfully obtained the biological features of the gene clusters with the quantitative biological assessment of clustering quality through GO Biological Process.

Bioinformatics, 2004 Feb 12, 20(3), 340 - 8
Functional topology in a network of protein interactions; Przulj N et al.; MOTIVATION: The building blocks of biological networks are individual protein-protein interactions (PPIs) . The cumulative PPI data set in Saccharomyces cerevisiae now exceeds 78 000 . Studying the network of these interactions will provide valuable insight into the inner workings of cells . RESULTS: We performed a systematic graph theory-based analysis of this PPI network to construct computational models for describing and predicting the properties of lethal mutations and proteins participating in genetic interactions, functional groups, protein complexes and signaling pathways . Our analysis suggests that lethal mutations are not only highly connected within the network, but they also satisfy an additional property: their removal causes a disruption in network structure . We also provide evidence for the existence of alternate paths that bypass viable proteins in PPI networks, while such paths do not exist for lethal mutations . In addition, we show that distinct functional classes of proteins have differing network properties . We also demonstrate a way to extract and iteratively predict protein complexes and signaling pathways . We evaluate the power of predictions by comparing them with a random model, and assess accuracy of predictions by analyzing their overlap with MIPS database . CONCLUSIONS: Our models provide a means for understanding the complex wiring underlying cellular function, and enable us to predict essentiality, genetic interaction, function, protein complexes and cellular pathways . This analysis uncovers structure-function relationships observable in a large PPI network.

Eukaryot Cell, 2004 Feb, 3(1), 180 - 9
An ste20 homologue in Ustilago maydis plays a role in mating and pathogenicity; Smith DG et al.; The mitogen-activated protein kinase (MAPK) pathways are conserved from fungi to humans and have been shown to play important roles in mating and filamentous growth for both Saccharomyces cerevisiae and dimorphic fungi and in infectivity for pathogenic fungi . STE20 encodes a protein kinase of the p21-activated protein kinase family that regulates more than one of these cascades in yeasts . We hypothesized that an Ste20p homologue would play a similar role in the dimorphic plant pathogen Ustilago maydis . The full-length copy of the U . maydis gene was obtained from a genomic library; it lacked introns and was predicted to encode a protein of 826 amino acids, whose sequence confirmed its identity as the first Ste20p homologue to be isolated from a plant pathogen . The predicted protein contained both an N-terminal regulatory Cdc42-Rac interactive binding domain and a C-terminal catalytic kinase domain . Disruption of the gene smu1 resulted in a delayed mating response in a mating-type-specific manner and also in a severe reduction in disease production on maize . Unlike the Ustilago bypass of cyclase (ubc) mutations previously identified in genes in the pheromone-responsive MAPK cascade, mutation of smu1 does not by itself act as an extragenic suppressor of the filamentous phenotype of a uac1 mutant . Thus, the direct connection of Smu1p to MAPK cascade function has yet to be established . Even so, Smu1, though not absolutely required for mating, is necessary for wild-type mating and pathogenicity.

Eukaryot Cell, 2004 Feb, 3(1), 144 - 56
Chromatin rearrangements in the prnD-prnB bidirectional promoter: dependence on transcription factors; Garcia I et al.; The prnD-prnB intergenic region regulates the divergent transcription of the genes encoding proline oxidase and the major proline transporter . Eight nucleosomes are positioned in this region . Upon induction, the positioning of these nucleosomes is lost . This process depends on the specific transcriptional activator PrnA but not on the general GATA factor AreA . Induction of prnB but not prnD can be elicited by amino acid starvation . A specific nucleosomal pattern in the prnB proximal region is associated with this process . Under conditions of induction by proline, metabolite repression depends on the presence of both repressing carbon (glucose) and nitrogen (ammonium) sources . Under these repressing conditions, partial nucleosomal positioning is observed . This depends on the CreA repressor's binding to two specific cis-acting sites . Three conditions (induction by the defective PrnA80 protein, induction by amino acid starvation, and induction in the presence of an activated CreA) result in similar low transcriptional activation . Each results in a different nucleosome pattern, which argues strongly for a specific effect of each signal on nucleosome positioning . Experiments with trichostatin A suggest that both default nucleosome positioning and partial positioning under induced-repressed conditions depend on deacetylated histones.

Eukaryot Cell, 2004 Feb, 3(1), 108 - 20
The Ras/protein kinase A pathway acts in parallel with the Mob2/Cbk1 pathway to effect cell cycle progression and proper bud site selection; Schneper L et al.; In Saccharomyces cerevisiae, Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity . Ras proteins also have PKA-independent functions in mitosis and actin repolarization . We have found that mutations in MOB2 or CBK1 confer a slow-growth phenotype in a ras2Delta background . The slow-growth phenotype of mob2Delta ras2Delta cells results from a G1 delay that is accompanied by an increase in size, suggesting a G1/S role for Ras not previously described . In addition, mob2Delta strains have imprecise bud site selection, a defect exacerbated by deletion of RAS2 . Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes . The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1 . From these observations, we conclude that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression.

Eukaryot Cell, 2004 Feb, 3(1), 52 - 60
C-terminal truncation of alpha-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha; Chechenova MB et al.; In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport . Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding alpha-COP, a subunit of the COPI protein complex . H . polymorpha ret1 mutants, which expressed truncated alpha-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of alpha-COP expression was lethal . The alpha-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase . Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion . The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed.

Genes Dev, 2004 Feb 1, 18(3), 333 - 43
In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer; Bhaumik SR et al.; Our understanding of eukaryotic transcriptional activation mechanisms has been hampered by an inability to identify the direct in vivo targets of activator proteins, primarily because of lack of appropriate experimental methods . To circumvent this problem, we have developed a fluorescence resonance energy transfer (FRET) assay to monitor interactions with transcriptional activation domains in living cells . We use this method to show that the Tra1 subunit of the SAGA (Spt/Ada/Gcn5/acetyltransferase) complex is the direct in vivo target of the yeast activator Gal4 . Chromatin-immunoprecipitation experiments demonstrate that the Gal4-Tra1 interaction is required for recruitment of SAGA to the upstream activating sequence (UAS), and SAGA, in turn, recruits the Mediator complex to the UAS . The UAS-bound Mediator is required for recruitment of the general transcription factors to the core promoter . Thus, our results identify the in vivo target of an activator and show how the activator-target interaction leads to transcriptional stimulation . The FRET assay we describe is a general method that can be used to identify the in vivo targets of other activators.

Biochim Biophys Acta, 2004 Feb 15, 1608(2-3), 190 - 9
Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase; Hsiao YY et al.; Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis . Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues . Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate {J . Protein Chem . 21 (2002) 51} . In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis . A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations . Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase . The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier . The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity . Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion . Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+) . Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.

Methods Mol Biol, 2004, 257, 17 - 28
Identifying phosphoCTD-associating proteins; Phatnani HP et al.; The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is hyperphosphorylated during transcription elongation . The phosphoCTD is known to bind to a subset of RNA processing factors and to several other nuclear proteins, thereby positioning them to efficiently carry out their elongation-linked functions . The authors propose that additional phosphoCTD-associating proteins (PCAPs) exist and describe a systematic biochemical approach for identifying such proteins . A binding probe is generated by using yeast CTD kinase I to exhaustively phosphorylate a CTD fusion protein . This phosphoCTD is used to probe fractionated yeast or mammalian extracts in a Far Western protein interaction assay . Putative PCAPs are further purified and identified by mass spectrometry.

Methods Mol Biol, 2004, 262, 223 - 37
Chromatin immunoprecipitation to investigate protein-DNA interactions during genetic recombination; Goldfarb T et al.; Chromatin immunoprecipitation is a technique that allows one to examine the in vivo localization of proteins to DNA . This technique is well suited for studying genetic recombination since it can provide both a temporal and spatial assessment of the dynamic association of proteins with DNA in both wild-type and mutant backgrounds . To perform this procedure, cells undergoing a synchronous recombination event are treated with a crosslinking agent . Following cell lysis and shearing of the DNA, immunoprecipitation is used to isolate the protein of interest, along with any DNA that is crosslinked to the protein . Polymerase chain reaction (PCR) is then used to determine the relative amounts of DNA associated with the protein of interest throughout the recombination event . This in vivo chemical crosslinking technique can be used to localize proteins to both double-strand breaks and recombination intermediates.

Methods Mol Biol, 2004, 262, 209 - 19
Enhancement of in vivo targeted nucleotide exchange by nonspecific carrier DNA; Maguire KK et al.; Targeted nucleotide exchange (TNE) is a process in which an oligonucleotide bearing sequence complementarity aligns with the sequence of a target gene and directs the alteration of a single base . This technique can be used to repair a point mutation or mediate site-specific mutagenesis . A critical factor in the development of this approach centers around the elevation and stabilization of the frequencies with which these events occur . Here we describe a protocol for increasing the frequency of TNE in the true yeast, Saccharomyces cerevisiae, through the use of nonspecific, carrier oligonucleotides . These molecules, when added to the reaction, increase the TNE frequency up to 25-fold in some cases, perhaps by providing a molecular trap to bind factors, which may inactivate the specific targeting oligos.

Methods Mol Biol, 2004, 262, 35 - 52
Analysis of recombinational repair of DNA double-strand breaks in mammalian cells with I-SceI nuclease; Nickoloff JA et al.; Eukaryotes repair DNA double-strand breaks (DSBs) by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) . DSBs are a natural consequence of DNA metabolism, occurring, for example, during DNA replication and meiosis . DSBs are also induced by chemicals and radiation . I-SceI endonuclease recognizes an 18-bp sequence with little degeneracy; therefore I-SceI is highly specific, and its recognition sequence is predicted to occur by chance less than once in even the largest known genomes . As such, I-SceI can be used to introduce a DSB into a defined (engineered) site in a mammalian chromosome, and this facilitates detailed studies of DSB repair . DSBs induced in repeated regions can be repaired by several different HR processes, including gene conversion with or without associated crossovers, or single-strand annealing . The specific types of HR events that can be scored depend on the configuration of the repeated regions and whether selection for recombinants is imposed . Nonselective assays detect both HR and NHEJ events . This chapter focuses on the systems for delivering I-SceI nuclease to mammalian cells and the strategies for detecting various outcomes of DSB repair.

Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 1858 - 62 Epub 2004 Feb 09.
Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex; Hurt E et al.; The TREX (transcription/export) complex couples transcription elongation to the nuclear export of mRNAs . In this article, we show that the poly(A)(+) RNA-binding proteins Gbp2 and Hrb1, which resemble the serine-arginine-rich (SR) family of splicing factors found in higher eukaryotes, are specifically associated with the yeast TREX complex . We also show that Gbp2 and Hrb1 interact with Ctk1, a kinase that phosphorylates the C-terminal domain of RNA polymerase II during transcription elongation . Consistent with these findings, Gbp2 and Hrb1 associate with actively transcribed genes throughout their entire lengths . By using an RNA immunoprecipitation assay, we show that Gbp2 and Hrb1 also are bound to transcripts that are derived from these genes . We conclude that recruitment of the SR-like proteins Gbp2 and Hrb1 to mRNA occurs cotranscriptionally by means of association with the TREX complex and/or Ctk1.

Mol Biol Cell, 2004 Apr, 15(4), 2038 - 47 Epub 2004 Feb 06.
Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex; de Graaf P et al.; Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex . In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11 . The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane . We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.

Endocrinology, 2004 May, 145(5), 2307 - 18 Epub 2004 Feb 05.
Cloning and characterization of granulosa cell high-mobility group (HMG)-box protein-1, a novel HMG-box transcriptional regulator strongly expressed in rat ovarian granulosa cells; Kajitani T et al.; Specific events in the ovary are dependent on gene expression in the tissue . By screening a rat ovarian granulosa cell cDNA library, a cDNA clone encoding a novel transcription factor-like protein containing a high-mobility group-box, referred to as granulosa cell high-mobility group-box protein-1 (GCX-1), was identified . The expression of GCX-1 is restricted to the hypothalamus, pituitary, testis, uterus, and ovary but was not detected in the adrenal gland . An in situ hybridization study revealed that the expression of GCX-1 was restricted to granulosa cell layers in early-stage follicles, and the expression was very low in large antral follicles and the corpus luteum, but localized expression in the testis or pituitary was not clear . Endogenous GCX-1 protein in the granulosa cells was identified by a Western blot analysis, and an analysis using the green fluorescence protein-GCX-1 fusion protein revealed that the GCX-1 protein was localized in the cell nucleus . GAL4 fusion protein-based assays demonstrated that GCX-1 is a potent transcriptional activator, and its putative transactivation domain was mapped to the region between amino acid residues 25 and 63 from the N terminus . These data strongly suggest that GCX-1 is likely a novel transcriptional activator that is exclusively expressed in reproductive tissues involving the hypothalamo-pituitary-gonadal axis, and functions as a specific regulator of follicular development, and may also participate in other specific events related to reproduction, particularly in the female.

Bioinformatics, 2004 May 1, 20(7), 1119 - 28 Epub 2004 Feb 05.
Predicting rules on organization of cis-regulatory elements, taking the order of elements into account; Terai G et al.; MOTIVATION: In eukaryotes, rules regarding organization of cis-regulatory elements are complex . They sometimes govern multiple kinds of elements and positional restrictions on elements . RESULTS: We propose a method for detecting rules, by which the order of elements is restricted . The order restriction is expressed as element patterns . We extract all the element patterns that occur in promoter regions of at least the specified number of genes . Then, we find significant patterns based on the expression similarity of genes with promoter regions containing each of the extracted patterns . When we applied our method to Saccharomyces cerevisiae, we detected significant patterns overlooked by previous methods, thus demonstrating the utility of our method for analyses of eukaryotic gene regulation . We also suggest that several types of element organization exist: (i) those in which only the order of elements is important, (ii) order and distance both are important and (iii) only the combination of elements is important . AVAILABILITY: The program for extracting element patterns is available upon request.

Mol Microbiol, 2004 Feb, 51(4), 1129 - 42
Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance; de Pinto B et al.; In yeast the UPF1, UPF2 and UPF3 genes encode three interacting factors involved in translation termination and nonsense-mediated mRNA decay (NMD) . UPF1 plays a central role in both processes . In addition, UPF1 was originally isolated as a multicopy suppressor of mitochondrial splicing deficiency, and its deletion leads to an impairment in respiratory growth . Here, we provide evidence that inactivation of UPF2 or UPF3, like that of UPF1, leads to an impairment in respiratory competence, suggesting that their products, Upf1p, Upf2p and Upf3p, are equivalently involved in mitochondrial biogenesis . In addition, however, we show that only Upf1p acts as a multicopy suppressor of mitochondrial splicing deficiency, and its activity does not require either Upf2p or Upf3p . Mutations in the conserved cysteine- and histidine-rich regions and ATPase and helicase motifs of Upf1p separate the ability of Upf1p to complement the respiratory impairment of a Deltaupf1 strain from its ability to act as a multicopy suppressor of mitochondrial splicing deficiency, indicating that distinct pathways express these phenotypes . In addition, we show that, when overexpressed, Upf1p is not detected within mitochondria, suggesting that its role as multicopy suppressor of mitochondrial splicing deficiency is indirect . Furthermore, we provide evidence that cells overexpressing certain upf1 alleles accumulate a phosphorylated isoform of Upf1p . Altogether, these results indicate that overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance, which relies on Upf1p-Upf2p-Upf3p functional interplay.

Nucleic Acids Res, 2004 Feb 03, 32(2), 784 - 90 Print 2004.
A programmed -1 ribosomal frameshift signal can function as a cis-acting mRNA destabilizing element; Plant EP et al.; Nonsense-mediated mRNA decay (NMD) directs rapid degradation of premature termination codon (PTC)-containing mRNAs, e.g . those containing frameshift mutations . Many viral mRNAs encode polycistronic messages where programmed -1 ribosomal frameshift (-1 PRF) signals direct ribosomes to synthesize polyproteins . A previous study, which identified consensus -1 PRF signals in the yeast genome, found that, in contrast to viruses, the majority of predicted -1 PRF events would direct translating ribosomes to PTCs . Here we tested the hypothesis that a -1 PRF signal can function as a cis-acting mRNA destabilizing element by inserting an L-A viral -1 PRF signal into a PGK1 reporter construct in the 'genomic' orientation . The results show that even low levels of -1 PRF are sufficient to target the reporter mRNA for degradation via the NMD pathway, with half-lives similar to messages containing in-frame PTCs . The demonstration of an inverse correlation between frameshift efficiency and mRNA half-lives suggests that modulation of -1 PRF frequencies can be used to post-transcriptionally regulate gene expression . Analysis of the mRNA decay profiles of the frameshift-signal- containing reporter mRNAs also supports the notion that NMD remains active on mRNAs beyond the 'pioneer round' of translation in yeast.

Mol Genet Genomics, 2004 Mar, 271(2), 121 - 9 Epub 2004 Jan 31.
An insertional mutation in the rice PAIR2 gene, the ortholog of Arabidopsis ASY1, results in a defect in homologous chromosome pairing during meiosis; Nonomura KI et al.; To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis . The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene . The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae . Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues . In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages . The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1 . The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene.

Nat Struct Mol Biol, 2004 Mar, 11(3), 249 - 56 Epub 2004 Feb 01.
Crystal structure of Dcp1p and its functional implications in mRNA decapping; She M et al.; A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5'-->3' exonucleolytic degradation . A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p . The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains . Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains . Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites . One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins . Moreover, a conserved hydrophobic patch is shown to be critical for decapping.

J Med Genet, 2004 Feb, 41(2), 120 - 4
Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12; De Meirleir L et al.; In patients with mitochondrial encephalomyopathies an increasing number of causative gene defects have been detected . The number of identified pathogenic mitochondrial DNA mutations has largely increased over the past 15 years . Recently, much attention has turned to the investigation of nuclear oxidative phosphorylation (OXPHOS) gene defects . Within the OXPHOS defects, complex V deficiency is rarely found and, so far, these defects have only been attributed to mutations in the mitochondrial MTATP6 gene . Mutation analysis of the complete coding regions at the cDNA level of the nuclear ATP11, ATP12, ATPalpha, ATPbeta and ATPgamma genes and the mitochondrial MTATP6 and MTAT8 genes was undertaken in two unrelated patients . Blue Native polyacrylamide gel electrophoresis followed by catalytic staining had already documented their complex V decreased activity . Extensive molecular analysis of five nuclear and two mitochondrial genes revealed a mutation in the ATP12 assembly gene in one patient . This mutation is believed to be the cause of the impaired complex V activity . To our knowledge, this is the first report of a pathogenic mutation in a human nuclear encoded ATPase assembly gene.

J Mol Biol, 2004 Feb 13, 336(2), 489 - 96
Cytochrome c and SDS: a molten globule protein with altered axial ligation; Bertini I et al.; Saccharomices cerevisiae (yeast iso-1) cytochrome c has been investigated in the presence of 100 mM SDS in order to simulate the interaction of cytochrome c with membrane . Under these circumstances, a high spin species with detached methionine axial ligand is observed through NMR, in analogy to findings on the horse heart protein . However, at variance with the latter system, for the yeast protein also a low spin species is detected, which appears to be present with a concentration of about 40% with respect to that of the high spin species . The R(1), R(2), {1H}-15N NOE of backbone amides which are not affected by paramagnetism are homogeneous and allow a simultaneous analysis of the data for the two species . The result is that the rotational correlation time is larger than in water and larger than expected on the basis of viscosity of the SDS-containing solution . This finding suggests interactions of cytochrome c with SDS . Furthermore, it appears that there is subnanosecond backbone mobility, which also accounts for the decreased intensity of NOE cross-peaks and may be associated with equilibria between helical and random coil structure . The dynamic behavior appears to be a common feature of the high spin and low spin species and is consistent with the presence of a molten globule state . The molten globule nature of the protein could account for the presence of the different axial coordination of the heme iron . Such findings are meaningful with respect to the physiology of cytochrome c as electron transfer protein and as promoter of apoptosis.

Biochemistry, 2004 Feb 10, 43(5), 1204 - 12
Transition state structure for ADP-ribosylation of eukaryotic elongation factor 2 catalyzed by diphtheria toxin; Parikh SL et al.; Bacterial protein toxins are the most powerful human poisons known, exhibiting an LD(50) of 0.1-1 ng kg(-)(1) . A major subset of such toxins is the NAD(+)-dependent ADP-ribosylating exotoxins, which include pertussis, cholera, and diphtheria toxin . Diphtheria toxin catalyzes the ADP ribosylation of the diphthamide residue of eukaryotic elongation factor 2 (eEF-2) . The transition state of ADP ribosylation catalyzed by diphtheria toxin has been characterized by measuring a family of kinetic isotope effects using (3)H-, (14)C-, and (15)N-labeled NAD(+) with purified yeast eEF-2 . Isotope trapping experiments yield a commitment to catalysis of 0.24 at saturating eEF-2 concentrations, resulting in suppression of the intrinsic isotope effects . Following correction for the commitment factor, intrinsic primary kinetic isotope effects of 1.055 +/- 0.003 and 1.022 +/- 0.004 were observed for {1(N)'-(14)C}- and {1(N)-(15)N}NAD(+), respectively; the double primary isotope effect was 1.066 +/- 0.004 for {1(N)'-(14)C, 1(N)-(15)N}NAD(+) . Secondary kinetic isotope effects of 1.194 +/- 0.002, 1.101 +/- 0.003, 1.013 +/- 0.005, and 0.988 +/- 0.002 were determined for {1(N)'-(3)H}-, {2(N)'-(3)H}-, {4(N)'-(3)H}-, and {5(N)'-(3)H}NAD(+), respectively . The transition state structure was modeled using density functional theory (B1LYP/6-31+G) as implemented in Gaussian 98, and theoretical kinetic isotope effects were subsequently calculated using Isoeff 98 . Constraints were varied in a systematic manner until the calculated kinetic isotope effects matched the intrinsic isotope effects . The transition state model most consistent with the intrinsic isotope effects is characterized by the substantial loss in bond order of the nicotinamide leaving group (bond order = 0.18, 1.99 A) and weak participation of the attacking imidazole nucleophile (bond order = 0.03, 2.58 A) . The transition state structure imparts strong oxacarbenium ion character to the ribose ring even though significant bond order remains to the nicotinamide leaving group . The transition state model presented here is asymmetric and consistent with a dissociative S(N)1 type mechanism in which attack of the diphthamide nucleophile lags behind departure of the nicotinamide.

Plant Mol Biol, 2003 Sep, 53(1-2), 227 - 36
Cyclin D-knockout uncouples developmental progression from sugar availability; Lorenz S et al.; Multicellular organisms need to modulate proliferation and differentiation in response to external conditions . An important role in these processes plays the mitogen-stimulated induction of cyclin D (cycD) gene expression . D-type cyclins have been identified as the crucial intracellular sensors for cell-cycle regulation in all eukaryotes . However, cycD deletions have been found to cause specific phenotypic alterations in animals but not yet in plants . An insertional mutation of a so far uncharacterized Arabidopsis cycD gene did not alter the plant phenotype . To gain new insights into CycD function of land plants, we generated targeted cycD gene knockouts in the moss Physcomitrella patens and observed a surprisingly limited disruption phenotype . While wild-type plants reacted to exogenous glucose sources with prolonged growth of juvenile stages and retarded differentiation, cycD knockouts exhibited developmental progression independent of sugar supply . On the other hand, growth rate, cell sizes or plant size were not affected . Thus, we conclude that Physcomitrella CycD might not be essential for cell-cycle regulation but is important for coupling the developmental progression to nutrient availability.

Plant Mol Biol, 2003 Sep, 53(1-2), 201 - 12
Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7; Ziemienowicz A et al.; We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1) . Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals . The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals . We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants . AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms . Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis . We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction . Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays . We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8 . Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal . In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1 . These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.

Biochem J, 2004 May 15, 380(Pt 1), 131 - 7
Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR {S6-(4-nitrobenzyl)-mercaptopurine riboside} and dilazep for the human equilibrative nucleoside transporter, hENT1; Endres CJ et al.; We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates . In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate . This growth was prevented when inhibitors of hENT1 {e.g . NBMPR {S6-(4-nitrobenzyl)-mercaptopurine riboside}, dilazep or dipyridamole} were included in the media . To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain . Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized . Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole . The IC50 values of NBMPR and dilazep for {3H}adenosine transport by one of these mutants L92Q (Leu92-->Gln) were approx . 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged . Additionally, when compared with the wild-type transporter, {3H}adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines . The Km value for inosine transport was approx . 4-fold greater for the L92Q mutant (260+/-16 mM) when compared with the wild-type transporter (65+/-7.8 mM) . We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.

J Cell Biochem, 2004 Feb 15, 91(3), 633 - 45
A LIM protein, Hic-5, functions as a potential coactivator for Sp1; Shibanuma M et al.; Hic-5 is a LIM protein with striking similarity to paxillin, and shuttles between focal adhesions and the nucleus . Our previous study suggested that Hic-5 participates in the transcriptional control of several genes such as the c-fos and p21 genes . In the present study, we examined the function of Hic-5 in the nucleus using the transcriptional promoter region of the p21 gene . When localized to the nucleus, Hic-5 was found to transactivate the p21 promoter through two of five Sp1 sites in the region proximal to the TATA box . The Hic-5 effect was mediated by a transactivation domain of Sp1 and functional interaction with p300 through the LIM4 domain . Hic-5 was also shown to interact functionally and physically with Smad3 through the LIM domains and to potentiate p21 promoter activity together with Smad3 and Sp1 . These properties were confirmed in an artificial system using GAL4-fusion protein . Thus, Hic-5 was suggested to have a potential function as a cofactor in the transcriptional complex that contains Sp1, playing a role in gene transcription in the nucleus as well as in integrin signaling at focal adhesion sites .

Oncogene, 2004 Mar 11, 23(10), 1801 - 8
An RNF11: Smurf2 complex mediates ubiquitination of the AMSH protein; Li H et al.; RING-finger proteins play crucial roles in ubiquitination events involved in diverse cellular processes including signal transduction, differentiation and apoptosis . Most of the RING-finger proteins have E3-ubiquitin ligase activity . RNF11 is a small RING-finger protein and harbors a RING-H2 domain and a PY motif that could facilitate protein:protein interaction(s) involved in oncogenesis . To isolate RNF11 protein partners and determine its role in normal and cancer cells, we performed yeast two-hybrid screening . Among 18 in-frame positive clones, three were found to be ZBRK1, Eps15 and AMSH (associated molecule with the SH3 domain of STAM) . ZBRK1 is a KRAB domain containing Zinc-finger protein and is known to repress target gene transcription in a BRCA1-dependent manner . Eps15 is monoubiquitinated and is part of an essential complex involved in the endocytosis of plasma membrane receptors via the clathrin-mediated internalization pathway . Recent studies have shown that AMSH protein is involved in BMP/TGF-beta signaling pathway by binding to Smad6 and Smad7 . The association of RNF11 with these binding partners suggests that it would be involved in biological processes such as gene transcription, BMP/TGF-beta signaling and ubiquitination-associated events . Previously, we have shown that RNF11 interacts with the HECT-type E3 ligases AIP4 and Smurf2 . Here, we show that RNF11 binds to AMSH in mammalian cells and that this interaction is independent of the RNF11 RING-finger domain and the PY motif . Our results also demonstrate that AMSH is ubiquitinated by Smurf2 E3 ligase in the presence of RNF11 and that a consequent reduction in its steady-state level requires both RNF11 and Smurf2 . RNF11 therefore recruits AMSH to Smurf2 for ubiquitination, leading to its degradation by the 26S proteasome . The potential functions of RNF11-mediated degradation of AMSH in breast cancer are discussed.

J Exp Bot, 2004 Mar, 55(397), 585 - 94 Epub 2004 Jan 30.
Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley; Fukuda A et al.; Two cDNA clones encoding vacuolar H+-inorganic pyrophosphatase (HVP1 and HVP10), one clone encoding the catalytic subunit (68 kDa) of vacuolar H+-ATPase (HvVHA-A), and one clone encoding vacuolar Na+/H+ antiporter (HvNHX1) were isolated from barley (Hordeum vulgare), a salt-tolerant crop . Salt stress increased the transcript levels of HVP1, HVP10, HvVHA-A, and HvNHX1, and osmotic stress also increased the transcript levels of HVP1 and HvNHX1 in barley roots . The transcription of HVP1 in response to salt stress was regulated differently from that of HVP10 . In addition, the HVP1 expression changed in a pattern similar to that of HvNHX1 expression . These results indicate that the expression of HVP1 is co-ordinated with that of HvNHX1 in barley roots in response to salt and osmotic stresses.

J Cell Mol Med, 2003 Oct-Dec, 7(4), 388 - 400
Peroxisome biogenesis and the role of protein import; Brown LA et al.; Peroxisomes are metabolic organelles with enzymatic content that are found in virtually all cells and are involved in beta-oxidation of fatty acids, hydrogen peroxide-based respiration and defence against oxidative stress . The steps of their biogenesis involves "peroxins", proteins encoded by PEX genes . Peroxins are involved in three key stages of peroxisome development: (1) . import of peroxisomal membrane proteins; (2) . import of peroxisomal matrix proteins and (3) . peroxisome proliferation . Of these three areas, peroxisomal matrix-protein import is by far the best understood and accounts for most of the available published data on peroxisome biogenesis . Defects in peroxisome biogenesis result in peroxisome biogenesis disorders (PBDs), which although rare, have no known cure to-date . This review explores current understanding of each key area in peroxisome biogenesis, paying particular attention to the role of protein import.

J Chromatogr A, 2004 Jan 23, 1024(1-2), 71 - 8
Effect of the detergent Tween-20 on the DNA affinity chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration; Robinson FD et al.; C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-affinity chromatography . When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times . Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase . The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads . This detergent effect is seen when either Sepharose or silica is used for the stationary phase . Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20 . Mg2+ or EDTA in the mobile phase gave similar chromatography for C/EBP; since EDTA protects columns from DNases, its inclusion in the mobile phase is preferred . After extended use, the DNA affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns . Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.

Cell Motil Cytoskeleton, 2004 Apr, 57(4), 233 - 45
The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers; Nikulina K et al.; The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle . In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis . Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers . This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes . In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1 . Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis . Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells . In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella .

Cell Cycle, 2004 Apr, 3(4), 401 - 3 Epub 2004 Apr 01.
Cdk1: unsung hero of S phase?
Pacek M, Prokhorova TA, Walter JC.
Cdk2 has been viewed as a key cell cycle regulator that is essential for S phase progression . The recent discovery that Cdk2 is not required for cell proliferation in mice now shows that other factors must be able to replace Cdk2 in stimulating DNA replication . Experiments performed in Xenopus egg extracts identify the mitotic protein kinases Cdk1/Cyclin B and Cdk1/Cyclin A as likely candidates . These observations raise the intriguing possibility that Cdk1 normally participates in genome duplication in wild type cells.

Science, 2004 Feb 27, 303(5662), 1367 - 70 Epub 2004 Jan 29.
A genome-wide screen identifies genes required for centromeric cohesion; Marston AL et al.; During meiosis, two chromosome segregation phases follow a single round of DNA replication . We identified factors required to establish this specialized cell cycle by examining meiotic chromosome segregation in a collection of yeast strains lacking all nonessential genes . This analysis revealed Sgo1, Chl4, and Iml3 to be important for retaining centromeric cohesin until the onset of anaphase II . Consistent with this role, Sgo1 localizes to centromeric regions but dissociates at the onset of anaphase II . The screen described here provides a comprehensive analysis of the genes required for the meiotic cell cycle and identifies three factors important for the stepwise loss of sister chromatid cohesion.

Genes Dev, 2004 Jan 15, 18(2), 184 - 95
Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B; Kao CF et al.; Covalent modifications of the histone N tails play important roles in eukaryotic gene expression . Histone acetylation, in particular, is required for the activation of a subset of eukaryotic genes through the targeted recruitment of histone acetyltransferases . We have reported that a histone C tail modification, ubiquitylation of H2B, is required for optimal expression of several inducible yeast genes, consistent with a role in transcriptional activation . H2B was shown to be ubiquitylated and then deubiquitylated at the GAL1 core promoter following galactose induction . We now show that the Rad6 protein, which catalyzes monoubiquitylation of H2B, is transiently associated with the GAL1 promoter upon gene activation, and that the period of its association temporally overlaps with the period of H2B ubiquitylation . Rad6 promoter association depends on the Gal4 activator and the Rad6-associated E3 ligase, Bre1, but is independent of the histone acetyltransferase, Gcn5 . The SAGA complex, which contains a ubiquitin protease that targets H2B for deubiquitylation, is recruited to the GAL1 promoter in the absence of H2B ubiquitylation . The data suggest that Rad6 and SAGA function independently during galactose induction, and that the staged recruitment of these two factors to the GAL1 promoter regulates the ubiquitylation and deubiquitylation of H2B . We additionally show that both Rad6 and ubiquitylated H2B are absent from two regions of transcriptionally silent chromatin but present at genes that are actively transcribed . Thus, like histone H3 lysine 4 and lysine 79 methylation, two modifications that it regulates, Rad6-directed H2B ubiquitylation defines regions of active chromatin.

Bioinformatics, 2004 Mar 22, 20(5), 750 - 7 Epub 2004 Jan 29.
Regression trees for regulatory element identification; Phuong TM et al.; MOTIVATION: The transcription of a gene is largely determined by short sequence motifs that serve as binding sites for transcription factors . Recent findings suggest direct relationships between the motifs and gene expression levels . In this work, we present a method for identifying regulatory motifs . Our method makes use of tree-based techniques for recovering the relationships between motifs and gene expression levels . RESULTS: We treat regulatory motifs and gene expression levels as predictor variables and responses, respectively, and use a regression tree model to identify the structural relationships between them . The regression tree methodology is extended to handle responses from multiple experiments by modifying the split function . The significance of regulatory elements is determined by analyzing tree structures and using a variable importance measure . When applied to two data sets of the yeast Saccharomyces cerevisiae, the method successfully identifies most of the regulatory motifs that are known to control gene transcription under the given experimental conditions, and suggests several new putative motifs . Analysis of the tree structures also reconfirms several pairs of motifs that are known to regulate gene transcription in combination . AVAILABILITY: http://if.kaist.ac.kr/~phuong/RegTree

Bioinformatics, 2004 Apr 12, 20(6), 895 - 902 Epub 2004 Jan 29.
Mapping Gene Ontology to proteins based on protein-protein interaction data; Deng M et al.; MOTIVATION: Gene Ontology (GO) consortium provides structural description of protein function that is used as a common language for gene annotation in many organisms . Large-scale techniques have generated many valuable protein-protein interaction datasets that are useful for the study of protein function . Combining both GO and protein-protein interaction data allows the prediction of function for unknown proteins . RESULT: We apply a Markov random field method to the prediction of yeast protein function based on multiple protein-protein interaction datasets . We assign function to unknown proteins with a probability representing the confidence of this prediction . The functions are based on three general categories of cellular component, molecular function and biological process defined in GO . The yeast proteins are defined in the Saccharomyces Genome Database (SGD) . The protein-protein interaction datasets are obtained from the Munich Information Center for Protein Sequences (MIPS), including physical interactions and genetic interactions . The efficiency of our prediction is measured by applying the leave-one-out validation procedure to a functional path matching scheme, which compares the prediction with the GO description of a protein's function from the abstract level to the detailed level along the GO structure . For biological process, the leave-one-out validation procedure shows 52% precision and recall of our method, much better than that of the simple guilty-by-association methods.

Toxicology, 2004 Feb 15, 195(2-3), 243 - 54
Antiandrogenic activity of extracts of diesel exhaust particles emitted from diesel-engine truck under different engine loads and speeds; Okamura K et al.; To clarify the alteration of androgenic and antiandrogenic activities by diesel engine conditions, we collected diesel exhaust particles (DEP) samples emitted from a diesel-engine truck under different conditions of engine loads and vehicle speeds, and DEP extract (DEPE) samples were prepared from each . The androgenic and antiandrogenic activities of the DEPE samples were examined using a prostate specific antigen (PSA) promoter-luciferase reporter gene assay in PC3/AR human prostate cancer cells . While all DEPE samples did not exhibit androgenic effects, the antiandrogenic effects were enhanced by higher engine load but not by higher vehicle speed . In this study, significant correlations between antiandrogenic and aryl hydrocarbon receptor (AhR) agonistic activities were demonstrated in PC3/AR cells by 16 polycyclic aromatic compounds and beta-naphthoflavone . Yeast two-hybrid assay and cytochrome P450 (CYP) 1A1 promoter-luciferase reporter gene assay showed that the antiandrogenic constituents acting as androgen receptor (AR) antagonists and AhR agonists were increased by only the higher engine load . In conclusion, the antiandrogenic effects of DEPE samples were enhanced by a higher engine load which resulted in DEPC samples with elevated AhR agonistic and AR antagonistic activities.

Biochem J, 2004 Apr 1, 379(Pt 1), 23 - 9
Repression of Smad4 transcriptional activity by SUMO modification; Long J et al.; Smad4 plays a key role in TGF-beta (transforming growth factor beta)/Smad-mediated transcriptional responses . We show that Smad4 is sumoylated both in vivo and in vitro . Recent studies showed that sumoylation of Smad4 regulated its stability, but the effect of sumoylation on the intrinsic transcriptional activity of Smad4 was not defined . We show that overexpression of SUMO (small ubiquitin-related modifier)-1 and Ubc9 can inhibit a TGF-beta-responsive reporter gene, whereas co-transfection with SUMO-1 protease-1 (SuPr-1) can increase the TGF-beta response . We show further that mutation of the Smad4 sumoylation sites or co-transfection with SuPr-1 greatly increases Smad4 transcriptional activity . Moreover, direct fusion of SUMO-1 to the sumoylation mutant Smad4 potently inhibits its transcriptional activity . Thus, as it is being rapidly discovered that sumoylation inhibits the activities of many transcription factors, sumoylation also represses Smad4 transcriptional activity . The net effect of sumoylation of Smad4 can therefore be either stimulatory or inhibitory, depending on the target promoter that is analysed.

Anal Chem, 2004 Feb 1, 76(3), 720 - 7
Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein; Amunugama R et al.; A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer . Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures . Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance . The identification of the protein was achieved by matching the uninterpreted product ion spectrum against protein sequence databases with varying degrees of annotation, coupled with a scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential sites . The protein was identified to be an acetylated yeast heat shock protein, HS12_Yeast (11.6 kDa), with the initiating methionine residue removed . This constitutes the first example of the identification of an a priori unknown protein that is not present in an annotated protein database using a "top-down" approach with a quadrupole ion trap . This example illustrates the utility of relatively low cost instrumentation with modest mass analysis characteristics for the identification of modified proteins without recourse to enzymatic digestion . It also illustrates how experimental data can be used interactively with protein databases when the modified protein of interest is not initially present in the database.

J Org Chem, 2004 Feb 6, 69(3), 613 - 8
The first direct evaluation of the two-active site mechanism for chitin synthase; Yeager AR et al.; Chitin synthase polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc) and is essential for fungal cell wall biosynthesis . The alternating orientation of the GlcNAc residues within the chitin chain has led to the proposal that chitin synthase possesses two active sites . We report the results of the first direct test of this possibility . Two simple uridine-derived dimeric inhibitors are shown to exhibit 10-fold greater inhibition than a monomeric control, consistent with the presence of two active sites . This observation has important implications for the development of antifungal agents, as well as the understanding of polymerizing glycosyltransferases.

Plant Mol Biol, 2003 Oct, 53(3), 383 - 97
Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress; Hegedus D et al.; Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors . The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration . Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A . thaliana nam and cuc mutants when expressed ectopically in A . thaliana . Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A . thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root . Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter . A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485 . The protein interaction and transcriptional activation domains were mapped by deletion analysis.

Plant Mol Biol, 2003 Oct, 53(3), 273 - 9
Identification of rice (Oryza sativa) proteins linked to the cyclin-mediated regulation of the cell cycle; Cooper B et al.; Yeast two-hybrid assays were used to identify rice proteins interacting with two rice cyclins and other proteins potentially involved in cell cycling . The DNA sequences encoding 119 protein fragments identified were then compared by BLAST against proteins in GenBank . The proteins found include myosin-like proteins, transcription factors, kinesins, centromere proteins and undefined proteins . Based on interactions with cyclins and other elements required for cycling, we believe the undefined proteins may be involved in associated cycling processes . The identification of proteins involved in cell cycle regulation in rice may allow for the control of agronomic traits involving plant growth or development.

EMBO J, 2004 Feb 11, 23(3), 616 - 26 Epub 2004 Jan 29.
Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase; Kaufmann I et al.; In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF) . CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal . Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF . hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA . Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner . hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA . These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.

Mol Cell Biol, 2004 Feb, 24(4), 1758 - 68
TRAM2 protein interacts with endoplasmic reticulum Ca2+ pump Serca2b and is necessary for collagen type I synthesis; Stefanovic B et al.; Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes . Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts . Activated HSCs are collagen-producing cells in the fibrotic liver . Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold . Likewise, expression of TRAM2 dramatically increases in activated HSCs . TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon . However, TRAM2 has a C terminus with only a 15% identity . The C-terminal part of TRAM2 interacts with the Ca(2+) pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells . TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact . Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs . The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect . Depletion of ER Ca(2+) with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation . We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon . This coupling may increase the local Ca(2+) concentration at the site of collagen synthesis, and a high Ca(2+) concentration may be necessary for the function of molecular chaperones involved in collagen folding.

Mol Cell Biol, 2004 Feb, 24(4), 1721 - 35
Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex; Liu Y et al.; Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD) . The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors . Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro . In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together . Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II . Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex . Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified . In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation.

Mol Cell Biol, 2004 Feb, 24(4), 1709 - 20
RNA polymerase II (Pol II)-TFIIF and Pol II-mediator complexes: the major stable Pol II complexes and their activity in transcription initiation and reinitiation; Rani PG et al.; Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found in Saccharomyces cerevisiae nuclear extracts . About 50% of Pol II is found associated with the general transcription factor TFIIF (Pol II-TFIIF), and about 20% of Pol II is associated with Mediator (Pol-Med) . No Pol II-Med-TFIIF complex was observed . The activity of Pol II and the purified Pol II complexes in transcription initiation and reinitiation was investigated by supplementing extracts depleted of either total Pol II or total TFIIF with purified Pol II or the Pol II complexes . We found that all three forms of Pol II can complement Pol II-depleted extracts for transcription initiation, but Pol II-TFIIF has the highest specific activity . Similarly, Pol II-TFIIF has a much higher specific activity than TFIIF for complementation of TFIIF transcription activity . Although the Pol II-TFIIF and Pol II-Med complexes were stable when purified, we found these complexes were dynamic in extracts under transcription conditions, with a single polymerase capable of exchanging bound Mediator and TFIIF . Using a purified system to examine transcription reinitiation, we found that Pol II-TFIIF was active in promoting multiple rounds of transcription while Pol II-Med was nearly inactive . These results suggest that both the Pol II-Med and Pol II-TFIIF complexes can be recruited for transcription initiation but that only the Pol II-TFIIF complex is competent for transcription reinitiation.

Org Lett, 2004 Feb 5, 6(3), 321 - 4
Water-soluble camptothecin derivatives that are intrinsic topoisomerase I poisons; Rahier NJ et al.; {structure: see text} In an effort to improve the water solubility of camptothecin, four 20-O-phosphate and phosphonate analogues have been prepared . These analogues are freely water soluble, stable at physiological pH, and stabilize the human topoisomerase I-DNA covalent binary complex with the same sequence selectivity as camptothecin itself . All four compounds inhibited the growth of yeast expressing human topoisomerase I in an enzyme-dependent fashion.

Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 317 - 9 Epub 2004 Jan 23.
Crystallization and preliminary crystallographic analysis of rat monoamine oxidase A complexed with clorgyline; Ma J et al.; Monoamine oxidase (MAO) is an FAD-containing mitochondrial outer-membrane protein which catalyzes the degradation of several neurotransmitters in the central nervous system . The two subtypes of MAO, MAOA and MAOB, have similar primary sequences but different substrate and inhibitor specificities . The structure of human MAOB has recently been determined, but the structure of MAOA remains unknown . To clarify the mechanisms underlying their unique substrate and inhibitor recognition and thereby facilitate the development of new specific inhibitors to treat MAO-related neurological disorders, rat MAOA was crystallized in a complex with the specific inhibitor clorgyline . Diffraction data were collected to 3.2 A resolution . The crystal belongs to the space group P4(3)2(1)2, with unit-cell parameters a = b = 158.2, c = 258.4 A.

Trends Genet, 2004 Feb, 20(2), 72 - 6
Conservation of protein-protein interactions - lessons from ascomycota; Pagel P et al.; Interacting proteins from Saccharomyces cerevisiae are evolutionarily conserved and their likelihood of having an ortholog in other ascomycota species correlates with the number of interaction partners . Moreover, interacting proteins show a clear preference to be conserved as a pair, indicating that nature maintains selection pressure on the interaction links between proteins . The conservation of interacting protein pairs between different organisms does not exhibit any bias with respect to protein functional roles.

Cell, 2004 Jan 23, 116(2), 153 - 66
The mechanisms of vesicle budding and fusion; Bonifacino JS et al.; Genetic and biochemical analyses of the secretory pathway have produced a detailed picture of the molecular mechanisms involved in selective cargo transport between organelles . This transport occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment . Vesicle budding and cargo selection are mediated by protein coats, while vesicle targeting and fusion depend on a machinery that includes the SNARE proteins . Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity.

Nat Cell Biol, 2004 Feb, 6(2), 138 - 45 Epub 2004 Jan 25.
The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression; Searle JS et al.; The conserved checkpoint kinases Chk1 and Rad53-Dun1 block the metaphase to anaphase transition by the phosphorylation and stabilization of securin, and block the mitotic exit network regulated by the Bfa1-Bub2 complex . However, both chk1 and rad53 mutants are able to exit from mitosis and initiate a new cell cycle, suggesting that both pathways have supporting functions in restraining anaphase and in blocking the inactivation of mitotic cyclin-Cdk1 complexes . Here we find that the cyclic-AMP-dependent protein kinase (PKA) pathway supports Chk1 in the regulation of mitosis by targeting the mitotic inducer Cdc20 . Cdc20 is phosphorylated on PKA consensus sites after DNA damage, and this phosphorylation requires the Atr orthologue Mec1 and the PKA catalytic subunits Tpk1 and Tpk2 . We show that the inactivation of PKA or expression of phosphorylation-defective Cdc20 proteins accelerates securin and Clb2 destruction in chk1 mutants and is sufficient to remove most of the DNA damage-induced delay . Mutation of the Cdc20 phosphorylation sites permitted the interaction of Cdc20 with Clb2 under conditions that should halt cell cycle progression . These data show that PKA pathways regulate mitotic progression through Cdc20 and support the DNA damage checkpoint pathways in regulating the destruction of Clb2 and securin.

Mol Biol Cell, 2004 Apr, 15(4), 1785 - 92 Epub 2004 Jan 23.
Sti1 and Cdc37 can stabilize Hsp90 in chaperone complexes with a protein kinase; Lee P et al.; Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown . We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase . Studies on signaling via this protein kinase pathway was measured by alpha-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1 . Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents . The greatest defects were in cpr7Delta, sse1Delta, and ydj1Delta mutants . Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies . Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Delta mutant back to wild-type levels . CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Delta mutant strain . These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes . Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7.

Mol Biol Cell, 2004 Apr, 15(4), 1736 - 45 Epub 2004 Jan 23.
Identification of protein complexes required for efficient sister chromatid cohesion; Mayer ML et al.; Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae . We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion . We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen . Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion . Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts . These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene . Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.

Mol Biol Cell, 2004 Apr, 15(4), 1623 - 34 Epub 2004 Jan 23.
Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response; Lin J et al.; Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres . We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1 . Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants . Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant . The mutants showed defects in sister chromatid separation at centromeres as well as telomeres, suggesting activation of a cell cycle checkpoint . Deletion of the DNA damage response genes DDC1, MEC3, or DDC2/SML1 failed to restore chromosome separation in the tlc1 template mutants . These results suggest that mutant telomere sequences elicit a checkpoint that is genetically distinct from those activated by deletion of telomerase or DNA damage.

Mol Biol Cell, 2004 Apr, 15(4), 1802 - 15 Epub 2004 Jan 23.
Positive and negative regulation of a SNARE protein by control of intracellular localization; Nakanishi H et al.; In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores . Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain . We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein . In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo . Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid . The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis . However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane . Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

Infect Immun, 2004 Feb, 72(2), 949 - 57
CpG oligodeoxynucleotide and Montanide ISA 51 adjuvant combination enhanced the protective efficacy of a subunit malaria vaccine; Kumar S et al.; Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans . We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of the Saccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP1(19)) of the murine malaria parasite Plasmodium yoelii . We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP1(19) in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP1(19) emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant) . In total, among mice immunized with yMSP1(19), 22 of 32 (68.7%) with ISA plus CpG 1826, 0 of 4 (0%) with CFA/incomplete Freund's adjuvant, 0 of 4 (0%) with CpG 1826 mixed with ISA (no yMSP1(19)), and 0 of 11 (0%) with CpG 1826 alone were completely protected against development of erythrocytic stage infection after sporozoite challenge . The adjuvant effect of CpG ODN 1826 was manifested as both significantly improved complete protection from malaria (defined as the absence of detectable erythrocytic form parasites) (P = 0.007, chi square) and reduced parasite burden in infected mice . In vivo depletions of interleukin-12 and gamma interferon cytokines and CD4+ and CD8+ T cells in vaccinated mice had no significant effect on immunity . On the other hand, immunoglobulin G (IgG) isotype levels appeared to correlate with protection . Inclusion of CpG ODN 1826 in the yMSP1(19) plus ISA vaccine contributed towards the induction of higher levels of IgG2a and IgG2b (Th1 type) antibodies, suggesting that CpG ODN 1826 caused a shift towards a Th1 type of immune response that could be responsible for the higher degree of protective immunity . Our results indicate that this potent adjuvant formulation should be further evaluated for use in clinical trials of recombinant malarial vaccine candidates.

Biochim Biophys Acta, 2004 Feb 2, 1644(1), 47 - 59
Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7; Fujimoto Y et al.; Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species . In mammalian cells, there exist proteins specifically localize in lipid droplets . However, the protein profile in the droplet remains yet to be clarified . In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques . Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction . The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11) . Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins . When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction . In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP . These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.

FEBS Lett, 2004 Jan 16, 557(1-3), 185 - 92
Impas 1 possesses endoproteolytic activity against multipass membrane protein substrate cleaving the presenilin 1 holoprotein; Moliaka YK et al.; Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the gamma-secretase complex regulating cleavage of type I proteins . Multiple mutations in PS1 are a major cause of familial early-onset Alzheimer's disease (AD) . We and others recently identified PS-related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)) . The functions of these proteins are yet to be determined . We found that intramembrane protease-associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins . Mutations in evolutionary invariant sites in hIMP1 or specific gamma-secretase inhibitors abolish the hIMP1-mediated endoproteolysis of PS1 . In contrast, neither AD-like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage . The data suggest that IMP1 is a bi-aspartic polytopic protease capable of cleaving transmembrane precursor proteins . These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis.

EMBO J, 2004 Jan 28, 23(2), 333 - 42 Epub 2004 Jan 22.
Nhp6 facilitates Aft1 binding and Ssn6 recruitment, both essential for FRE2 transcriptional activation; Fragiadakis GS et al.; We found Nhp6a/b yeast HMG-box chromatin-associated architectural factors and Ssn6 (Cyc8) corepressor to be crucial transcriptional coactivators of FRE2 gene . FRE2 encoding a plasma membrane ferric reductase is induced by the iron-responsive, DNA-binding, transcriptional activator Aft1 . We have shown that Nhp6 interacts directly with the Aft1 N-half, including the DNA-binding region, to facilitate Aft1 binding at FRE2 UAS . Ssn6 also interacts directly with the Aft1 N-half and is recruited on FRE2 promoter only in the presence of both Aft1 and Nhp6 . This Nhp6/Ssn6 role in Aft1-mediated transcription is FRE2 promoter context specific, and both regulators are required for activation-dependent chromatin remodeling . Our results provide the first in vivo biochemical evidence for nonsequence-specific HMG-box protein-facilitated recruitment of a yeast gene-specific transactivator to its DNA target site and for Nhp6-mediated Ssn6 promoter recruitment . Ssn6 has an explicitly coactivating role on FRE2 promoter only upon induction . Therefore, transcriptional activation in response to iron availability involves multiple protein interactions between the Aft1 iron-responsive DNA-binding factor and global regulators such as Nhp6 and Ssn6.

J Biomol NMR, 2004 Jan, 28(1), 59 - 67
New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes; Peterson RD et al.; NMR spectra of large RNAs are difficult to assign because of extensive spectral overlap and unfavorable relaxation properties . Here we present a new approach to facilitate assignment of RNA spectra using a suite of four 2D-filtered/edited NOESY experiments in combination with base-type-specific isotopically labeled RNA . The filtering method was developed for use in 3D filtered NOESY experiments (Zwahlen et al., 1997), but the 2D versions are both more sensitive and easier to interpret for larger RNAs than their 3D counterparts . These experiments are also useful for identifying intermolecular NOEs in RNA-protein complexes . Applications to NOE assignment of larger RNAs and an RNA-protein complex are presented.

Nucleic Acids Res, 2004 Jan 22, 32(2), 447 - 55 Print 2004.
Statistical resynchronization and Bayesian detection of periodically expressed genes; Lu X et al.; We propose a periodic-normal mixture (PNM) model to fit transcription profiles of periodically expressed (PE) genes in cell cycle microarray experiments . The model leads to a principled statistical estimation procedure that produces more accurate estimates of the mean cell cycle length and the gene expression periodicity than existing heuristic approaches . A central component of the proposed procedure is the resynchronization of the observed transcription profile of each PE gene according to the PNM with estimated periodicity parameters . By using a two-component mixture-Beta model to approximate the PNM fitting residuals, we employ an empirical Bayes method to detect PE genes . We estimate that about one-third of the genes in the genome of Saccharomyces cerevisiae are likely to be transcribed periodically, and identify 822 genes whose posterior probabilities of being PE are greater than 0.95 . Among these 822 genes, 540 are also in the list of 800 genes detected by Spellman . Gene ontology annotation analysis shows that many of the 822 genes were involved in important cell cycle-related processes, functions and components . When matching the 822 resynchronized expression profiles of three independent experiments, little phase shifts were observed, indicating that the three synchronization methods might have brought cells to the same phase at the time of release.

Curr Biol, 2004 Jan 20, 14(2), R70 - 2
Cell division: AAAtacking the mitotic spindle; Cheeseman IM et al.; Cell division requires the assembly of a microtubule-based spindle which captures and segregates sister chromatids . But how is this spindle broken down once chromosome segregation is complete? New evidence implicates a highly conserved AAA-ATPase in spindle disassembly at the end of mitosis.

Curr Biol, 2004 Jan 20, 14(2), 125 - 30
Condensin regulates rDNA silencing by modulating nucleolar Sir2p; Machin F et al.; The tandem array of ribosomal DNA (rDNA) in Saccharomyces cerevisiae is subjected to transcriptional silencing of RNA polymerase II-transcribed genes . This form of silencing depends on SIR2 and has been tightly linked to the suppression of rDNA recombination and the control of cellular lifespan . Paradoxically, rDNA silencing takes place in the context of an extremely high rate of RNA polymerase I transcription . Because rDNA silencing requires different factors than HMR and telomere silencing, the chromatin structure and the mechanisms of silencing must be fundamentally different . Here we show that yeast condensin organizes the specialized topology of rDNA chromatin . We then demonstrate that this function is necessary for maintaining the correct balance of telomeric and nucleolar Sir2p . Condensin mutants relocalize telomeric Sir2p to rDNA and show histone hyperacetylation at telomeres . Our data reveal the implication of yeast condensin in the arrangement of rDNA repeats into a heterochromatic-like structure that is important for the correct delineation of silencing domains in the nucleus.

Nature, 2004 Jan 22, 427(6972), 370 - 4
The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes; De Nadal E et al.; Regulation of gene expression by mitogen-activated protein kinases (MAPKs) is essential for proper cell adaptation to extracellular stimuli . Exposure of yeast cells to high osmolarity results in rapid activation of the MAPK Hog1, which coordinates the transcriptional programme required for cell survival on osmostress . The mechanisms by which Hog1 and MAPKs in general regulate gene expression are not completely understood, although Hog1 can modify some transcription factors . Here we propose that Hog1 induces gene expression by a mechanism that involves recruiting a specific histone deacetylase complex to the promoters of genes regulated by osmostress . Cells lacking the Rpd3-Sin3 histone deacetylase complex are sensitive to high osmolarity and show compromised expression of osmostress genes . Hog1 interacts physically with Rpd3 in vivo and in vitro and, on stress, targets the deacetylase to specific osmostress-responsive genes . Binding of the Rpd3-Sin3 complex to specific promoters leads to histone deacetylation, entry of RNA polymerase II and induction of gene expression . Together, our data indicate that targeting of the Rpd3 histone deacetylase to osmoresponsive promoters by the MAPK Hog1 is required to induce gene expression on stress.

Nucleic Acids Res, 2004 Jan 21, 32(2), 415 - 21 Print 2004.
The major 5' determinant in stop codon read-through involves two adjacent adenines; Tork S et al.; The aim of this approach was to identify the major determinants, located at the 5' end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae . We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene . Variations at these positions modulated translational read-through efficiency approximately 16-fold . The major effect was imposed by the two nucleotides immediately upstream of the stop codon . We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site . We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5' determinant of read-through efficiency.

Biol Reprod, 2004 May, 70(5), 1527 - 33 Epub 2004 Jan 21.
Estrogenic activities of nitrophenols in diesel exhaust particles; Furuta C et al.; We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators . Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by using recombinant yeast screens, myometrial contractility assays, and in vivo uterotrophic assays . Recombinant yeast screen assays showed that both PNMC and PNMPP possess estrogenic activity . Furthermore, ovariectomized 25-day-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 days showed significant increases in uterine weight among those receiving 100 mg/kg PNMC and 0.1 and 1.0 mg/kg PNMPP . To clarify further the estrogenic activity of PNMC and PNMPP, rat uterine horns were monitored in organ bath chambers for myometrial contractility in response to oxytocin (OT) . Significant differences occurred in the initial and maximum contractilities to OT at 0.25 and 25 mIU/ml in uterine horns obtained from animals treated with 100 mg/kg PNMC and in the maximum contractilities to OT at 0.025, 0.25, and 25 mIU/ml in those from rats treated with 0.1 mg/kg PNMPP . These results clearly demonstrated that PNMC and PNMPP in DEP have estrogenic activity both in vitro and in vivo and might therefore be considered as endocrine-disrupting chemicals.

Proc Natl Acad Sci U S A, 2004 Feb 3, 101(5), 1200 - 5 Epub 2004 Jan 20.
A genomewide oscillation in transcription gates DNA replication and cell cycle; Klevecz RR et al.; Microarray analysis from a yeast continuous synchrony culture system shows a genomewide oscillation in transcription . Maximums in transcript levels occur at three nearly equally spaced intervals in this approximately 40-min cycle of respiration and reduction . Two temporal clusters (4,679 of 5,329) are maximally expressed during the reductive phase of the cycle, whereas a third cluster (650) is maximally expressed during the respiratory phase . Transcription is organized functionally into redox-state superclusters with genes known to be important in respiration or reduction being synthesized in opposite phases of the cycle . The transcriptional cycle gates synchronous bursts in DNA replication in a constant fraction of the population at 40-min intervals . Restriction of DNA synthesis to the reductive phase of the cycle may be an evolutionarily important mechanism for reducing oxidative damage to DNA during replication.

J Biol Chem, 2004 Apr 2, 279(14), 13616 - 23 Epub 2004 Jan 20.
Systematic mutagenesis of the leucine-rich repeat (LRR) domain of CCR4 reveals specific sites for binding to CAF1 and a separate critical role for the LRR in CCR4 deadenylase activity; Clark LB et al.; CCR4, a poly(A) deadenylase of the exonuclease III family, is a component of the multiprotein CCR4-NOT complex of Saccharomyces cerevisiae that is involved in mRNA degradation . CCR4, unlike all other exonuclease III family members, contains a leucine-rich repeat (LRR) motif through which it makes contact to CAF1 and other factors . The LRR residues important in contacting CAF1 were identified by constructing 29 CCR4 mutations encompassing a majority (47 of 81) of residues interstitial to the conserved structural residues . Two-hybrid and immunoprecipitation data revealed that physical contact between CAF1 and the LRR is blocked by mutation of just two alpha-helix/beta-helix strand loop residues linking the first and second repeats . In contrast, CAF16, a potential ligand of CCR4, was abrogated in its binding to the LRR by mutations in the N terminus of the second beta-strand . The LRR domain was also found to contact the deadenylase domain of CCR4, and deletion of the LRR region completely inhibited CCR4 enzymatic activity . Mutations throughout the beta-sheet surface of the LRR, including those that did not specifically interfere with contacts to CAF1 or CAF16, significantly reduced CCR4 deadenylase activity . These results indicate that the CCR4-LRR, in addition to binding to CAF1, plays an essential role in the CCR4 deadenylation of mRNA.

J Cell Biol, 2004 Jan 19, 164(2), 219 - 31
Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2; Hofken T et al.; The guanine nucleotide exchange factor Cdc24, the GTPase Cdc42, and the Cdc42 effectors Cla4 and Ste20, two p21-activated kinases, form a signal transduction cascade that promotes mitotic exit in yeast . We performed a genetic screen to identify components of this pathway . Two related bud cortex-associated Cdc42 effectors, Gic1 and Gic2, were obtained as factors that promoted mitotic exit independently of Ste20 . The mitotic exit function of Gic1 was dependent on its activation by Cdc42 and on the release of Gic1 from the bud cortex . Gic proteins became essential for mitotic exit when activation of the mitotic exit network through Cdc5 polo kinase and the bud cortex protein Lte1 was impaired . The mitotic exit defect of cdc5-10 Deltalte1 Deltagic1 Deltagic2 cells was rescued by inactivation of the inhibiting Bfa1-Bub2 GTPase-activating protein . Moreover, Gic1 bound directly to Bub2 and prevented binding of the GTPase Tem1 to Bub2 . We propose that in anaphase the Cdc42-regulated Gic proteins trigger mitotic exit by interfering with Bfa1-Bub2 GTPase-activating protein function.

J Cell Biol, 2004 Jan 19, 164(2), 207 - 18
Regulation of polarized growth initiation and termination cycles by the polarisome and Cdc42 regulators; Bidlingmaier S et al.; The dynamic regulation of polarized cell growth allows cells to form structures of defined size and shape . We have studied the regulation of polarized growth using mating yeast as a model . Haploid yeast cells treated with high concentration of pheromone form successive mating projections that initiate and terminate growth with regular periodicity . The mechanisms that control the frequency of growth initiation and termination under these conditions are not well understood . We found that the polarisome components Spa2, Pea2, and Bni1 and the Cdc42 regulators Cdc24 and Bem3 control the timing and frequency of projection formation . Loss of polarisome components and mutation of Cdc24 decrease the frequency of projection formation, while loss of Bem3 increases the frequency of projection formation . We found that polarisome components and the cell fusion proteins Fus1 and Fus2 are important for the termination of projection growth . Our results define the first molecular regulators that control the timing of growth initiation and termination during eukaryotic cell differentiation.

FEMS Yeast Res, 2004 Jan, 4(4-5), 459 - 65
ATG23, a novel gene required for maturation of proaminopeptidase I, but not for autophagy; Meiling-Wesse K et al.; In rich media proaminopeptidase I is targeted to the vacuole via the Cvt pathway and during starvation via autophagy . We here identify Atg23 (Ylr431c), a protein of so far unknown function, as a novel component essential for proaminopeptidase I maturation under non-starvation conditions . Maturation of proaminopeptidase I takes place in starved atg23Delta cells . Selective vacuolar targeting of the autophagosomal marker GFP-Aut7 and the accumulation of autophagic bodies during starvation in the presence of phenylmethylsulfonyl fluoride suggest that autophagy occurs in atg23Delta cells but at a reduced rate . In atg23Delta cells mature vacuolar carboxypeptidase Y is present and accumulation of quinacrine suggests no significant defect in vacuolar acidification . Furthermore, growth of atg23Delta cells on nitrocellulose detects no significant secretion of carboxypeptidase Y.

Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 586 - 96
Sorting pathway and molecular targeting signals for the Arabidopsis peroxin 3; Hunt JE et al.; Peroxin 3 (Pex3p) has been identified and characterized as a peroxisomal membrane protein in yeasts and mammals . We identified two putative homologs in Arabidopsis (AtPex3p, forms 1 and 2), both with an identical cluster of positively charged amino acid residues (RKHRRK) immediately preceding one of the two predicted transmembrane domains (TMD1) . In transiently transformed Arabidopsis and tobacco BY-2 suspension-cultured cells, epitope-tagged AtPex3p (form 2) sorted post-translationally from the cytosol directly to peroxisomes, the first sorting pathway described for any peroxin in plants . TMD1 and RKHRRK were necessary for targeting form 2 to peroxisomes and sufficient for directing chloramphenicol acetyltransferase to peroxisomes in both cell types . The N and C termini of AtPex3p (form 2) extend into the peroxisomal matrix, different from mammal and yeast Pex3 proteins . Thus, two authentic peroxisomal membrane-bound Pex3p homologs possessing a membrane peroxisomal targeting signal, the first one defined for a plant peroxin and for any Pex3p homolog, exist in plant cells.

Neurosci Lett, 2004 Jan 30, 355(3), 217 - 20
Expression of the mammalian homologue of vacuolar protein sorting 16 (Vps16p) in the mouse and rat brain; Kim BY et al.; Synaptic vesicle fusion events are essential for synaptic transmission . Membrane docking and fusion events are highly regulated processes requiring the participation of a large number of soluble N-ethylmaleimide-sensitive factors (SNAREs) and SNARE-interacting proteins . We report the neuronal expression of mammalian homologue of vacuolar protein sorting 16 (mVps16p) which exhibits a high homology to the yeast Vps16p, a component of Class C Vps . Western blot and immunohistochemical analyzes revealed that mVps16p is highly expressed in the various brain areas and developmental stages tested . The immunoreactivities of mVps16p colocalized with microtubule associated protein 2, but not glial fibrillary acidic protein . In the primary culture of cortical neurons, mVps16p immunoreactivities were observed in the cell body and the neuronal processes, and highly enriched in the axonal outgrowths.

Fungal Genet Biol, 2004 Feb, 41(2), 148 - 56
A putative high affinity hexose transporter, hxtA, of Aspergillus nidulans is induced in vegetative hyphae upon starvation and in ascogenous hyphae during cleistothecium formation; Wei H et al.; Fungi employ different carbohydrate uptake systems to adapt to certain environmental conditions and to different carbon source concentrations . The hydrolysis of polymeric carbohydrates and the subsequent uptake of monomeric forms may also play a role in development . Aspergillus nidulans accumulates cell wall components during vegetative growth and degrades them during sexual development . We have identified the hxtA (high affinity hexose transporter) gene in a differential library, which was enriched for sexual-specific genes . The hxtA gene is disrupted by 6 introns and predicted to encode a 531 amino acid protein with high similarity to major facilitator superfamily members including the high affinity hexose transporter Gtt1 from Trichoderma harzianum . A . nidulans HxtA contains the 12 predicted transmembrane domains characteristic for this family . Deletion of hxtA did not impair growth of A . nidulans on a variety of carbon sources nor did it inhibit sexual development suggesting redundant sugar uptake systems . We found at least 17 putative hexose transporters in the genome of A . nidulans . Despite the high similarity of HxtA to fungal high affinity glucose transporters, the hxtA gene did not restore growth on glucose of a Saccharomyces cerevisiae mutant, in which all hexose transporters were deleted . Northern blot analysis revealed that the A . nidulans hxtA gene was repressed under high glucose conditions and expressed in vegetative hyphae upon carbon starvation and during sexual development . We found hxtA(p)::sgfp expression in developing cleistothecia specifically in ascogenous hyphae and propose that HxtA is a high affinity glucose transporter involved in sugar metabolism during sexual development.

Trends Cell Biol, 1995 Jul, 5(7), 278 - 82
Nuclear migration advances in fungi; Morris NR et al.; Nuclear migration encompasses three areas: separation of daughter nuclei during mitosis, congress of parental nuclei before they fuse during fertilization, and positioning of nuclei in interphase cells . This review deals primarily with interphase nuclear migration, which is crucial for events as disparate as vertebrate embryonic development and growth of fungal mycelia . Mutants of Aspergillus nidulans, Neurospora crassa and Saccharomyces cerevisiae have been particularly informative, and a detailed molecular analysis of this process is now well under way.

Trends Cell Biol, 1995 Aug, 5(8), 322 - 7
Ste5: a meeting place for MAP kinases and their associates; Elion EA; Growth and differentiation of the budding yeast Saccharomyces cerevisiae is regulated by six functionally distinct but structurally similar MAP kinase cascades . Three of the protein kinases in the cascade that regulates G1-phase arrest and mating have recently been shown to form a multikinase complex with a LIM-domain-containing protein called Ste5 . These studies implicate Ste5 as a tethering protein that physically links protein kinases operating sequentially in a cascade . The significance of this complex for the regulation and specificity of signal transduction is explored in this review.

Trends Cell Biol, 1994 May, 4(5), 160 - 6
The MCM2-3-5 proteins: are they replication licensing factors?
Tye BK.
DNA replication occurs only once in each normal mitotic cell cycle . To explain this strict control, a 'licensing factor' was proposed to enter the nucleus periodically as the nuclear envelope disintegrates and reassembles at the end of mitosis . Inactivation of licensing factor immediately following initiation of DNA synthesis would prevent reinitiation until after the next mitosis . The MCM2-3-5 proteins of Saccharomyces cerevisiae may be yeast's equivalent of licensing factor: they are present in the nucleus only between M and S phase, bind to chromatin and are important for the initiation of DNA replication.

Mol Cell, 2004 Jan 16, 13(1), 67 - 76
Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing; Ahn SH et al.; The largest subunit of RNA polymerase II contains a unique C-terminal domain important for coupling of transcription and mRNA processing . This domain consists of a repeated heptameric sequence (YSPTSPS) phosphorylated at serines 2 and 5 . Serine 5 is phosphorylated during initiation and recruits capping enzyme . Serine 2 is phosphorylated during elongation by the Ctk1 kinase, a protein similar to mammalian Cdk9/P-TEFb . Chromatin immunoprecipitation was used to map positions of transcription elongation and mRNA processing factors in strains lacking Ctk1 . Ctk1 is not required for association of elongation factors with transcribing polymerase . However, in ctk1Delta strains, the recruitment of polyadenylation factors to 3' regions of genes is disrupted and changes in 3' ends are seen . Therefore, Serine 2 phosphorylation by Ctk1 recruits factors for cotranscriptional 3' end processing in vivo.

Mol Cell, 2004 Jan 16, 13(1), 7 - 18
Indecent exposure: when telomeres become uncapped; Ferreira MG et al.; The protective "cap" that assembles at chromosome ends recruits and controls an intricate network of biochemical activities, each one critical for telomere structure and the maintenance of genomic stability . Recent studies have uncovered the components of telomere caps and have started to define the pathways that lead from telomere dysfunction to chromosomal catastrophe.

Biochemistry, 2004 Jan 27, 43(3), 718 - 27
Monomeric and dimeric bZIP transcription factor GCN4 bind at the same rate to their target DNA site; Cranz S et al.; Basic leucine zipper (bZIP) transcription factors are dimeric proteins that recognize dyadic and mostly palindromic DNA sites . Dimerization of bZIP transcription factor GCN4 is linked to the folding of its C-terminal leucine zipper domain . However, monomeric GCN4, lacking a folded leucine zipper, also recognizes the DNA site with dimerization taking place on the DNA . Here we report the kinetics of DNA recognition by unfolded monomeric and folded dimeric derivatives of GCN4 using a 19 bp double-stranded DNA containing a palindromic CRE site . The rate of DNA binding of both monomeric and dimeric GCN4 has a bimolecular rate constant of 3-5 x 10(8) M(-1) s(-1), which is near the diffusion limit . Because the rate of dimerization of GCN4 is slower (1.7 x 10(7) M(-1) s(-1)) than the rate of DNA association, the formation of the dimeric GCN4-DNA complex through consecutive binding of two monomers (monomer pathway) is faster when starting from free monomers . Thus, the results presented here support facilitated and rapid target recognition by the monomeric transcription factor . However, DNA binding of preformed folded dimeric GCN4 is as rapid as complex formation through the monomer pathway . Therefore, the monomer and dimer pathways are kinetically equivalent if monomeric and dimeric GCN4 are at equilibrium . Hence, the dimer pathway may also have a role under in vivo conditions . The lower affinity of GCN4 in which two DNA contacting residues have been mutated is due exclusively to the faster dissociation of the mutant protein-DNA complex and not to slower complex formation.

Nature, 2004 Feb 5, 427(6974), 510 - 7 Epub 2004 Jan 18.
The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis; Kitajima TS et al.; Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells . To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres . The factors that protect centromeric cohesion during meiosis I have remained elusive . Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast . We also identify a homologue of Sgo1 in budding yeast . We provide evidence that shugoshin is widely conserved among eukaryotes . Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation . Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1 . These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.

Nat Genet, 2004 Feb, 36(2), 197 - 204 Epub 2004 Jan 18.
Comparing genomic expression patterns across species identifies shared transcriptional profile in aging; McCarroll SA et al.; We developed a method for systematically comparing gene expression patterns across organisms using genome-wide comparative analysis of DNA microarray experiments . We identified analogous gene expression programs comprising shared patterns of regulation across orthologous genes . Biological features of these patterns could be identified as highly conserved subpatterns that correspond to Gene Ontology categories . Here, we demonstrate these methods by analyzing a specific biological process, aging, and show that similar analysis can be applied to a range of biological processes . We found that two highly diverged animals, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, implement a shared adult-onset expression program of genes involved in mitochondrial metabolism, DNA repair, catabolism, peptidolysis and cellular transport . Most of these changes were implemented early in adulthood . Using this approach to search databases of gene expression data, we found conserved transcriptional signatures in larval development, embryogenesis, gametogenesis and mRNA degradation.

Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2591 - 7
SS33410, an inhibitor of V-ATPase, blocks intracellular protein transport of the VSV-G protein in the Golgi compartment; Seog DH; An earlier report suggested that SS33410, structurally related to folimycin and bafilomycin A(1), blocked secretion of the glycoprotein (G protein) of vesicular stomatitis virus (VSV) into the medium and, instead, G protein was accumulated intracellulary . To identify the inhibition site of SS33410 in intracellular protein transport, I have analyzed the oligosaccharide chain structure of the intracellularly accumulated G protein . In SS33410-treated VSV-infected cells, G protein oligosaccharide was suggested to have a composition of GlcNAc-Man(5)-GlcNAc(2) as analyzed by Bio-Gel P-4 column chromatography following digestion with alpha-mannosidase, beta-N-acetylhexosaminidase, and then with alpha-mannosidase . SS33410 specifically inhibited vacuolar-type ATPase (V-ATPase) . These studies thus suggest that SS33410 blocks the intracellular protein transport before the step of trimming by mannosidase II, which is confined to the medial Golgi compartment.

Plant Physiol, 2004 Feb, 134(2), 726 - 34 Epub 2004 Jan 15.
Localization of an ascorbate-reducible cytochrome b561 in the plant tonoplast; Griesen D et al.; As a free radical scavenger, and cofactor, ascorbate (ASC) is a key player in the regulation of cellular redox processes . It is involved in responses to biotic and abiotic stresses and in the control of enzyme activities and metabolic reactions . Cytochromes (Cyts) b561 catalyze ASC-driven trans-membrane electron transport and contribute to ASC-mediated redox reactions in subcellular compartments . Putative Cyts b561 have been identified in Arabidopsis (ecotype Columbia) on the basis of sequence similarity to their mammalian counterparts . However, little is known about the function or subcellular localization of this unique class of membrane proteins . We have expressed one of the putative Arabidopsis Cyt b561 genes (CYBASC1) in yeast and we demonstrate that this protein encodes an ASC-reducible b-type Cyt with absorbance characteristics similar to that of other members of this family . Several lines of independent evidence demonstrate that CYBASC1 is localized at the plant tonoplast (TO) . Isoform-specific antibodies against CYBASC1 indicate that this protein cosediments with the TO marker on sucrose gradients . Moreover, CYBASC1 is strongly enriched in TO-enriched membrane fractions, and TO fractions contain an ASC-reducible b-type Cyt with alpha-band absorbance maximum near 561 nm . The TO ASC-reducible Cyt has a high specific activity, suggesting that it is a major constituent of this membrane . These results provide evidence for the presence of trans-membrane redox components in this membrane type, and they suggest the coupling of cytoplasmic and vacuolar metabolic reactions through ASC-mediated redox activity.

Plant Physiol, 2004 Jan, 134(1), 482 - 91
A novel family of transporters mediating the transport of glutathione derivatives in plants; Zhang MY et al.; Uptake and compartmentation of reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione conjugates are important for many functions including sulfur transport, resistance against biotic and abiotic stresses, and developmental processes . Complementation of a yeast (Saccharomyces cerevisiae) mutant (hgt1) deficient in glutathione transport was used to characterize a glutathione transporter cDNA (OsGT1) from rice (Oryza sativa) . The 2.58-kb full-length cDNA (AF393848, gi 27497095), which was obtained by screening of a cDNA library and 5'-rapid amplification of cDNA ends-polymerase chain reaction, contains an open reading frame encoding a 766-amino acid protein . Complementation of the hgt1 yeast mutant strain with the OsGT1 cDNA restored growth on a medium containing GSH as the sole sulfur source . The strain expressing OsGT1 mediated {3H}GSH uptake, and this uptake was significantly competed not only by unlabeled GSSG and GS conjugates but also by some amino acids and peptides, suggesting a wide substrate specificity . OsGT1 may be involved in the retrieval of GSSG, GS conjugates, and nitrogen-containing peptides from the cell wall.

Plant Physiol, 2004 Jan, 134(1), 204 - 14
AmSUT1, a sucrose transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis; Knop C et al.; A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae . This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C . Knop, O . Voitsekhovskaja, G . Lohaus {2001} Planta 213: 80-91) . These are typical properties of symplastic phloem loaders . For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae) . Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders . A polyclonal antiserum against the 17 N-terminal amino acids of the A . meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein . Using fluorescence labeling on sections from A . meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells . Further histological characterization identified these cells as companion cells and sieve elements . p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased . The data presented indicate that phloem loading of Suc and retrieval of Suc in A . meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.

RNA, 2004 Feb, 10(2), 308 - 20
Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA; Dobbyn HC et al.; Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins . One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing . As such, it is the first splicing factor to be identified in two functionally distinct particles . 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA . Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro . Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae . Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion . These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing . In addition, these mutants also display rRNA processing defects that are variable in nature . Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs . The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.

J Cell Sci, 2004 Feb 1, 117(Pt 4), 521 - 6
Model organisms lead the way to protein palmitoyltransferases; Linder ME et al.; The acylation of proteins with palmitate and related fatty acids has been known for over 30 years, but the molecular machinery that carries out palmitoylation has only recently emerged from studies in the model organisms Saccharomyces cerevisiae and Drosophila . Two classes of protein acyltransferases (PATs) have been proposed . In yeast, members of a family of integral membrane proteins harboring a cysteine-rich domain (CRD) containing a conserved DHHC (Asp-His-His-Cys) motif are PATs for cytoplasmic signaling molecules . The DHHC-CRD protein Erf2p, together with an associated subunit Erf4p, palmitoylates yeast Ras proteins, and Akr1p catalyzes the palmitoylation of the yeast casein kinase Yck2p . The existence of a second class of PATs that modify secreted signaling proteins has been suggested from work in Drosophila . Rasp is required in vivo for the production of functional Hedgehog and shares sequence identity with membrane-bound O-acyltransferases, which suggests that it catalyzes the palmitoylation of Hedgehog . With the identification of PATs in model genetic organisms, the field is now poised to uncover their mammalian counterparts and to understand the enzymology of protein palmitoylation.

Mol Cell Biol, 2004 Feb, 24(3), 1301 - 12
Nicotinamide clearance by Pnc1 directly regulates Sir2-mediated silencing and longevity; Gallo CM et al.; The Saccharomyces cerevisiae Sir2 protein is an NAD(+)-dependent histone deacetylase (HDAC) that functions in transcriptional silencing and longevity . The NAD(+) salvage pathway protein, Npt1, regulates Sir2-mediated processes by maintaining a sufficiently high intracellular NAD(+) concentration . However, another NAD(+) salvage pathway component, Pnc1, modulates silencing independently of the NAD(+) concentration . Nicotinamide (NAM) is a by-product of the Sir2 deacetylase reaction and is a natural Sir2 inhibitor . Pnc1 is a nicotinamidase that converts NAM to nicotinic acid . Here we show that recombinant Pnc1 stimulates Sir2 HDAC activity in vitro by preventing the accumulation of NAM produced by Sir2 . In vivo, telomeric, rDNA, and HM silencing are differentially sensitive to inhibition by NAM . Furthermore, PNC1 overexpression suppresses the inhibitory effect of exogenously added NAM on silencing, life span, and Hst1-mediated transcriptional repression . Finally, we show that stress suppresses the inhibitory effect of NAM through the induction of PNC1 expression . Pnc1, therefore, positively regulates Sir2-mediated silencing and longevity by preventing the accumulation of intracellular NAM during times of stress.

Mol Cell Biol, 2004 Feb, 24(3), 1188 - 99
BRG1 controls the activity of the retinoblastoma protein via regulation of p21CIP1/WAF1/SDI; Kang H et al.; The ubiquitous mammalian chromatin-remodeling SWI/SNF-like BAF complexes play critical roles in tumorigenesis . It was suggested that the direct interaction of BRG1 with the retinoblastoma protein pRB is required for regulation of cell cycle progression by pRB . We present evidence that the BRG1-containing complexes regulate the expression of the cdk inhibitor p21(CIP1/WAF1/SDI) . Furthermore, we show that the physical interaction between BRG1 and pRB is not required for induction of cell growth arrest and transcriptional repression of E2F target genes by pRB . Instead, BRG1 activates pRB by inducing its hypophosphorylation through up-regulation of the cdk inhibitor p21 . The hypophosphorylation of pRB is reinforced by down-regulation of critical components, including cdk2, cyclin E, and cyclin D, in the pRB regulatory network . We demonstrate that up-regulation of p21 by BRG1 is necessary to induce formation of flat cells, growth arrest, and finally, cell senescence . Our results suggest that the BRG1-containing complexes control cellular proliferation and senescence by modulating the pRB pathway via multiple mechanisms.

Mol Cell Biol, 2004 Feb, 24(3), 945 - 53
RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation; Gill T et al.; RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay . Here we describe the molecular defect behind this delay . CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants . This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability . We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay . In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products . Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome . Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease . Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery . In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.

Plant Cell, 2004 Feb, 16(2), 379 - 93 Epub 2004 Jan 16.
MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana; Tatematsu K et al.; We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation . Yet other aspects of growth and development of msg2 plants are almost normal . These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000) . Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain II of an Auxin/Indole-3-Acetic Acid protein, IAA19 . Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7 . Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) two-hybrid assay and to the whole latter protein in vitro by pull-down assay . These results suggest that MSG2/IAA19 and NPH4/ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.

J Biol Chem, 2004 Apr 9, 279(15), 14686 - 93 Epub 2004 Jan 16.
Domain organization of phytochelatin synthase: functional properties of truncated enzyme species identified by limited proteolysis; Ruotolo R et al.; Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms . No structural information on this enzyme is as yet available . It is generally believed, however, that the active site region is located in the more conserved N-terminal portion of PCS, whereas various, as yet unidentified (but supposedly less critical) roles have been proposed for the C-terminal region . To gain insight into the structural/functional organization of PCS, we have conducted a limited proteolysis analysis of the enzyme from Arabidopsis (AtPCS1), followed by functional characterization of the resulting polypeptide fragments . Two N-terminal fragments ending at positions 372 (PCS_Nt1) and 283 (PCS_Nt2) were produced sequentially upon V8 protease digestion, without any detectable accumulation of the corresponding C-terminal fragments . As revealed by the results of in vivo and in vitro functional assays, the core PCS_Nt2 fragment is biosynthetically active in the presence of cadmium ions and supports phytochelatin formation at a rate that is only approximately 5-fold lower than that of full-length AtPCS1 . The loss of the C-terminal region, however, substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals (e.g . mercury and zinc, but not cadmium or copper) . The latter phenotype was shared by PCS_Nt2 and by its precursor fragment PCS_Nt1, which, on the other hand, was almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme . AtPCS1 thus appears to be composed of a protease-resistant (and hence presumably highly structured) N-terminal domain, flanked by an intrinsically unstable C-terminal region . The most upstream part of such a region (positions 284-372) is important for enzyme stabilization, whereas its most terminal part (positions 373-485) appears to be required to determine enzyme responsiveness to a broader range of heavy metals.

Cancer Res, 2004 Jan 1, 64(1), 102 - 7
Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene; Fenton SL et al.; Epigenetic inactivation of the candidate tumor suppressor gene RASSF1A is a frequent and critical event in the pathogenesis of many human cancers . The RASSF1A protein contains a Ras association domain, suggesting a role in Ras-like signaling pathways, and has also been implicated in cell cycle progression . However, the preliminary data suggests that the RASSF1A gene product is likely to have multiple functions . To identify novel RASSF1A functions, we have sought to identify interacting proteins by yeast two-hybrid analysis in a human brain cDNA library . We identified the E1A-regulated transcription factor p120(E4F) as a RASSF1A interacting partner in yeast and mammalian cells, and demonstrated that RASSF1A protein and p120(E4F) form a complex in vivo . The interaction between RASSF1A and p120(E4F) was confirmed by both in vitro and in vivo pull downs and coimmunoprecipitation assays . In addition, specific inactivation of RASSF1A by short interfering RNA disrupts binding of RASSF1A to p120(E4F) in coimmunoprecipitation assays . In addition, we demonstrated enhanced G(1) cell cycle arrest and S phase inhibition by propidium iodide staining of p120(E4F) in the presence of RASSF1A . As p120(E4F) has been reported previously to interact with p14ARF, retinoblastoma, and p53, these findings provide an important link between the function of RASSF1A and other major human tumor suppressor genes.

Genes Dev, 2004 Jan 15, 18(2), 196 - 209 Epub 2004 Jan 16.
A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits; Oeffinger M et al.; Rrp12p (Ypl012w) is unusual among characterized ribosome synthesis factors in being associated with late precursors to both the 40S and 60S subunits . Rrp12p is predominantly nuclear with nucleolar enrichment at steady state, but shuttled between the nucleus and cytoplasm in a heterokaryon assay . Strains depleted of Rrp12p are impaired in the nuclear export of both ribosomal subunits . Sequence analysis combined with fold recognition and modeling showed that Rrp12p is a member of a family of pre-ribosome-associated HEAT-repeat proteins . Like other HEAT-repeat transport factors, Rrp12p binds in vitro to nucleoporin FG-repeats of both the GLFG and FXFG families and to the GTPase Gsp1p (yeast RAN) . Rrp12p also showed robust in vitro binding to a pre-rRNA transcript, in addition to poly(A) and poly(U) . We propose that Rrp12p binds to the RNA components of the pre-ribosomes and promotes export of both subunits via its interactions with the nucleoporins and Gsp1p.

J Inorg Biochem, 2004 Feb, 98(2), 189 - 98
Strong protective action of Copper(II) N-substituted sulfonamide complexes against reactive oxygen species; Gonzalez-Alvarez M et al.; Copper(II) complexes of N-benzothiazolsulfonamides, {Cu(N-2-(5,6-dimethylbenzothiazole)toluenesulfonamidate)(2)(dmso)(2)} (1), {Cu(N-2-(6-chlorobenzothiazole)benzenesulfonamidate)(2)(dmso)(2)} (2) and {Cu(N-2-(6-chlorobenzothiazole)toluenesulfonamidate)(2)(dmso)(2)} (3) with interesting protective properties against superoxide radicals have been prepared . The compounds have been characterized by X-ray diffraction and their chemical properties have been studied by spectroscopic methods . The crystal structure of 1 shows that the copper(II) is surrounded by two benzothiazole N atoms from the sulfonamide ligands and two O atoms from the dimethylsulfoxide molecules in a square planar arrangement . The coordination polyhedron around copper(II) in 2 and 3 is distorted square pyramidal being the metal ion linked to benzothiazole N and sulfonamidate O atoms of the ligand and to two dimethylsulfoxide O atoms . The three complexes have a strong protective action over Delta sod1 mutant of Saccharomyces cerevisiae against reactive oxygen radicals derived from respiration and against those generated by hydrogen peroxide and menadione.

Gene, 2004 Feb 4, 326, 107 - 16
Characterization of the Drosophila Rae1 protein as a G1 phase regulator of the cell cycle; Sitterlin D; Messengers RNA (mRNA) are thought to be export from the nucleus as ribonucleoprotein complexes (mRNP), whose exact protein composition remains not completely determined . The Gle2/RAE1 protein, a highly conserved member of the WD40 repeat protein family, was first shown to be involved in mRNA export . More recently, a role in the cell cycle was also suggested . To get new insights into the functions of the metazoan protein, the Drosophila melanogaster rae1 (dmrae1) cDNA was first cloned, then the corresponding protein characterized and its function investigated by RNA interference . This paper shows that dmRae1 mainly localises to the nuclear membrane like its homologue in Saccharomyces cerevisiae . However, unlike its homologue and despite this particular sub-cellular distribution, its depletion does not impair the export of polyA+ RNAs . Interestingly, the presence of dmRae1 is important for normal proliferation and, more importantly, for the progression through the G1 phase of the cell cycle . Given that dmRae1 is closely related to the human form, results suggest that the human homologue, hsRAE1, may also play a similar role during the cell cycle.

Trends Plant Sci, 2004 Jan, 9(1), 7 - 9
Vacuolar compartmentalization: a second-generation approach to engineering plants for phytoremediation; Tong YP et al.; Engineering plants with greater metal tolerance and accumulation properties is the key to developing phytoremediators . A recent study by Won-Yong Song et al . has shown that overexpressing the yeast vacuolar transporter YCF1 increases Pb and Cd tolerance and consequently increases the accumulation of these metals in shoots of transgenic Arabidopsis plants even though expression levels of YCF1 were relatively low . This technology can be used to engineer advanced phytoremediators, increasing their ability to pump heavy metals into a safe compartment while requiring only a small amount of transporters rather than a large amount of chelating peptide material.

Cell Res, 2003 Dec, 13(6), 499 - 502
Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog; Pan GJ et al.; Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells . However, the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown . Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family . The rest of nanog contains no apparent homology to any known proteins characterized so far . It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes . To test this hypothesis, we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs . Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities . Despite the fact that it contains no apparent transactivation motifs, the C-terminal domain is about 7 times as active as the N-terminal one . This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.

AMIA Annu Symp Proc . 2003;:958.
Social network analysis of gene expression data; Ohn JH et al.; To investigate the structure of genomic interaction network, social affiliation network analysis was performed for the yeast gene expression compendium dataset of hundreds of systematic perturbations . Network density and centrality indices of genes and groups of genes revealed the core-peripheral and the significant intermediary players that may be critical for the control of the biological system.

AMIA Annu Symp Proc . 2003;:733-7.
A computer-based microarray experiment design-system for gene-regulation pathway discovery; Yoo C et al.; This paper reports the methods and evaluation of a computer-based system that recommends microarray experimental design for biologists - causal discovery in Gene Expression data using Expected Value of Experimentation (GEEVE) . The GEEVE system uses causal Bayesian networks and generates a decision tree for recommendations . To evaluate the GEEVE system, we first built an expression simulation model based on a gene regulation model assessed by an expert biologist . Using the simulation model, we conducted a controlled study that involved 10 biologists, some of whom used GEEVE and some of whom did not . The results show that biologists who used GEEVE reached correct causal assessments about gene regulation more often than did those biologists who did not use GEEVE.

EMBO J, 2004 Jan 28, 23(2), 429 - 38 Epub 2004 Jan 15.
Physical and functional interactions between nucleotide excision repair and DNA damage checkpoint; Giannattasio M et al.; The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins . Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated cdu1-1, which is allelic to RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER) . Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co-immunoprecipitation . We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA . Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes . Conversely, elimination of transcription-coupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients.

EMBO J, 2004 Jan 28, 23(2), 343 - 53 Epub 2004 Jan 15.
Sin mutations alter inherent nucleosome mobility; Flaus A et al.; Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones . Here we characterise the biochemical properties of nucleosomes bearing these mutations . We find that sin mutant nucleosomes have a high inherent thermal mobility . As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function . The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation . We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimer-tetramer interface influence nucleosome mobility . Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility . Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility.

Cell Cycle, 2004 Mar, 3(3), 276 - 9 Epub 2004 Mar 01.
Cdc20 in S-phase: the Banquo at replication's banquet; Andrews CA et al.; The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase that covalently attaches ubiquitins onto proteins to target them for proteolysis by the 26S proteasome . During mitosis, the APC/C is instrumental in allowing the cell to enter and exit from mitosis . The APC/C accomplishes this by using different specificity factors to recognize, interact with, and ubiquitylate key proteins that block cell cycle progression . The specificity factors, Cdc20p and Cdh1p, are not always associated with the APC/C and indeed they have the ability to interact with substrates in isolation . The molecular events that take place in order for Cdc20p and Cdh1p to couple substrates and APC/C are currently being resolved . Meanwhile, evidence has emerged suggesting that at least one of the specificity factors, Cdc20p, might be capable of functioning independently of the APC/C.

Cell Cycle, 2004 Mar, 3(3), 273 - 5 Epub 2004 Mar 01.
Genome organization in three dimensions: thinking outside the line; Haeusler RA et al.; Gene organization on nuclear chromosomes is usually depicted as a linear array, but at least some regions of the genome are localized to specific subnuclear positions in interphase nuclei . Studies in yeast have found that centromeres and telomeres are found around the nuclear periphery, and that tRNA genes are gathered at the nucleolus, along with the ribosomal RNA gene cluster . These 325 loci alone impose significant constraints on the three dimensional organization of chromosomes in the nucleus, and there is mounting experimental evidence that transcription by RNA polymerase II is strongly affected by proximity to these regions . Given these observations, one consideration in understanding nuclear gene regulation might be the degree to which spatial positioning affects at least a subset of gene families.

J Biol Chem, 2004 Apr 9, 279(15), 15339 - 47 Epub 2004 Jan 15.
An extensive requirement for transcription factor IID-specific TAF-1 in Caenorhabditis elegans embryonic transcription; Walker AK et al.; The general transcription factor TFIID sets the mRNA start site and consists of TATA-binding protein and associated factors (TAF(II)s), some of which are also present in SPT-ADA-GCN5 (SAGA)-related complexes . In yeast, results of multiple studies indicate that TFIID-specific TAF(II)s are not required for the transcription of most genes, implying that intact TFIID may have a surprisingly specialized role in transcription . Relatively little is known about how TAF(II)s contribute to metazoan transcription in vivo, especially at developmental and tissue-specific genes . Previously, we investigated functions of four shared TFIID/SAGA TAF(II)s in Caenorhabditis elegans . Whereas TAF-4 was required for essentially all embryonic transcription, TAF-5, TAF-9, and TAF-10 were dispensable at multiple developmental and other metazoan-specific promoters . Here we show evidence that in C . elegans embryos transcription of most genes requires TFIID-specific TAF-1 . TAF-1 is not as universally required as TAF-4, but it is essential for a greater proportion of transcription than TAF-5, -9, or -10 and is important for transcription of many developmental and other metazoan-specific genes . TAF-2, which binds core promoters with TAF-1, appears to be required for a similarly substantial proportion of transcription . C . elegans TAF-1 overlaps functionally with the coactivator p300/CBP (CBP-1), and at some genes it is required along with the TBP-like protein TLF(TRF2) . We conclude that during C . elegans embryogenesis TAF-1 and TFIID have broad roles in transcription and development and that TFIID and TLF may act together at certain promoters . Our findings imply that in metazoans TFIID may be of widespread importance for transcription and for expression of tissue-specific genes.

Arch Biochem Biophys, 2004 Feb 1, 422(1), 71 - 80
Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation; Gravot A et al.; Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant . The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae . Mechanism-based inactivation was investigated using various psoralen derivatives . Only psoralen and 8-methoxypsoralen were found to inactivate C4H . The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration . Inactivation stoichiometry was 0.9 (+/-0.2) for CYP73A1 and 1.1 (+/-0.2) for CYP73A32 . SDS-PAGE analysis demonstrated that {3H}psoralen was irreversibly bound to the C4H apoprotein . K(i) and k(inact) for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1 . Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum . This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation . Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included . Our results suggest a possible evolution of R . graveolens and P . crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.

Nature, 2004 Jan 15, 427(6971), 247 - 52
Functional genomic hypothesis generation and experimentation by a robot scientist; King RD et al.; The question of whether it is possible to automate the scientific process is of both great theoretical interest and increasing practical importance because, in many scientific areas, data are being generated much faster than they can be effectively analysed . We describe a physically implemented robotic system that applies techniques from artificial intelligence to carry out cycles of scientific experimentation . The system automatically originates hypotheses to explain observations, devises experiments to test these hypotheses, physically runs the experiments using a laboratory robot, interprets the results to falsify hypotheses inconsistent with the data, and then repeats the cycle . Here we apply the system to the determination of gene function using deletion mutants of yeast (Saccharomyces cerevisiae) and auxotrophic growth experiments . We built and tested a detailed logical model (involving genes, proteins and metabolites) of the aromatic amino acid synthesis pathway . In biological experiments that automatically reconstruct parts of this model, we show that an intelligent experiment selection strategy is competitive with human performance and significantly outperforms, with a cost decrease of 3-fold and 100-fold (respectively), both cheapest and random-experiment selection.

Nucleic Acids Res, 2004 Jan 14, 32(1), 328 - 37 Print 2004.
TopNet: a tool for comparing biological sub-networks, correlating protein properties with topological statistics; Yu H et al.; Biological networks are a topic of great current interest, particularly with the publication of a number of large genome-wide interaction datasets . They are globally characterized by a variety of graph-theoretic statistics, such as the degree distribution, clustering coefficient, characteristic path length and diameter . Moreover, real protein networks are quite complex and can often be divided into many sub-networks through systematic selection of different nodes and edges . For instance, proteins can be sub-divided by expression level, length, amino-acid composition, solubility, secondary structure and function . A challenging research question is to compare the topologies of sub- networks, looking for global differences associated with different types of proteins . TopNet is an automated web tool designed to address this question, calculating and comparing topological characteristics for different sub-networks derived from any given protein network . It provides reasonable solutions to the calculation of network statistics for sub-networks embedded within a larger network and gives simplified views of a sub-network of interest, allowing one to navigate through it . After constructing TopNet, we applied it to the interaction networks and protein classes currently available for yeast . We were able to find a number of potential biological correlations . In particular, we found that soluble proteins had more interactions than membrane proteins . Moreover, amongst soluble proteins, those that were highly expressed, had many polar amino acids, and had many alpha helices, tended to have the most interaction partners . Interestingly, TopNet also turned up some systematic biases in the current yeast interaction network: on average, proteins with a known functional classification had many more interaction partners than those without . This phenomenon may reflect the incompleteness of the experimentally determined yeast interaction network.

Genes Dev, 2004 Jan 1, 18(1), 88 - 98
Loss of the anaphase-promoting complex in quiescent cells causes unscheduled hepatocyte proliferation; Wirth KG et al.; The anaphase-promoting complex or cyclosome (APC/C) is an ubiquitin protein ligase that together with Cdc20 and Cdh1 targets mitotic proteins for degradation by the proteosome . APC-Cdc20 activity during mitosis triggers anaphase by destroying securin and cyclins . APC-Cdh1 promotes degradation of cyclins and other proteins during G(1) . We show that loss of APC/C during embryogenesis is early lethal before embryonic day E6.5 (E6.5) . To investigate the role of APC/C in quiescent cells, we conditionally inactivated the subunit Apc2 in mice . Deletion of Apc2 in quiescent hepatocytes caused re-entry into the cell cycle and arrest in metaphase, resulting in liver failure . Re-entry into the cell cycle either occurred without any proliferative stimulus or could be easily induced . We demonstrate that the APC has an additional function to prevent hepatocytes from unscheduled re-entry into the cell cycle.

Dev Cell, 2004 Jan, 6(1), 79 - 90
The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure; Reggiori F et al.; To survive extreme environmental conditions, and in response to certain developmental and pathological situations, eukaryotic organisms employ the catabolic process of autophagy . Structures targeted for destruction are enwrapped by double-membrane vesicles, then delivered into the interior of the lysosome/vacuole . Despite the identification of many specific components, the molecular mechanism that directs formation of the sequestering vesicles remains largely unknown . We analyzed the trafficking of Atg23 and the integral membrane protein Atg9 in the yeast Saccharomyces cerevisiae . These components localize both to the pre-autophagosomal structure (PAS) and other cytosolic punctate compartments . We show that Atg9 and Atg23 cycle through the PAS in a process governed by the Atg1-Atg13 signaling complex . Atg1 kinase activity is essential only for retrograde transport of Atg23, while recycling of Atg9 requires additional factors including Atg18 and Atg2 . We postulate that Atg9 employs a recycling system mechanistically similar to that used at yeast early and late endosomes.

Nucleic Acids Res . 2004 Jan 13;32(1):e12.
Biological detection of low radiation doses by combining results of two microarray analysis methods; Mercier G et al.; The accurate determination of the biological effects of low doses of pollutants is a major public health challenge . DNA microarrays are a powerful tool for investigating small intracellular changes . However, the inherent low reliability of this technique, the small number of replicates and the lack of suitable statistical methods for the analysis of such a large number of attributes (genes) impair accurate data interpretation . To overcome this problem, we combined results of two independent analysis methods (ANOVA and RELIEF) . We applied this analysis protocol to compare gene expression patterns in Saccharomyces cerevisiae growing in the absence and continuous presence of varying low doses of radiation . Global distribution analysis highlights the importance of mitochondrial membrane functions in the response . We demonstrate that microarrays detect cellular changes induced by irradiation at doses that are 1000-fold lower than the minimal dose associated with mutagenic effects.

J Biol Chem, 2004 Apr 2, 279(14), 14323 - 30 Epub 2004 Jan 13.
Role of C-terminal extensions of subunits beta2 and beta7 in assembly and activity of eukaryotic proteasomes; Ramos PC et al.; A close inspection of the crystal structure of the yeast 20 S proteasome revealed that a prominent connection between the two beta-rings is mediated by the subunit beta7/Pre4 . Its C-terminal extension intercalates between the beta1/Pre3 and beta2/Pup1 subunits on the opposite ring . We show that the interactions promoted by the beta7/Pre4 tail are important to facilitate the formation of 20 S particles from two half-proteasome precursor complexes and/or to stabilize mature 20 S proteasomes . The deletion of 19 residues from the beta7/Pre4 C terminus leads to an accumulation of half-proteasome precursor complexes containing the maturation factor Ump1 . The C-terminal extension of beta7/Pre4, which forms several hydrogen bonds with beta1/Pre3, is in addition required for the post-acidic activity mediated by the latter subunit . Deletion of the C-terminal tail of beta7/Pre4 results in an inhibition of beta1/Pre3 propeptide processing and abrogation of post-acidic activity . Our data obtained with yeast strains that expressed the mature form of Pre3 lacking its propeptide suggest that interactions between the Pre4 C terminus and Pre3 stabilize a conformation of its active site, which is essential for post-acidic activity . Deletion of the C-terminal extension of beta2/Pup1, which wraps around beta3/Pup3 within the same beta-ring, is lethal, indicating that this extension serves an essential function in proteasome assembly or stability.

J Biol Chem, 2004 Mar 19, 279(12), 11222 - 8 Epub 2004 Jan 13.
Analysis of the regulation of the molecular chaperone Hsp26 by temperature-induced dissociation: the N-terminal domail is important for oligomer assembly and the binding of unfolding proteins; Stromer T et al.; Small heat shock proteins (sHsps) are molecular chaperones that efficiently bind non-native proteins . All members of this family investigated so far are oligomeric complexes . For Hsp26, an sHsp from the cytosol of Saccharomyces cerevisiae, it has been shown that at elevated temperatures the 24-subunit complex dissociates into dimers . This dissociation seems to be required for the efficient interaction with unfolding proteins that results in the formation of large, regular complexes comprising Hsp26 and the non-native proteins . To gain insight into the molecular mechanism of this chaperone, we analyzed the dynamics and stability of the two oligomeric forms of Hsp 26 (i.e . the 24-mer and the dimer) in comparison to a construct lacking the N-terminal domain (Hsp26DeltaN) . Furthermore, we determined the stabilities of complexes between Hsp26 and non-native proteins . We show that the temperature-induced dissociation of Hsp26 into dimers is a completely reversible process that involves only a small change in energy . The unfolding of the dissociated Hsp26 dimer or Hsp26DeltaN, which is a dimer, requires a much higher energy . Because Hsp26DeltaN was inactive as a chaperone, these results imply that the N-terminal domain is of critical importance for both the association of Hsp26 with non-native proteins and the formation of large oligomeric complexes . Interestingly, complexes of Hsp26 with non-native proteins are significantly stabilized against dissociation compared with Hsp26 complexes . Taken together, our findings suggest that the quaternary structure of Hsp26 is determined by two elements, (i) weak, regulatory interactions required to form the shell of 24 subunits and (ii) a strong and stable dimerization of the C-terminal domain.

J Biol Chem, 2004 Mar 26, 279(13), 12396 - 405 Epub 2004 Jan 13.
The Tim8-Tim13 complex of Neurospora crassa functions in the assembly of proteins into both mitochondrial membranes; Hoppins SC et al.; The Tim8 and Tim13 proteins in yeast are known to exist in the mitochondrial intermembrane space and to form a hetero-oligomeric complex involved in the import of the mitochondrial inner membrane protein Tim23, the central component of the TIM23 translocase . Here, we have isolated tim8 and tim13 mutants in Neurospora crassa and have shown that mitochondria lacking the Tim8-Tim13 complex were deficient in the import of the outer membrane beta-barrel proteins Tom40 and porin . Cross-linking studies showed that the Tom40 precursor contacts the Tim8-Tim13 complex . The complex is involved at an early point in the Tom40 assembly pathway because cross-links can only be detected during the initial stages of Tom40 import . In mitochondria lacking the Tim8-Tim13 complex, the Tom40 precursor appears in a previously characterized early intermediate of Tom40 assembly more slowly than in wild type mitochondria . Thus, our data suggest a model in which one of the first steps in Tom40 assembly may be interaction with the Tim8-Tim13 complex . As in yeast, the N . crassa Tim23 precursor was imported inefficiently into mitochondria lacking the Tim8-Tim13 complex when the membrane potential was reduced . Tim23 import intermediates could also be cross-linked to the complex, suggesting a dual role for the Tim8-Tim13 intermembrane space complex in the import of proteins found in both the outer and inner mitochondrial membranes.

Mol Biol Cell, 2004 Mar, 15(3), 1479 - 86 Epub 2004 Jan 12.
Adenylyl cyclase G is activated by an intramolecular osmosensor; Saran S et al.; Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor . The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation . To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGDeltacat) and was driven by a UV-inducible promoter . After UV induction of ACGDeltacat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced . Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGDeltacat . However, high osmolality did not alter the dimer/monomer ratio . This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality . To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant . In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium . This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.

J Biol Chem, 2004 Mar 26, 279(13), 12495 - 502 Epub 2004 Jan 12.
Dimorphecolic acid is synthesized by the coordinate activities of two divergent Delta12-oleic acid desaturases; Cahoon EB et al.; Dimorphecolic acid (9-OH-18:2Delta(10)(trans)(,12)(trans)) is the major fatty acid of seeds of Dimorphotheca species . This fatty acid contains structural features that are not typically found in plant fatty acids, including a C-9 hydroxyl group, Delta(10),Delta(12)-conjugated double bonds, and trans-Delta(12) unsaturation . Expressed sequence tag analysis was conducted to determine the biosynthetic origin of dimorphecolic acid . cDNAs for two divergent forms of Delta(12)-oleic acid desaturase, designated DsFAD2-1 and Ds-FAD2-2, were identified among expressed sequence tags generated from developing Dimorphotheca sinuata seeds . Expression of DsFAD2-1 in Saccharomyces cerevisiae and soybean somatic embryos resulted in the accumulation of the trans-Delta(12) isomer of linoleic acid (18: 2Delta(9)(cis)(,12)(trans)) rather than the more typical cis-Delta(12) isomer . When co-expressed with DsFAD2-1 in soybean embryos or yeast, DsFAD2-2 converted 18:2Delta(9)(cis)(,12)(trans) into dimorphecolic acid . When DsFAD2-2 was expressed alone in soybean embryos or together with a typical cis-Delta(12)-oleic acid desaturase in yeast, trace amounts of the cis-Delta(12) isomer of dimorphecolic acid (9-OH-18:2Delta(10)(trans,)(12)(cis)) were formed from DsFAD2-2 activity with cis-Delta(12)-linoleic acid {corrected} . These results indicate that DsFAD2-2 catalyzes the conversion of the Delta(9) double bond of linoleic acid into a C-9 hydroxyl group and Delta(10)(trans) double bond and displays a substrate preference for the trans-Delta(12), rather than the cis-Delta(12), isomer of linoleic acid . Overall these data are consistent with a biosynthetic pathway of dimorphecolic acid involving the concerted activities of DsFAD2-1 and DsFAD2-2 . The evolution of two divergent Delta(12)-oleic acid desaturases for the biosynthesis of an unusual fatty acid is unprecedented in plants.

J Cell Biol, 2004 Jan 19, 164(2), 291 - 300 Epub 2004 Jan 12.
GADD34-PP1c recruited by Smad7 dephosphorylates TGFbeta type I receptor; Shi W et al.; The cascade of phosphorylation is a pivotal event in transforming growth factor beta (TGFbeta) signaling . Reversible phosphorylation regulates fundamental aspects of cell activity . TGFbeta-induced Smad7 binds to type I receptor (TGFbeta type I receptor; TbetaRI) functioning as a receptor kinase antagonist . We found Smad7 interacts with growth arrest and DNA damage protein, GADD34, a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme, which subsequently recruits catalytic subunit of PP1 (PP1c) to dephosphorylate TbetaRI . Blocking Smad7 expression by RNA interference inhibits association of GADD34-PP1c complex with TbetaRI, indicating Smad7 acts as an adaptor protein in the formation of the PP1 holoenzyme that targets TbetaRI for dephosphorylation . SARA (Smad anchor for receptor activation) enhances the recruitment PP1c to the Smad7-GADD34 complex by controlling the specific subcellular localization of PP1c . Importantly, GADD34-PP1c recruited by Smad7 inhibits TGFbeta-induced cell cycle arrest and mediates TGFbeta resistance in responding to UV light irradiation . The dephosphorylation of TbetaRI mediated by Smad7 is an effective mechanism for governing negative feedback in TGFbeta signaling.

Cell, 2004 Jan 9, 116(1), 75 - 86
Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery; Rodriguez-Navarro S et al.; Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus followed by mRNA export to the cytoplasm for translation . Here we report the identification of a protein, Sus1, which functions in both transcription and mRNA export . Sus1 is a nuclear protein with a concentration at the nuclear pores . Biochemical analyses show that Sus1 interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, and with the Sac3-Thp1 complex, which functions in mRNA export with specific nuclear pore proteins at the nuclear basket . DNA macroarray analysis revealed that Sus1 is required for transcription regulation . Moreover, chromatin immunoprecipitation showed that Sus1 is associated with the promoter of a SAGA-dependent gene during transcription activation . Finally, mRNA export is impaired in sus1 mutants . These data provide an unexpected connection between the SAGA histone acetylase complex and the mRNA export machinery.

Cell, 2004 Jan 9, 116(1), 39 - 50
Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair; Chapados BR et al.; Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair . To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results . FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity . Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity . The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.

Cell, 2004 Jan 9, 116(1), 3 - 4
Initiation of DNA replication: a new hint from archaea; Liberi G et al.; In this issue, Robinson and coworkers provide new insights into the mechanisms of initiation of chromosome replication in Archea . This and other studies, focused on model organisms, will certainly help to understand how the replication process has evolved in Eukaryotes.

BMC Genomics . 2004 Jan 13;5(1):4.
Inter-species differences of co-expression of neighboring genes in eukaryotic genomes; Fukuoka Y et al.; BACKGROUND: There is increasing evidence that gene order within the eukaryotic genome is not random . In yeast and worm, adjacent or neighboring genes tend to be co-expressed . Clustering of co-expressed genes has been found in humans, worm and fruit flies . However, in mice and rats, an effect of chromosomal distance (CD) on co-expression has not been investigated yet . Also, no cross-species comparison has been made so far . We analyzed the effect of CD as well as normalized distance (ND) using expression data in six eukaryotic species: yeast, fruit fly, worm, rat, mouse and human . RESULTS: We analyzed 24 sets of expression data from the six species . Highly co-expressed pairs were sorted into bins of equal sized intervals of CD, and a co-expression rate (CoER) in each bin was calculated . In all datasets, a higher CoER was obtained in a short CD range than a long distance range . These results show that across all studied species, there was a consistent effect of CD on co-expression . However, the results using the ND show more diversity . Intra- and inter-species comparisons of CoER reveal that there are significant differences in the co-expression rates of neighboring genes among the species . A pair-wise BLAST analysis finds 8-30 % of the highly co-expressed pairs are duplicated genes . CONCLUSION: We confirmed that in the six eukaryotic species, there was a consistent tendency that neighboring genes are likely to be co-expressed . Results of pair-wised BLAST indicate a significant effect of non-duplicated pairs on co-expression . A comparison of CD and ND suggests the dominant effect of CD.

Biochem J, 2004 Apr 15, 379(Pt 2), 375 - 83
Aspergillus niger mstA encodes a high-affinity sugar/H+ symporter which is regulated in response to extracellular pH; Vankuyk PA et al.; A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger . To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake . Uptake experiments using 14C-labelled monosaccharides demonstrated that although able to transport D-fructose ( K(m), 4.5+/-1.0 mM), D-xylose ( K(m), 0.3+/-0.1 mM) and D-mannose ( K(m), 60+/-20 microM), MSTA has a preference for D-glucose (K(m), 25+/-10 microM) . pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport . Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for D-glucose, D-mannose and D-xylose . Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC . A . niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1-60 mM D-glucose, D-mannose, D-fructose or D-xylose . This indicates that A . niger possesses other transporters capable of compensating for the absence of MSTA.

J Clin Endocrinol Metab, 2004 Jan, 89(1), 49 - 60
Two prevalent CYP17 mutations and genotype-phenotype correlations in 24 Brazilian patients with 17-hydroxylase deficiency; Costa-Santos M et al.; We performed molecular genetic analysis of 24 subjects from 19 families with 17-hydroxylase deficiency in Brazil . Of 7 novel CYP17 mutations, 2 (W406R and R362C) account for 50% and 32% of the mutant alleles, respectively . Both mutations were completely inactive when studied in COS-7 cells and yeast microsomes; however, phenotypic features varied among subjects . Some 46,XY individuals with these genotypes had ambiguous genitalia, and other subjects had normal blood pressure and/or serum potassium . We found mutations W406R and R362C principally in families with Spanish and Portuguese ancestry, respectively, suggesting that two independent founder effects contribute to the increased prevalence of 17-hydroxylase deficiency in Brazil . Mutations Y329D and P428L retained a trace of activity, yet the two individuals with these mutations had severe hypertension and hypokalemia . The 46,XX female with mutation Y329D reached Tanner stage 5, whereas the 46,XY subject with mutation P428L remained sexually infantile . The severity of hypertension, hypokalemia, 17-deoxysteroid excess, and sex steroid deficiency varied, even among patients with completely inactive CYP17 protein(s) . Spontaneous sexual development occurred only in 46,XX females with partial deficiencies . We conclude that other factors, in addition to CYP17 genotype, contribute to the phenotype of individual patients with 17-hydroxylase deficiency.

J Biol Chem, 2004 Apr 2, 279(14), 13435 - 46 Epub 2004 Jan 07.
S-(2-chloroethyl)glutathione-generated p53 mutation spectra are influenced by differential repair rates more than sites of initial dna damage; Valadez JG et al.; Several steps occur between the reaction of a chemical with DNA and a mutation, and each may influence the resulting mutation spectrum, i.e . nucleotides at which the mutations occur . The half-mustard S-(2-bro-moethyl)glutathione is the reactive conjugate implicated in ethylene dibromide-induced mutagenesis attributed to the glutathione-dependent pathway . A human p53-driven Ade reporter system in yeast was used to study the factors involved in producing mutations . The synthetic analog S-(2-chloroethyl)glutathione was used to produce DNA damage; the damage to the p53 exons was analyzed using a new fluorescence-based modification of ligation-mediated polymerase chain reaction and an automated sequencer . The mutation spectrum was strongly dominated by the G to A transition mutations seen in other organisms with S-(2-chloroethyl)glutathione or ethylene dibromide . The mutation spectrum clearly differed from the spontaneous spectrum or that derived from N-ethyl,N-nitrosourea . Distinct differences were seen between patterns of modification of p53 DNA exposed to the mutagen in vitro versus in vivo . In the four p53 exons in which mutants were analyzed, the major sites of mutation matched the sites with long half-lives of repair much better than the sites of initial damage . However, not all slowly repaired sites yielded mutations in part because of the lack of effect of mutations on phenotype . We conclude that the rate of DNA repair at individual nucleotides is a major factor in influencing the mutation spectra in this system . The results are consistent with a role of N(7)-guanyl adducts in mutagenesis.

Cell Cycle, 2004 Feb, 3(2), 119 - 22
Remodelling the Rad9 checkpoint complex: preparing Rad53 for action; van den Bosch M et al.; DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity . The Rad9 protein functions in the DNA damage checkpoint pathway in Saccharomyces cerevisiae and is essential for the Mec1-dependent activation of the effector kinase Rad53 . We recently described the purification of two soluble distinct Rad9 complexes . The large 850 kDa complex consists of hypophosphorylated Rad9 and the chaperone proteins Ssa1/2 . This complex is found both in undamaged cells as well as in cells treated with DNA damaging agents . The smaller 560 kDa complex contains hyperphosphorylated Rad9, Ssa1/2 and, in addition, Rad53 . This complex forms only in cells with compromised DNA integrity . Once bound to the smaller complex, Rad53 can be activated by in trans autophosphorylation . Here, we propose a model in which the large Rad9 complex is remodelled after a genomic insult by chaperone activity to a smaller Rad53 activating complex.

Cell Cycle, 2004 Feb, 3(2), 100 - 3
A new kind of prion: a modified protein necessary for its own modification; Roberts BT et al.; We recently described an infectious protein (prion) unrelated to amyloid formation, that is an enzyme whose precursor can only be activated by the active form of the enzyme . All previously described infectious proteins are self-propagating amyloid forms of chromosomally encoded proteins . The infectious enzyme, vacuolar protease B (PrB), can activate its own precursor in an indefinitely self-propagating process . Transfer from cell to cell of cytoplasm containing active protease B transmits this non-chromosomal gene . The importance of this system is that many protein-modifying enzymes may act on themselves, and if conditions are right, may become prions as well.

Appl Environ Microbiol, 2004 Jan, 70(1), 459 - 67
Characterization of secretory genes ypt1/yptA and nsf1/nsfA from two filamentous fungi: induction of secretory pathway genes of Trichoderma reesei under secretion stress conditions; Saloheimo M et al.; Two genes involved in protein secretion, encoding the Rab protein YPT1/YPTA and the general fusion factor NSFI/NSFA, were characterized from two filamentous fungi, Trichoderma reesei and Aspergillus niger var . awamori . The isolated genes showed a high level of conservation with their Saccharomyces cerevisiae and mammalian counterparts, and T . reesei ypt1 was shown to complement yeast Ypt1p depletion . The transcriptional regulation of the T . reesei ypt1, nsf1, and sar1 genes, involved in protein trafficking, was studied with mycelia treated with the folding inhibitor dithiothreitol (DTT) and with brefeldin A, which inhibits membrane traffic between the endoplasmic reticulum and Golgi complex . The well-known inducer of the yeast and T . reesei unfolded protein response (UPR), DTT, induced the nsf1 gene and the protein disulfide isomerase gene, pdi1, in both of the experiments, and sar1 mRNA increased in only one experiment under strong UPR induction . The ypt1 mRNA did not show a clear increase during DTT treatment . Brefeldin A strongly induced pdi1 and all of the intracellular trafficking genes studied . These results suggest the possibility that the whole secretory pathway of T . reesei could be induced at the transcriptional level by stress responses caused by protein accumulation in the secretory pathway.

J Biotechnol, 2004 Jan 22, 107(2), 135 - 49
Expression, purification and characterization of recombinant sucrose synthase 1 from Solanum tuberosum L . for carbohydrate engineering; Romer U et al.; The gene sus1 from Solanum tuberosum L . encoding for sucrose synthase 1 was cloned into the plasmid pDR195 under the control of the PMA1 promotor . After transformation of Saccharomyces cerevisiae strain 22574d sus1 was constitutively expressed giving a specific activity of 0.3Umg(-1) protein in the crude extract . A one-step purification by Q-Sepharose resulted in an 14-fold purified enzyme preparation in 74% yield . SuSy1 was subsequently purified by immobilized metal ion affinity chromatography and characterized for its utilization in synthesizing different nucleotide sugars and sucrose analogues . The kinetic constants for the cleavage and synthesis reaction were determined: K(m) (UDP) 4microM; K(iS) (UDP) 0.11mM; K(m) (sucrose) 91.6mM; K(m) (UDP-Glc) 0.5mM; K(iS) (UDP-Glc) 2.3mM; K(m) (D-fructose) 2.1mM; K(iS) (D-fructose) 35.9mM . Different nucleoside diphosphates as well as different donor substrate were accepted as follows: UDP>dTDP>ADP>CDP>GDP in the cleavage reaction and UDP-Glc>dTDP-Glc>ADP-Glc>CDP-Glc in the synthesis reaction . SuSy1 shows also a broad acceptance of D- and L-ketoses and D- and L-aldoses . The acceptance of aldoses was deduced from the binding of the inhibitor 5-deoxy-D-fructose (K(i) 0.3mM), an analogue of the natural substrate D-fructopyranoside . The broad substrate spectrum renders SuSy1 from potato a versatile biocatalyst for carbohydrate engineering.

Curr Biol, 2004 Jan 6, 14(1), R27 - 9
Centrosomes: Sfi1p and centrin unravel a structural riddle; Salisbury JL; The discovery of Sfi1p as a novel binding partner for the Ca2+-binding protein centrin has provided new insight into the dynamic behavior of centrosomes . Sfi1 binds to multiple centrin molecules along a series of internal repeats, and the complex forms Ca2+-sensitive contractile fibers that function to reorient centrioles and alter centrosome structure.

Curr Biol, 2004 Jan 6, 14(1), R11 - 3
Cellular differentiation: the violin strikes up another tune; Cook MA et al.; A switch in cellular identity in budding yeast requires the ubiquitin-dependent elimination of pre-existing master regulators encoded by the MAT locus . Failure to disassemble the prior state not only impairs the cell type transition but imparts a hybrid cellular fate . This theme will undoubtedly arise in many developmental and disease contexts.

Biochem J, 2004 May 1, 379(Pt 3), 777 - 84
Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal; Morillas M et al.; CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of L-carnitine, facilitating the entry of fatty acids into mitochondria . We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase . The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis . Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues . Mutants D477A, D567A and E590D showed reduced L-CPT I activity . In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered K(m) and the catalytic efficiency for carnitine in mutant S685A . We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state . Several conserved lysines, i.e . Lys455, Lys505, Lys560 and Lys561, were also mutated . Only mutants K455A and K560A showed decreases in activity of 50% . The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies.

Acta Biol Hung, 2003, 54(3-4), 347 - 55
Quantitative evaluation of macrophage phagocytosing capacity by a fluorometric assay; Miliukiene V et al.; This paper reviews sensitive and simple quantitative evaluation of macrophage phagocytosing capacity by applying fluoresecin-labeled Sacharomyces cerevisiae cells . Yeast cells were conjugated with fluoresceinisothiocyanate (FITC) and used as fluorescent particles . A time course analysis within this method showed that phagocytosis of yeast cells was temperature dependent and that the number of that ones ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C . Free fluorescent cells can be effectively removed by aspiration from the well . Furthermore, yeast cells required preopsonization with serum to achieve optimal uptake of the cells . The uptake of nonopsonized yeast cells by macrophages was significantly lower than that of opsonized cells (P < 0.05) . We propose that about 50% of mouse macrophages can carry functionally active FcR responsible for phagocytosis.

Hum Mol Genet, 2004 Mar 1, 13(5), 535 - 42 Epub 2004 Jan 06.
Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik; Grubenmann CE et al.; Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG) . In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum . The profiling of {3H}mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders . However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects . To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation . By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient . The accumulation pattern suggested a deficiency of the ALG1 beta1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides . This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other . The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants . The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-Ik.

Genome Biol . 2003;5(1):R6 . Epub 2003 Dec 15.
Functional classification of proteins for the prediction of cellular function from a protein-protein interaction network; Brun C et al.; We here describe PRODISTIN, a new computational method allowing the functional clustering of proteins on the basis of protein-protein interaction data . This method, assessed biologically and statistically, enabled us to classify 11% of the Saccharomyces cerevisiae proteome into several groups, the majority of which contained proteins involved in the same biological process(es), and to predict a cellular function for many otherwise uncharacterized proteins.

Nat Rev Mol Cell Biol, 2004 Jan, 5(1), 23 - 32
The GGA proteins: adaptors on the move; Bonifacino JS; The GGA proteins are a family of ubiquitously expressed, Arf-dependent clathrin adaptors that mediate the sorting of mannose-6-phosphate receptors between the trans-Golgi network and endosomes . Recent studies have elucidated the biochemical and structural bases for the interaction of the GGA proteins with many binding partners, and have shed light on the molecular and cellular mechanisms by which the GGA proteins participate in protein sorting.

Med Sci Sports Exerc, 2004 Jan, 36(1), 28 - 34
AMP-activated protein kinase: a key system mediating metabolic responses to exercise; Hardie DG; The finding that AMP-activated protein kinase (AMPK) was activated by exercise in skeletal muscle, reported by Winder and Hardie in 1996, provided the first hint that this signaling pathway might represent the elusive and long-sought system that was responsible for the metabolic changes associated with exercise . It triggered an increasing volume of research that has now largely vindicated this hypothesis although, in the usual manner of these things, it is not the whole story . As discussed in this article, it is becoming clear that the AMPK system is partly, but not entirely, responsible for the acute metabolic responses of muscle to acute exercise . It is particularly involved in the switch from the anaerobic metabolism of glycogen to oxidative metabolism of blood glucose and fatty acids . It also appears to be responsible for most, if not all, of the long-term metabolic adaptations to aerobic exercise (i.e., to endurance training), particularly the up-regulation of mitochondrial content and oxidative metabolism . Interestingly, this role is a reflection of the evolutionary origins of the kinase, because the homolog of AMPK in a single-celled eukaryote, the brewer's yeast Saccharomyces cerevisiae, is also involved in the switch from anaerobic to aerobic metabolism.

J Neurocytol, 2003 Feb, 32(2), 161 - 83
Expression of regeneration-related molecules in injured and regenerating striatal and nigral neurons; Chaisuksunt V et al.; Peripheral nerve grafts in the neostriatum promote axonal regeneration from restricted classes of CNS neuron, principally cells in the substantia nigra pars compacta (SNpc) and striatal cholinergic interneurons . We have examined the molecular responses of CNS neurons induced to regenerate axons by tibial nerve grafting to the neostriatum of adult rats . Brain sections were probed for mRNAs for the transcription factor c-jun, and the cell recognition molecule CHL1, or immunoreacted for TrkA or p75, 1 day to 29 weeks after grafting (dpo; wpo) . In unoperated rats, scattered neurons throughout the neostriatum showed weak signals for CHL1 mRNA and slightly stronger signals for c-jun mRNA . Cells of similar appearance strongly expressed TrkA but possessed little p75 . By 1 dpo, many neostriatal neurons of various sizes and GFAP + glial cells near the host/graft interface had upregulated CHL1 mRNA, c-jun mRNA and p75 . Most of the larger (20-25 microm diameter) CHL1 mRNA+ cells were also TrkA+, indicating that they were NGF-sensitive cholinergic interneurons . From two weeks postgrafting, high levels of CHL1 and c-jun mRNAs and p75 in the neostriatum were confined to a few presumptive cholinergic interneurons; p75+ cells were also TrkA+ and were larger than TrkA+ neurons on the contralateral side . Retrograde labelling showed that most p75+ and some TrkA+ neurons regenerated axons through the graft . Neurons in the SNpc showed a moderate to strong signal for CHL1 mRNA, weaker signal for c-jun mRNA, and no p75 or TrkA . Some SNpc cells upregulated c-jun mRNA after graft implantation, although they did not upregulate CHL1 mRNA, p75 or TrkA . Since neostriatal neurons which regenerate axons into grafts express receptors for NGF, and grafts mimic the effects of NGF treatment on these cells, sensitivity to graft-derived NGF may be a determinant of their high regenerative capacity . The finding that c-jun and CHL1 are consistently expressed by CNS neurons induced to regenerate their axons strongly supports the idea that these molecules are directly involved in axonal regeneration.

Genome Res, 2004 Jan, 14(1), 29 - 36
Coelomata and not Ecdysozoa: evidence from genome-wide phylogenetic analysis; Wolf YI et al.; Relative positions of nematodes, arthropods, and chordates in animal phylogeny remain uncertain . The traditional tree topology joins arthropods with chordates in a coelomate clade, whereas nematodes, which lack a coelome, occupy a basal position . However, the current leading hypothesis, based on phylogenetic trees for 18S ribosomal RNA and several proteins, joins nematodes with arthropods in a clade of molting animals, Ecdysozoa . We performed a phylogenetic analysis of over 500 sets of orthologous proteins, which are represented in plants, animals, and fungi, using maximum likelihood, maximum parsimony, and distance methods . Additionally, to increase the statistical power of topology tests, the same methods were applied to concatenated alignments of subunits of eight conserved macromolecular complexes . The majority of the methods, when applied to most of the orthologous clusters, both concatenated and individual, grouped the fly with humans to the exclusion of the nematode, in support of the coelomate phylogeny . Trees were also constructed using information on insertions and deletions in orthologous proteins, combinations of domains in multidomain proteins, and presence-absence of species in clusters of orthologs . All of these approaches supported the coelomate clade and showed concordance between evolution of protein sequences and higher-level evolutionary events, such as domain fusion or gene loss.

J Biol Chem, 2004 Mar 19, 279(12), 11129 - 36 Epub 2004 Jan 05.
Characterization of OSR1, a member of the mammalian Ste20p/germinal center kinase subfamily; Chen W et al.; In examining the protein kinase components of mitogen-activated protein (MAP) kinase (MAPK) cascades that regulate the c-Jun N-terminal kinase (JNK) in Drosophila S2 cells, we previously found that distinct upstream kinases were involved in responses to sorbitol and lipopolysaccharide . Here we have extended that analysis to the possible MAPK kinase kinase kinases (MAP4Ks) in the JNK pathway . Fray, a putative Drosophila MAP4K, provided a major contribution to JNK activation by sorbitol . To explore the possible link to JNK in mammalian cells, we isolated and characterized OSR1 (oxidative stress-responsive 1), one of two human Fray homologs . OSR1 is a 58-kDa protein of 527 amino acids that is widely expressed in mammalian tissues and cell lines . Of potential regulators surveyed, endogenous OSR1 is activated only by osmotic stresses, notably sorbitol and to a lesser extent NaCl . However, OSR1 did not increase the activity of coexpressed JNK, nor did it activate three other MAPKs, p38, ERK2, and ERK5 . A two-hybrid screen implicated another Ste20p family member, the p21-activated protein kinase PAK1, as an OSR1 target . OSR1 phosphorylated threonine 84 in the N-terminal regulatory domain of PAK1 . Replacement of threonine 84 with glutamate reduced the activation of PAK1 by an active form of the small G protein Cdc42, suggesting that phosphorylation by OSR1 modulates the G protein sensitivity of PAK isoforms.

FEBS Lett, 2004 Jan 2, 556(1-3), 81 - 5
Identification and characterization of a novel Delta6-fatty acid desaturase gene from Rhizopus arrhizus; Zhang Q et al.; A cDNA sequence putatively encoding a Delta(6)-fatty acid desaturase was isolated from Rhizopus arrhizus using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods . Sequence analysis indicated that this cDNA sequence had an open reading frame of 1377 bp encoding 458 amino acids of 52 kDa . The deduced amino acid sequence showed high similarity to those of fungal Delta(6)-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich motifs and hydropathy profile . A cytochrome b(5)-like domain was observed at the N-terminus . To elucidate the function of this novel putative desaturase, the coding sequence was expressed heterologously in Saccharomyces cerevisiae strain INVScl . The result demonstrated that the coding product of the sequence exhibited Delta(6)-fatty acid desaturase activity by the accumulation of gamma-linolenic acid.

FEBS Lett, 2004 Jan 2, 556(1-3), 1 - 6
Recent developments in the analysis of protein complexes; Dziembowski A et al.; The goal of this review is to analyse how recent technical developments contributed to the biochemical characterisation of protein complexes . Improvement of tags used for protein purification, including in our own laboratory, and the development of new strategies have allowed the use of generic procedures for the purification of a wide variety of protein complexes . Together with increased mass spectrometry sensitivity and automation, this made high throughput studies of protein complexes possible and allowed proteome-wide analyses of protein complexes . However, knowledge of protein complex composition, even at the cellular level, will not be sufficient to understand their function . We suggest that the next level of analysis in this area will be the definition of internal subunit arrangement in complexes as a first step toward more detailed structural analyses.

Biochemistry, 2004 Jan 13, 43(1), 46 - 54
Extension of the binding motif of the Sin3 interacting domain of the Mad family proteins; van Ingen H et al.; Sin3 forms the scaffold for a multiprotein corepressor complex that silences transcription via the action of histone deacetylases . Sin3 is recruited to the DNA by several DNA binding repressors, such as the helix-loop-helix proteins of the Mad family . Here, we elaborate on the Mad-Sin3 interaction based on a binding study, solution structure, and dynamics of the PAH2 domain of mSin3 in complex to an extended Sin3 interacting domain (SID) of 24 residues of Mad1 . We show that SID residues Met7 and Glu23, outside the previously defined minimal binding motif, mediate additional hydrophobic and electrostatic interactions with PAH2 . On the basis of these results we propose an extended consensus sequence describing the PAH2-SID interaction specifically for the Mad family, showing that residues outside the hydrophobic core of the SID interact with PAH2 and modulate binding affinity to appropriate levels.

Anal Biochem, 2003 Nov 1, 322(1), 116 - 23
A continuous assay of myristoyl-CoA:protein N-myristoyltransferase for proteomic analysis; Boisson B et al.; Protein N-myristoylation is an important lipid modification that affects the activity and membrane-binding properties of crucial proteins belonging to signal transduction cascades . The aim of this work was to develop a rapid and easy diagnostic method to check for (i) effective N-myristoylation of any given protein and (ii) easy proteome annotation . The N-myristoylation reaction was coupled to that of pyruvate dehydrogenase, and NADH was continuously detected spectrophotometrically . This method was optimized for and applied to full-length Saccharomyces cerevisiae and Arabidopsis thaliana N-myristoyltransferases and two A . thaliana enzyme derivatives . The data were validated by comparison with a previously described discontinuous assay, modification of the chemical nature of the substrates, and use of specific inhibitors . The kinetics of N-myristoylation were determined in vitro with various compounds including a full-length polypeptide substrate, a small G protein of the RAB family already known to be N-myristoylated in vivo . This automated assay can be used for proteomic studies to determine the N-myristoylation state of any protein.

Science, 2004 Jan 2, 303(5654), 98 - 101
Coordinated activation of Hsp70 chaperones; Steel GJ et al.; Hsp70s are a ubiquitous family of molecular chaperones involved in many cellular processes . Two Hsp70s, Lhs1p and Kar2p, are required for protein biogenesis in the yeast endoplasmic reticulum . Here, we found that Lhs1p and Kar2p specifically interacted to couple, and coordinately regulate, their respective activities . Lhs1p stimulated Kar2p by providing a specific nucleotide exchange activity, whereas Kar2p reciprocally activated the Lhs1p adenosine triphosphatase (ATPase) . The two ATPase activities are coupled, and their coordinated regulation is essential for normal function in vivo.

Nucleic Acids Res, 2004 Jan 02, 32(1), 179 - 88 Print 2004.
Computational inference of transcriptional regulatory networks from expression profiling and transcription factor binding site identification; Haverty PM et al.; We have developed a computational method for transcriptional regulatory network inference, CARRIE (Computational Ascertainment of Regu latory Relationships Inferred from Expression), which combines microarray and promoter sequence analysis . CARRIE uses sources of data to identify the transcription factors (TFs) that regulate gene expression changes in response to a stimulus and generates testable hypotheses about the regulatory network connecting these TFs to the genes they regulate . The promoter analysis component of CARRIE, ROVER (Relative OVER-abundance of cis-elements), is highly accurate at detecting the TFs that regulate the response to a stimulus . ROVER also predicts which genes are regulated by each of these TFs . CARRIE uses these transcriptional interactions to infer a regulatory network . To demonstrate our method, we applied CARRIE to six sets of publicly available DNA microarray experiments on Saccharomyces cerevisiae . The predicted networks were validated with comparisons to literature sources, experimental TF binding data, and gene ontology biological process information.

Genetics, 2003 Dec, 165(4), 1831 - 42
The homologous chromosome is an effective template for the repair of mitotic DNA double-strand breaks in Drosophila; Rong YS et al.; In recombinational DNA double-strand break repair a homologous template for gene conversion may be located at several different genomic positions: on the homologous chromosome in diploid organisms, on the sister chromatid after DNA replication, or at an ectopic position . The use of the homologous chromosome in mitotic gene conversion is thought to be limited in the yeast Saccharomyces cerevisiae and mammalian cells . In contrast, by studying the repair of double-strand breaks generated by the I-SceI rare-cutting endonuclease, we find that the homologous chromosome is frequently used in Drosophila melanogaster, which we suggest is attributable to somatic pairing of homologous chromosomes in mitotic cells of Drosophila . We also find that Drosophila mitotic cells of the germ line, like yeast, employ the homologous recombinational repair pathway more often than imperfect nonhomologous end joining.

Nat Genet, 2004 Jan, 36(1), 46 - 54 Epub 2003 Dec 21.
RPA regulates telomerase action by providing Est1p access to chromosome ends; Schramke V et al.; Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein involved in DNA replication, recombination and repair . We show here that RPA is present at the telomeres of the budding yeast Saccharomyces cerevisiae, with a maximal association in S phase . A truncation of the N-terminal region of Rfa2p (associated with the rfa2Delta40 mutated allele) results in severe telomere shortening caused by a defect in the in vivo regulation of telomerase activity . Cells carrying rfa2Delta40 show impaired binding of the protein Est1p, which is required for telomerase action . In addition, normal telomere length can be restored by expressing a Cdc13-Est1p hybrid protein . These findings indicate that RPA activates telomerase by loading Est1p onto telomeres during S phase . We propose a model of in vivo telomerase action that involves synergistic action of RPA and Cdc13p at the G-rich 3' overhang of telomeric DNA.

Genes Dev, 2004 Jan 1, 18(1), 35 - 47 Epub 2003 Dec 30.
GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs; Hilioti Z et al.; The conserved RCN family of proteins can bind and directly regulate calcineurin, a Ca(2+)-activated protein phosphatase involved in immunity, heart growth, muscle development, learning, and other processes . Whereas high levels of RCNs can inhibit calcineurin signaling in fungal and animal cells, RCNs can also stimulate calcineurin signaling when expressed at endogenous levels . Here we show that the stimulatory effect of yeast Rcn1 involves phosphorylation of a conserved serine residue by Mck1, a member of the GSK-3 family of protein kinases . Mutations at the GSK-3 consensus site of Rcn1 and human DSCR1/MCIP1 abolish the stimulatory effects on calcineurin signaling . RCNs may therefore oscillate between stimulatory and inhibitory forms in vivo in a manner similar to the Inhibitor-2 regulators of type 1 protein phosphatase . Computational modeling indicates a biphasic response of calcineurin to increasing RCN concentration such that protein phosphatase activity is stimulated by low concentrations of phospho-RCN and inhibited by high concentrations of phospho- or dephospho-RCN . This prediction was verified experimentally in yeast cells expressing Rcn1 or DSCR1/MCIP1 at different concentrations . Through the phosphorylation of RCNs, GSK-3 kinases can potentially contribute to a positive feedback loop involving calcineurin-dependent up-regulation of RCN expression . Such feedback may help explain the large induction of DSCR1/MCIP1 observed in brain of Down syndrome individuals.

Genes Dev, 2004 Jan 1, 18(1), 76 - 87 Epub 2003 Dec 30.
In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding; Lavoie BD et al.; Chromosome condensation plays an essential role in the maintenance of genetic integrity . Using genetic, cell biological, and biochemical approaches, we distinguish two cell-cycle-regulated pathways for chromosome condensation in budding yeast . From G(2) to metaphase, we show that the condensation of the approximately 1-Mb rDNA array is a multistep process, and describe condensin-dependent clustering, alignment, and resolution steps in chromosome folding . We functionally define a further postmetaphase chromosome assembly maturation step that is required for the maintenance of chromosome structural integrity during segregation . This late step in condensation requires the conserved mitotic kinase Ipl1/aurora in addition to condensin, but is independent of cohesin . Consistent with this, the late condensation pathway is initiated during the metaphase-to-anaphase transition, supports de novo condensation in cohesin mutants, and correlates with the Ipl1/aurora-dependent phosphorylation of condensin . These data provide insight into the molecular mechanisms of higher-order chromosome folding and suggest that two distinct condensation pathways, one involving cohesins and the other Ipl1/aurora, are required to modulate chromosome structure during mitosis.

J Biol Chem, 2004 Mar 5, 279(10), 9016 - 22 Epub 2003 Dec 29.
The molecular chaperone, Atp12p, from Homo sapiens . In vitro studies with purified wild type and mutant (E240K) proteins; Hinton A et al.; Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled alpha subunits of F(1) and in so doing prevents the alpha subunit from associating with itself in non-productive complexes during assembly of the F(1) moiety of the mitochondrial ATP synthase . We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells . The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate . Wild type HuAtp12p suppresses the aggregation of thermally inactivated citrate synthase . In contrast, the mutant protein HuAtp12p(E240K), which harbors a lysine at the position of the highly conserved Glu-240, fails to prevent citrate synthase aggregation at 43 degrees C . No significant differences were observed between the wild type and the mutant proteins as judged by sedimentation analysis, cysteine titration, tryptophan emission spectra, or limited proteolysis, which suggests that the E240K mutation alters the activity of HuAtp12p with minimal effects on the physical integrity of the protein . An additional important finding of this work is that the equilibrium chemical denaturation curve of HuAtp12p shows two components, the first of which is associated with protein aggregation . This result is consistent with a model for Atp12p structure in which there is a hydrophobic chaperone domain that is buried within the protein interior.

Mol Cell Biol, 2004 Jan, 24(2), 561 - 72
Analysis of a mutant histone H3 that perturbs the association of Swi/Snf with chromatin; Duina AA et al.; We have isolated new histone H3 mutants in Saccharomyces cerevisiae that confer phenotypes indicative of transcriptional defects . Here we describe the characterization of one such mutant, encoded by the hht2-11 allele, which contains the single amino acid change L61W in the globular domain of H3 . Whole-genome expression analyses show that the hht2-11 mutation confers pleiotropic transcriptional defects and that many of the genes it affects are normally controlled by the Swi/Snf chromatin remodeling complex . Furthermore, we show that Swi/Snf occupancy at two promoters, PHO84 and SER3, is reduced in hht2-11 mutants . Detailed studies of the PHO84 promoter suggest that the hht2-11 mutation impairs Swi/Snf association with chromatin in a direct fashion . Taken together, our results strongly suggest that the integrity of the globular domain of histone H3 is an important determinant in the ability of Swi/Snf to associate with chromatin.

J Biol Chem, 2004 Mar 12, 279(11), 10136 - 41 Epub 2003 Dec 29.
Coronavirus replication complex formation utilizes components of cellular autophagy; Prentice E et al.; The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell . However, the mechanism by which these DMVs form has not been determined . Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated . The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12 . MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition . MHV replication is impaired in autophagy knockout, APG5-/-, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5-/-cells . In MHV-infected APG5-/-cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen . The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication . Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes.

Mol Biol Cell, 2004 Mar, 15(3), 1101 - 11 Epub 2003 Dec 29.
In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker; Mizushima N et al.; Macroautophagy mediates the bulk degradation of cytoplasmic components . It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs . Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods . To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes . Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues . In some tissues, autophagy even occurs actively without starvation treatments . Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response . This transgenic mouse model is a useful tool to study mammalian autophagy.

Mol Biol Cell, 2004 Mar, 15(3), 1356 - 63 Epub 2003 Dec 29.
Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae; Pena-Diaz J et al.; 3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial . Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum . In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens . Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T . cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment . Furthermore, T . cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells . Thus, when the gene encoding T . cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms.

Mol Biol Cell, 2004 Mar, 15(3), 1011 - 23 Epub 2003 Dec 29.
The alpha- and beta'-COP WD40 domains mediate cargo-selective interactions with distinct di-lysine motifs; Eugster A et al.; Coatomer is required for the retrieval of proteins from an early Golgi compartment back to the endoplasmic reticulum . The WD40 domain of alpha-COP is required for the recruitment of KKTN-tagged proteins into coatomer-coated vesicles . However, lack of the domain has only minor effects on growth in yeast . Here, we show that the WD40 domain of beta'-COP is required for the recycling of the KTKLL-tagged Golgi protein Emp47p . The protein is degraded more rapidly in cells with a point mutation in the WD40 domain of beta'-COP (sec27-95) or in cells lacking the domain altogether, whereas a point mutation in the Clathrin Heavy Chain Repeat (sec27-1) does not affect the turnover of Emp47p . Lack of the WD40 domain of beta'-COP has only minor effects on growth of yeast cells; however, absence of both WD40 domains of alpha- and beta'-COP is lethal . Two hybrid studies together with our analysis of the maturation of KKTN-tagged invertase and the turnover of Emp47p in alpha- and beta'-COP mutants suggest that the two WD40 domains of alpha- and beta'-COP bind distinct but overlapping sets of di-lysine signals and hence both contribute to recycling of proteins with di-lysine signals.

Mol Biol Cell, 2004 Mar, 15(3), 1417 - 24 Epub 2003 Dec 29.
Endoplasmic reticulum quality control of unassembled iron transporter depends on Rer1p-mediated retrieval from the golgi; Sato M et al.; Endoplasmic reticulum (ER) quality control is a conserved process by which misfolded or unassembled proteins are selectively retained in the endoplasmic reticulum (ER) . Failure in oligomerization of multisubunit membrane proteins is one of the events that triggers ER quality control . The transmembrane domains (TMDs) of unassembled subunits are determinants of ER retention in many cases, although the mechanism of the TMD-mediated sorting of unassembled subunits remains elusive . We studied a yeast iron transporter complex on the cell surface as a new model system for ER quality control . When Fet3p, a transmembrane subunit, is not assembled with the other membrane subunit, Ftr1p, unassembled Fet3p is exclusively localized to the ER at steady state . The TMD of Fet3p contains a determinant for this process . However, pulse-chase analysis and in vitro budding assays indicate that unassembled Fet3p rapidly escapes from the ER . Furthermore, Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi, is responsible for the TMD-dependent ER retrieval of unassembled Fet3p . These findings provide clear evidence that the ER quality control of unassembled membrane proteins can be achieved by retrieval from the Golgi and that Rer1p serves as a specific sorting receptor in this process.

Mutat Res, 2004 Jan 12, 545(1-2), 23 - 35
Genetic and metabolic effects of gluconasturtiin, a glucosinolate derived from cruciferae; Canistro D et al.; It is thought that induction of detoxifying phase-II drug metabolizing enzymes or inhibition of bioactivating phase-I by phytoalexins could protect against mutagens and neoplasia . In the search for potential naturally occurring molecular chemoprevention agents, particular attention has been devoted to isothiocyanates, which are breakdown products-via myrosinase-of glucosinolates such as gluconasturtiin (GNST), a natural constituent of cruciferae . Here, we first investigated the ability of GNST to modulate metabolizing enzymes in male Swiss Albino CD1 mice injected by gavage (24 mg/kg or 48 mg/kg b.w.) with GNST either in single or repeated (daily for four consecutive days) dose . Using selected probes to various cytochrome P450 (CYP) isoforms, a marked and generalized decrease of CYP content, NADPH-(CYP)-c-reductase and various CYP-linked monooxygenases (measuring CYP1A1, CYP2B1/2, CYP3A1/2, CYP1A2 and CYP2E1), was observed in hepatic, renal and pulmonary subcellular preparations (up to approximately 66% loss, liver) . Similar behavior was recorded using the regio- and stereo-selective hydroxylation of testosterone as multibiomarker (CYP2A1 and CYP2B9, up to approximately 96% loss), as well as with the phase-II marker glutathione S-transferase (up to approximately 50% loss, liver) . We also performed genotoxicity investigations, using the diploid D7 strain of yeast Saccharomyces cerevisiae as a biological test system . GNST was able to significantly induce point reverse mutation in growing cells without myrosinase, thus suggesting either a direct GNST or a CYP-linked metabolite role in the genotoxic response . On the contrary, in suspension test, the addition of myrosinase significantly increased mitotic gene conversion, probably due to the formation of GNST-derived phenylethyl isothiocyanate (PEITC) breakdown product . Taken together, our data suggest that GNST exerts a dual effect: while strongly inhibiting the microsomal (bioactivating) metabolism, GNST also possesses genotoxic activity . This concomitant mutagenic activity underlines the necessity of overall toxicological characterization of this (or any other molecule) prior to mass chemopreventive use.

J Mol Biol, 2004 Jan 23, 335(4), 937 - 51
The rad50 signature motif: essential to ATP binding and biological function; Moncalian G et al.; The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies . One of the most critical of these is the Mre11/Rad50 (M/R) complex, which is present in all three biological kingdoms, but is not well-understood at the biochemical level . Previous structural analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases . Here, we present the crystallographic structure of the Rad50 mutant S793R . This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases . The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis . We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops . This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization . The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities . This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo . These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms.

J Mol Biol, 2004 Jan 23, 335(4), 923 - 36
GTP-dependent recognition of the methionine moiety on initiator tRNA by translation factor eIF2; Kapp LD et al.; Eukaryotic translation initiation factor 2 (eIF2) is a G-protein that functions as a central switch in the initiation of protein synthesis . In its GTP-bound state it delivers the methionyl initiator tRNA (Met-tRNA(i)) to the small ribosomal subunit and releases it upon GTP hydrolysis following the recognition of the initiation codon . We have developed a complete thermodynamic framework for the assembly of the Saccharomyces cerevisiae eIF2.GTP.Met-tRNA(i) ternary complex and have determined the effect of the conversion of GTP to GDP on eIF2's affinity for Met-tRNA(i) in solution . In its GTP-bound state the factor forms a positive interaction with the methionine moiety on Met-tRNA(i) that is disrupted when GTP is replaced with GDP, while contacts between the factor and the body of the tRNA remain intact . This positive interaction with the methionine residue on the tRNA may serve to ensure that only charged initiator tRNA enters the initiation pathway . The toggling on and off of the factor's interaction with the methionine residue is likely to play an important role in the mechanism of initiator tRNA release upon initiation codon recognition . In addition, we show that the conserved base-pair A1:U72, which is known to be a critical identity element distinguishing initiator from elongator methionyl tRNA, is required for recognition of the methionine moiety by eIF2 . Our data suggest that a role of this base-pair is to orient the methionine moiety on the initiator tRNA in its recognition pocket on eIF2.

Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 771 - 4
A hitherto unknown transketolase-catalyzed reaction; Sevostyanova IA et al.; Yeast transketolase, in addition to catalyzing the transferase reaction through utilization of two substrates--the donor substrate (ketose) and the acceptor substrate (aldose)--is also able to catalyze a one-substrate reaction with only aldose (glycolaldehyde) as substrate . The interaction of glycolaldehyde with holotransketolase results in formation of the transketolase reaction intermediate, dihydroxyethyl-thiamin diphosphate . Then the glycolaldehyde residue is transferred from dihydroxyethyl-thiamin diphosphate to free glycolaldehyde . As a result, the one-substrate transketolase reaction product, erythrulose, is formed . The specific activity of transketolase was found to be 0.23 U/mg and the apparent Km for glycolaldehyde was estimated as 140 mM.

Cell, 2003 Dec 26, 115(7), 879 - 91
A neuronal isoform of the aplysia CPEB has prion-like properties; Si K et al.; Prion proteins have the unusual capacity to fold into two functionally distinct conformations, one of which is self-perpetuating . When yeast prion proteins switch state, they produce heritable phenotypes . We report prion-like properties in a neuronal member of the CPEB family (cytoplasmic polyadenylation element binding protein), which regulates mRNA translation . Compared to other CPEB family members, the neuronal protein has an N-terminal extension that shares characteristics of yeast prion-determinants: a high glutamine content and predicted conformational flexibility . When fused to a reporter protein in yeast, this region confers upon it the epigenetic changes in state that characterize yeast prions . Full-length CPEB undergoes similar changes, but surprisingly it is the dominant, self-perpetuating prion-like form that has the greatest capacity to stimulate translation of CPEB-regulated mRNA . We hypothesize that conversion of CPEB to a prion-like state in stimulated synapses helps to maintain long-term synaptic changes associated with memory storage.

Cell, 2003 Dec 26, 115(7), 813 - 23
Cell cycle regulated transport controlled by alterations in the nuclear pore complex; Makhnevych T et al.; Eukaryotic cells have developed mechanisms for regulating the nuclear transport of macromolecules that control various cellular events including movement through defined stages of the cell cycle . In yeast cells, where the nuclear envelope remains intact throughout the cell cycle, these transport regulatory mechanisms must also function during mitosis . We have uncovered a mechanism for regulating transport that is controlled by M phase specific molecular rearrangements in the nuclear pore complex (NPC) . These changes allow a transport inhibitory nucleoporin, Nup53p, to bind the karyopherin Kap121p specifically during mitosis, slowing its movement through the NPC and inducing cargo release . Yeast strains that possess defects in the function of Kap121p or the fidelity of the inhibitory pathway are delayed in mitosis . We propose that fluctuations in Kap121p transport mediated by the NPC contribute to controlling the subcellular distribution of molecules that direct progression through mitosis.

Cancer Res, 2003 Dec 15, 63(24), 8777 - 83
Expression of prolyl-hydroxylase-1 (PHD1/EGLN2) suppresses hypoxia inducible factor-1alpha activation and inhibits tumor growth; Erez N et al.; Hypoxic stress is one of the major selective pressures in the microenvironment of solid tumors, and overcoming this restriction is essential for tumor progression . One of the key factors driving the cellular response to lack of oxygen is hypoxia inducible factor (HIF), a key transcriptional factor . The level of the alpha subunit of HIF-1 is regulated by rapid degradation that is controlled by a family of prolyl hydroxylases (PHDs/EGLNs), the activity of which depends on oxygen availability . Our study shows that ectopic expression of mPHD1 suppressed accumulation of HIF-1alpha and secretion of Vascular Endothelial Growth Factor after treatment of cells with a hypoxia-mimetic drug . Furthermore, when colon carcinoma cells expressing mPHD1 were injected into nude mice, tumor growth was inhibited, and the inhibition of tumor growth was correlated with increased necrosis and a striking decrease in microvessel density . These data demonstrate that inhibition of hypoxia-induced activation of HIF-1alpha through activation of HIF-hydroxylase can provide a novel therapeutic strategy for inhibition of tumor growth and neovascularization and support the development of gene transfer approaches based on the activation of HIF-prolyl hydroxylases.

J Am Soc Nephrol, 2004 Jan, 15(1), 41 - 51
The death domain of kidney ankyrin interacts with Fas and promotes Fas-mediated cell death in renal epithelia; Del Rio M et al.; Ankyrins are a ubiquitously expressed family of conserved proteins that mediate the linkage of integral membrane proteins such as transporters and channels with the underlying cytoskeleton . Ankyrins possess a conserved death domain, the functional significance of which has remained puzzling . In this study, the death domain of AnkG190, the isoform of ankyrin expressed in kidney tubules, was used as bait in a yeast two-hybrid screen to identify interacting partners . One of these interactions was with the proapoptotic molecule Fas . This was confirmed by coimmunoprecipitation, colocalization, and glutathione S-transferase pull-down assays in cultured renal epithelial (MDCK) cells . Site-directed mutagenesis of a conserved arginine (R1496 in AnkG190), previously shown to be critical for the binding of Fas (R234 in Fas) to FADD, abolished the interaction of ankyrin's death domain with Fas . Overexpression of constructs containing ankyrin's death domain promoted Fas-mediated apoptosis in MDCK cells . The linkage between ankyrin and Fas was confirmed in vivo in mouse kidney tubule cells by coimmunoprecipitation and colocalization . In an established mouse model of renal ischemia-reperfusion injury characterized by apoptotic tubule cell death, the expression of both ankyrin and Fas was markedly induced, and the interaction between these molecules remained intact . The results identify a novel tethering interaction between ankyrin and Fas in kidney epithelia and suggest that AnkG190 may play a role as an adapter molecule in renal tubule cell death.

Bioinformatics, 2004 Jan 1, 20(1), 21 - 8
Prediction of protein subcellular locations using fuzzy k-NN method; Huang Y et al.; MOTIVATION: Protein localization data are a valuable information resource helpful in elucidating protein functions . It is highly desirable to predict a protein's subcellular locations automatically from its sequence . RESULTS: In this paper, fuzzy k-nearest neighbors (k-NN) algorithm has been introduced to predict proteins' subcellular locations from their dipeptide composition . The prediction is performed with a new data set derived from version 41.0 SWISS-PROT databank, the overall predictive accuracy about 80% has been achieved in a jackknife test . The result demonstrates the applicability of this relative simple method and possible improvement of prediction accuracy for the protein subcellular locations . We also applied this method to annotate six entirely sequenced proteomes, namely Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Oryza sativa, Arabidopsis thaliana and a subset of all human proteins . AVAILABILITY: Supplementary information and subcellular location annotations for eukaryotes are available at http://166.111.30.65/hying/fuzzy_loc.htm

Crit Rev Biochem Mol Biol, 2003, 38(5), 433 - 52
The protein components and mechanism of eukaryotic Okazaki fragment maturation; Kao HI et al.; An initiator RNA (iRNA) is required to prime cellular DNA synthesis . The structure of double-stranded DNA allows the synthesis of one strand to be continuous but the other must be generated discontinuously . Frequent priming of the discontinuous strand results in the formation of many small segments, designated Okazaki fragments . These short pieces need to be processed and joined to form an intact DNA strand . Our knowledge of the mechanism of iRNA removal is still evolving . Early reconstituted systems suggesting that the removal of iRNA requires sequential action of RNase H and flap endonuclease 1 (FEN1) led to the RNase H/FEN1 model . However, genetic analyses implied that Dna2p, an essential helicase/nuclease, is required . Subsequent biochemical studies suggested sequential action of RPA, Dna2p, and FEN1 for iRNA removal, leading to the second model, the Dna2p/RPA/FEN1 model . Studies of strand-displacement synthesis by polymerase delta indicated that in a reconstituted system, FEN1 could act as soon as short flaps are created, giving rise to a third model, the FEN1-only model . Each of the three pathways is supported by different genetic and biochemical results . Properties of the major protein components in this process will be discussed, and the validity of each model as a true representation of Okazaki fragment processing will be critically evaluated in this review.

Lab Invest, 2003 Dec, 83(12), 1859 - 66
APC haploinsufficiency, but not CTNNB1 or CDH1 gene mutations, accounts for a fraction of familial adenomatous polyposis patients without APC truncating mutations; Venesio T et al.; Familial adenomatous polyposis (FAP) is an autosomal dominant condition characterized by the development of hundreds to thousands of colorectal adenomatous polyps . In addition to the classic form, there is also attenuated polyposis (attenuated adenomatous polyposis coli; AAPC), which is characterized by a milder phenotype . FAP/AAPC is caused by germline mutations in the adenomatous polyposis coli (APC) gene . Very recently, germline mutations in the base-excision repair gene MYH have been associated with recessive inheritance of multiple colorectal adenomas in a subset of patients . APC pathogenic alterations are mostly (>95%) represented by frameshift or nonsense mutations leading to the synthesis of a truncated protein . We identified 20 APC truncating mutation carriers out of 30 FAP/AAPC patients from different Italian kindreds . In the remaining 10 patients, we searched for alterations other than truncating mutations by enzymatic mutation detection, real-time quantitative RT-PCR, and genotyping of polymorphic markers encompassing the APC locus . Moreover, to assess whether mutations of genes interacting with APC can substitute or act in association with APC alterations, we sequenced both CTNNB1 (beta-catenin) and CDH1 (E-cadherin) genes . No CTNNB1 or CDH1 mutations were found . On the contrary, four patients showed a reduced APC gene expression compared with healthy subjects . In three of the four cases, genotyping results were compatible with a constitutive allelic deletion . In one case this conclusion was confirmed by haplotype segregation analysis . Our results support the notion that FAP/AAPC can result from APC constitutive haploinsufficiency, with gene deletion being a possible cause of reduced gene expression.

J Cell Biol, 2003 Dec 22, 163(6), 1281 - 90
Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes; Janvier K et al.; The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins . A subset of these signals conform to the {DE}XXXL{LI} consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes . However, the identity of the AP subunits that recognize these signals remains controversial . We have used a yeast three-hybrid assay to demonstrate that {DE}XXXL{LI}-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits . The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo . These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.

J Cell Biol, 2003 Dec 22, 163(6), 1193 - 6
Myosin V and the endoplasmic reticulum: the connection grows; Wagner W et al.; In this issue, Estrada et al . (2003) provide new and important insights into how the endoplasmic reticulum (ER) of budding yeast cells is inherited . Together with other studies in plant and animal cells, the results of Estrada et al . (2003) support the idea that myosin V acts as a universal motor for the transport of ER membranes.

Mol Cell, 2003 Dec, 12(6), 1607 - 13
TOR and PKA signaling pathways converge on the protein kinase Rim15 to control entry into G0; Pedruzzi I et al.; The highly conserved Tor kinases (TOR) and the protein kinase A (PKA) pathway regulate cell proliferation in response to growth factors and/or nutrients . In Saccharomyces cerevisiae, loss of either TOR or PKA causes cells to arrest growth early in G(1) and to enter G(0) by mechanisms that are poorly understood . Here we demonstrate that the protein kinase Rim15 is required for entry into G(0) following inactivation of TOR and/or PKA . Induction of Rim15-dependent G(0) traits requires two discrete processes, i.e., nuclear accumulation of Rim15, which is negatively regulated both by a Sit4-independent TOR effector branch and the protein kinase B (PKB/Akt) homolog Sch9, and release from PKA-mediated inhibition of its protein kinase activity . Thus, Rim15 integrates signals from at least three nutrient-sensory kinases (TOR, PKA, and Sch9) to properly control entry into G(0), a key developmental process in eukaryotic cells.

Mol Cell, 2003 Dec, 12(6), 1565 - 76
A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1; Krogan NJ et al.; Deletions of three yeast genes, SET2, CDC73, and DST1, involved in transcriptional elongation and/or chromatin metabolism were used in conjunction with genetic array technology to screen approximately 4700 yeast deletions and identify double deletion mutants that produce synthetic growth defects . Of the five deletions interacting genetically with all three starting mutations, one encoded the histone H2A variant Htz1 and three encoded components of a novel 13 protein complex, SWR-C, containing the Snf2 family ATPase, Swr1 . The SWR-C also copurified with Htz1 and Bdf1, a TFIID-interacting protein that recognizes acetylated histone tails . Deletions of the genes encoding Htz1 and seven nonessential SWR-C components caused a similar spectrum of synthetic growth defects when combined with deletions of 384 genes involved in transcription, suggesting that Htz1 and SWR-C belong to the same pathway . We show that recruitment of Htz1 to chromatin requires the SWR-C . Moreover, like Htz1 and Bdf1, the SWR-C promotes gene expression near silent heterochromatin.

Mol Cell, 2003 Dec, 12(6), 1499 - 510
Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms; Lopes M et al.; Cells overcome intra-S DNA damage and replication impediments by coupling chromosome replication to sister chromatid-mediated recombination and replication-bypass processes . Further, molecular junctions between replicated molecules have been suggested to assist sister chromatid cohesion until anaphase . Using two-dimensional gel electrophoresis, we have identified, in yeast cells, replication-dependent X-shaped molecules that appear during origin activation, branch migrate, and distribute along the replicon through a mechanism influenced by the rate of fork progression . Their formation is independent of Rad51- and Rad52-mediated homologous recombination events and is not affected by DNA damage or replication blocks . Further, in hydroxyurea-treated rad53 mutants, altered in the replication checkpoint, the branched molecules progressively degenerate and likely contribute to generate pathological structures . We suggest that cells couple sister chromatid tethering with replication initiation by generating specialized joint molecules resembling hemicatenanes: this process might prime cohesion and assist sister chromatid-mediated recombination and replication events.

Plant J, 2004 Jan, 37(2), 251 - 68
Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri; Becher M et al.; Arabidopsis halleri ssp . halleri (accession Langelsheim) is a naturally selected zinc (Zn)- and cadmium-tolerant Zn hyperaccumulator . This plant differs strikingly from its close relative A . thaliana by accumulating Zn specifically in above-ground tissues . A . thaliana GeneChips were used in order to identify, on a transcriptome-wide scale, genes with a potential involvement in cellular metal uptake or detoxification in the shoots of A . halleri . Compared to A . thaliana, transcript abundance of several genes was found and confirmed to be substantially higher in A . halleri after 4 days of exposure to low as well as high Zn concentrations in the hydroponic culture medium . The identified candidate genes encode proteins closely related to the following A . thaliana proteins: AtZIP6, a putative cellular Zn uptake system and member of the zinc-regulated transporter (ZRT)-iron regulated transporter (IRT)-like protein (ZIP)-family of metal transporters, the putative P-type metal ATPase AtHMA3, the cation diffusion facilitator ZAT/AtCDF1, and the nicotianamine synthase AtNAS3 . Heterologous expression in mutant strains of the yeast Saccharomyces cerevisiae suggested that AhHMA3, AhCDF1-3, and AhNAS3 can function in cellular Zn detoxification . Our data indicate that, at the transcript level, the Zn tolerance strategy of A . halleri involves high constitutive expression of metal homeostasis genes in the shoots to accommodate higher basal levels of Zn accumulation, and possibly to prepare for sudden increases in Zn influx into shoot cells . Furthermore, profiling of metal homeostasis gene transcripts in shoot and root tissues by real-time RT-PCR indicated that A . halleri and A . thaliana respond differently to changes in plant Zn status.

Plant J, 2004 Jan, 37(2), 156 - 73
Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini; McCartney AW et al.; Fatty acid desaturases (FADs) play a prominent role in plant lipid metabolism and are located in various subcellular compartments, including the endoplasmic reticulum (ER) . To investigate the biogenesis of ER-localized membrane-bound FADs, we characterized the mechanisms responsible for insertion of Arabidopsis FAD2 and Brassica FAD3 into ER membranes and determined the molecular signals that maintain their ER residency . Using in vitro transcription/translation reactions with ER-derived microsomes, we show that both FAD2 and FAD3 are efficiently integrated into membranes by a co-translational, translocon-mediated pathway . We also demonstrate that while the C-terminus of FAD3 (-KSKIN) contains a functional prototypic dilysine ER retrieval motif, FAD2 contains a novel C-terminal aromatic amino acid-containing sequence (-YNNKL) that is both necessary and sufficient for maintaining localization in the ER . Co-expression of a membrane-bound reporter protein containing the FAD2 C-terminus with a dominant-negative mutant of ADP-ribosylation factor (Arf)1 abolished transient localization of the reporter protein in the Golgi, indicating that the FAD2 peptide signal acts as an ER retrieval motif . Mutational analysis of the FAD2 ER retrieval signal revealed a sequence-specific motif consisting of Phi-X-X-K/R/D/E-Phi-COOH, where -Phi- are large hydrophobic amino acid residues . Interestingly, this aromatic motif was present in a variety of other known and putative ER membrane proteins, including cytochrome P450 and the peroxisomal biogenesis factor Pex10p . Taken together, these data describe the insertion and retrieval mechanisms of FADs and define a new ER localization signal in plants that is responsible for the retrieval of escaped membrane proteins back to the ER.

Biochemistry, 2003 Dec 30, 42(51), 15208 - 14
Solvent and primary deuterium isotope effects show that lactate CH and OH bond cleavages are concerted in Y254F flavocytochrome b2, consistent with a hydride transfer mechanism; Sobrado P et al.; Yeast flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate; because of the wealth of structural and mechanistic information available, this enzyme has served as the model for the family of flavoproteins catalyzing oxidation of alpha-hydroxy acids . Primary deuterium and solvent isotope effects have now been used to analyze the effects of mutating the active site residue Tyr254 to phenylalanine . Both the V(max) and the V/K(lactate) values decrease about 40-fold in the mutant enzyme . The primary deuterium isotope effects on the V(max) and the V/K(lactate) values increase to 5.0, equivalent to the intrinsic isotope effect for the wild-type enzyme . In addition, both the V(max) and the V/K(lactate) values exhibit solvent isotope effects of 1.5 . Measurement of the solvent isotope effect with deuterated lactate establishes that the primary and solvent isotope effects arise from the same chemical step, consistent with concerted cleavage of the lactate OH and CH bonds . The pH dependence of the mutant enzyme is not significantly different from that of the wild-type enzyme; this is most consistent with a requirement that the side chain of Tyr254 be uncharged for catalysis . The results support a hydride transfer mechanism for the mutant protein and, by extension, wild-type flavocytochrome b(2) and the other flavoproteins catalyzing oxidation of alpha-hydroxy acids.

Biochemistry, 2003 Dec 30, 42(51), 15092 - 101
Limited set of amino acid residues in a class Ia aminoacyl-tRNA synthetase is crucial for tRNA binding; Geslain R et al.; The aim of this work was to characterize crucial amino acids for the aminoacylation of tRNA(Arg) by yeast arginyl-tRNA synthetase . Alanine mutagenesis was used to probe all the side chain mediated interactions that occur between tRNA(Arg2)(ICG) and ArgRS . The effects of the substitutions were analyzed in vivo in an ArgRS-knockout strain and in vitro by measuring the aminoacylation efficiencies for two distinct tRNA(Arg) isoacceptors . Nine mutants that generate lethal phenotypes were identified, suggesting that only a limited set of side chain mediated interactions is essential for tRNA recognition . The majority of the lethal mutants was mapped to the anticodon binding domain of ArgRS, a helix bundle that is characteristic for class Ia synthetases . The alanine mutations induce drastic decreases in the tRNA charging rates, which is correlated with a loss in affinity in the catalytic site for ATP . One of those lethal mutations corresponds to an Arg residue that is strictly conserved in all class Ia synthetases . In the known crystallographic structures of complexes of tRNAs and class Ia synthetases, this invariant Arg residue stabilizes the idiosyncratic conformation of the anticodon loop . This paper also highlights the crucial role of the tRNA and enzyme plasticity upon binding . Divalent ions are also shown to contribute to the induced fit process as they may stabilize the local tRNA-enzyme interface . Furthermore, one lethal phenotype can be reverted in the presence of high Mg(2+) concentrations . In contrast with the bacterial system, in yeast arginyl-tRNA synthetase, no lethal mutation has been found in the ArgRS specific domain recognizing the Dhu-loop of the tRNA(Arg) . Mutations in this domain have no effects on tRNA(Arg) aminoacylation, thus confirming that Saccharomyces cerevisiae and other fungi belong to a distinct class of ArgRS.

J Biol Chem, 2004 Mar 12, 279(11), 9847 - 56 Epub 2003 Dec 19.
Stimulation of flap endonuclease-1 by the Bloom's syndrome protein; Sharma S et al.; Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer . Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange . Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive . We report here a novel interaction of the BLM protein with the human 5'-flap endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair . BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity . BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts . The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM . This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme . We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.

J Biol Chem, 2004 Mar 5, 279(10), 8617 - 26 Epub 2003 Dec 19.
Motifs IV and V in the DEAH box splicing factor Prp22 are important for RNA unwinding, and helicase-defective Prp22 mutants are suppressed by Prp8; Schneider S et al.; The yeast pre-mRNA splicing factor Prp22 is a member of the DEAH box family of nucleic acid-stimulated ATPases and RNA helicases . Here we report a mutational analysis of 16 conserved residues in motifs Ia ((534)TQPRRVAA(541)), IV ((695)LVFLTG(700)), and V ((757)TNIAETSIT(765)) . Mutants T757A, I764A, and T765A were lethal, and F697A cells did not grow at < or =30 degrees C . The mutant proteins failed to catalyze mRNA release from the spliceosome in vitro, and they were deficient for RNA unwinding . The F697A, I764A, and T765A proteins were active for ATP hydrolysis in the presence of RNA cofactor . The T757A mutant retained basal ATPase activity but was not stimulated by RNA, whereas ATP hydrolysis by T765A was strictly dependent on the RNA cofactor . Thus Thr-757 and Thr-765 in motif V link ATP hydrolysis to the RNA cofactor . To illuminate the mechanism of Prp22-catalyzed mRNA release, we performed a genetic screen to identify extragenic suppressors of the cold-sensitive growth defect of a helicase/release-defective Prp22 mutant . We identified one of the suppressors as a missense mutation of PRP8 (R1753K), a protein component of the U5 small nuclear ribonucleoprotein . We show that PRP8-R1753K suppressed multiple helicase-deficient prp22 mutations, including the lethal I764A mutation . Replacing Arg-1753 of Prp8 by either Lys, Ala, Gln, or Glu resulted in suppression of helicase-defective Prp22 mutants . Prp8-Arg1753 mutations by themselves caused temperature-sensitive growth defects in a PRP22 strain . These findings suggest a model whereby Prp22 disrupts an RNA/protein or RNA/RNA interaction in the spliceosome that is normally stabilized by Prp8.

J Biol Chem, 2004 Apr 9, 279(15), 14587 - 94 Epub 2003 Dec 19.
Hydrogen peroxide induces topoisomerase I-mediated DNA damage and cell death; Daroui P et al.; Reactive oxygen species modify DNA, generating various DNA lesions including modified bases such as 8-oxoguanine (8-oxoG) . These base-modified DNA lesions have been shown to trap DNA topoisomerase I (TOP1) into covalent cleavage complexes . In this study, we have investigated the role of TOP1 in hydrogen peroxide toxicity . We showed that ectopic expression of TOP1 in Saccharomyces cerevisiae conferred sensitivity to hydrogen peroxide, and this sensitivity was dependent on RAD9 checkpoint function . Moreover, in the mammalian cell culture system, hydrogen peroxide-induced growth inhibition and apoptosis were shown to be partly TOP1-dependent as evidenced by a specific increase in resistance to hydrogen peroxide in TOP1-deficient P388/CPT45 murine leukemia cells as compared with their TOP1-proficient parental cell line P388 . In addition, hydrogen peroxide was shown to induce TOP1-DNA cross-links . These results support a model in which hydrogen peroxide promotes the trapping of TOP1 on oxidative DNA lesions to form TOP1-DNA cleavage complexes that contribute to hydrogen peroxide toxicity.

J Biol Chem, 2004 Mar 5, 279(10), 8635 - 41 Epub 2003 Dec 19.
Repeat sequence of Epstein-Barr virus-encoded nuclear antigen 1 protein interrupts proteasome substrate processing; Zhang M et al.; The Epstein-Barr virus thwarts immune surveillance through a Gly-Ala repeat (GAr) within the viral Epstein-Barr virus-encoded nuclear antigen 1 protein . The GAr inhibits proteasome processing, an early step in antigen peptide presentation, but the mechanism of proteasome inhibition has been unclear . By embedding a GAr within ornithine decarboxylase, a natural proteasome substrate that does not require ubiquitin conjugation, we now demonstrate inhibition in a purified system, excluding involvement of ubiquitin conjugation or of proteins extraneous to substrate and proteasome . We show further that the GAr acts as a stop-transfer signal in proteasome substrate processing, resulting in vivo in partial proteolysis that halts just short of the GAr . Similarly, introducing a GAr into green fluorescent protein destabilized by the ornithine decarboxylase degradation domain also stops the progress of proteolysis, leading to the accumulation of partial degradation products . We postulate that the ATP motor of the proteasome slips when it encounters the GAr, impeding further insertion and, in this way, halting degradation.

J Biol Chem, 2004 Mar 5, 279(10), 8946 - 56 Epub 2003 Dec 19.
The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro; Fraser CS et al.; Eukaryotic initiation factor 3 (eIF3) is a 12-subunit protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 40 S initiation complex . The molecular mechanisms by which eIF3 exerts these functions are poorly understood . To learn more about the structure and function of eIF3 we have expressed and purified individual human eIF3 subunits or complexes of eIF3 subunits using baculovirus-infected Sf9 cells . The results indicate that the subunits of human eIF3 that have homologs in Saccharomyces cerevisiae form subcomplexes that reflect the subunit interactions seen in the yeast eIF3 core complex . In addition, we have used an in vitro 40 S ribosomal subunit binding assay to investigate subunit requirements for efficient association of the eIF3 subcomplexes to the 40 S ribosomal subunit . eIF3j alone binds to the 40 S ribosomal subunit, and its presence is required for stable 40 S binding of an eIF3bgi subcomplex . Furthermore, purified eIF3 lacking eIF3j binds 40 S ribosomal subunits weakly, but binds tightly when eIF3j is added . Cleavage of a 16-residue C-terminal peptide from eIF3j by caspase-3 significantly reduces the affinity of eIF3j for the 40 S ribosomal subunit, and the cleaved form provides substantially less stabilization of purified eIF3-40S complexes . These results indicate that eIF3j, and especially its C terminus, play an important role in the recruitment of eIF3 to the 40 S ribosomal subunit.

J Mol Biol, 2004 Jan 16, 335(3), 723 - 31
Asymmetric removal of supercoils suggests how topoisomerase II simplifies DNA topology; Trigueros S et al.; Type-IIA topoisomerases consume ATP as they catalyse the interconversion of DNA topoisomers by transporting one DNA segment through a transient break in another . It remains unclear how their activity simplifies the topology of DNA below equilibrium values . Here we report that eukaryotic topoisomerase II narrows the thermal distribution of DNA supercoils, by mainly removing negative DNA crossings . Surprisingly, this asymmetry in supercoil removal is not due to deformation of the DNA before strand passage . Topoisomerase II neither bends nor alters the helical conformation of the interacting DNA . Rather, it appears to interact with a third DNA segment, in addition to the gated and the transported segments . Remarkably, the simultaneous interaction with three DNA segments accounts for the asymmetric removal of supercoils in relaxed DNA and gives a clue to how topoisomerase II simplifies the topology of DNA against the thermal drive.

BMC Bioinformatics . 2003 Dec 19;4(1):64.
The PAM domain, a multi-protein complex-associated module with an all-alpha-helix fold; Ciccarelli FD et al.; BACKGROUND: Multimeric protein complexes have a role in many cellular pathways and are highly interconnected with various other proteins . The characterization of their domain composition and organization provides useful information on the specific role of each region of their sequence . RESULTS: We identified a new module, the PAM domain (PCI/PINT associated module), present in single subunits of well characterized multiprotein complexes, like the regulatory lid of the 26S proteasome, the COP-9 signalosome and the Sac3-Thp1 complex . This module is an around 200 residue long domain with a predicted TPR-like all-alpha-helical fold . CONCLUSIONS: The occurrence of the PAM domain in specific subunits of multimeric protein complexes, together with the role of other all-alpha-helical folds in protein-protein interactions, suggest a function for this domain in mediating transient binding to diverse target proteins.

EMBO J, 2004 Jan 14, 23(1), 45 - 53 Epub 2003 Dec 11.
The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion; Dietrich LE et al.; The NSF homolog Sec18 initiates fusion of yeast vacuoles by disassembling cis-SNARE complexes during priming . Sec18 is also required for palmitoylation of the fusion factor Vac8, although the acylation machinery has not been identified . Here we show that the SNARE Ykt6 mediates Vac8 palmitoylation and acts during a novel subreaction of vacuole fusion . This subreaction is controlled by a Sec17-independent function of Sec18 . Our data indicate that Ykt6 presents Pal-CoA via its N-terminal longin domain to Vac8, while transfer to Vac8's SH4 domain occurs spontaneously and not enzymatically . The conservation of Ykt6 and its localization to several organelles suggest that its acyltransferase activity may also be required in other intracellular fusion events.

EMBO J, 2004 Jan 14, 23(1), 180 - 90 Epub 2003 Dec 18.
Recruitment of Cdc28 by Whi3 restricts nuclear accumulation of the G1 cyclin-Cdk complex to late G1; Wang H et al.; The G1 cyclin Cln3 is a key activator of cell-cycle entry in budding yeast . Here we show that Whi3, a negative G1 regulator of Cln3, interacts in vivo with the cyclin-dependent kinase Cdc28 and regulates its localization in the cell . Efficient interaction with Cdc28 depends on an N-terminal domain of Whi3 that is also required for cytoplasmic localization of Cdc28, and for proper regulation of G1 length and filamentous growth . On the other hand, nuclear accumulation of Cdc28 requires the nuclear localization signal of Cln3, which is also found in Whi3 complexes . Both Cln3 and Cdc28 are mainly cytoplasmic during early G1, and become nuclear in late G1 . However, Whi3-deficient cells show a distinct nuclear accumulation of Cln3 and Cdc28 already in early G1 . We propose that Whi3 constitutes a cytoplasmic retention device for Cln3-Cdc28 complexes, thus defining a key G1 event in yeast cells.

EMBO J, 2004 Jan 14, 23(1), 234 - 43 Epub 2003 Dec 18.
Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments; Koszul R et al.; There is growing evidence that duplications have played a major role in eucaryotic genome evolution . Sequencing data revealed the presence of large duplicated regions in the genomes of many eucaryotic organisms, and comparative studies have suggested that duplication of large DNA segments has been a continuing process during evolution . However, little experimental data have been produced regarding this issue . Using a gene dosage assay for growth recovery in Saccharomyces cerevisiae, we demonstrate that a majority of the revertant strains (58%) resulted from the spontaneous duplication of large DNA segments, either intra- or interchromosomally, ranging from 41 to 655 kb in size . These events result in the concomitant duplication of dozens of genes and in some cases in the formation of chimeric open reading frames at the junction of the duplicated blocks . The types of sequences at the breakpoints as well as their superposition with the replication map suggest that spontaneous large segmental duplications result from replication accidents . Aneuploidization events or suppressor mutations that do not involve large-scale rearrangements accounted for the rest of the reversion events (in 26 and 16% of the strains, respectively).

EMBO J, 2004 Jan 14, 23(1), 33 - 44 Epub 2003 Dec 18.
Identification of a family of animal sphingomyelin synthases; Huitema K et al.; Sphingomyelin (SM) is a major component of animal plasma membranes . Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product . This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively . SM synthesis occurs in the lumen of the Golgi as well as on the cell surface . As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations . Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase . Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes . Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane . Collectively, these findings open up important new avenues for studying sphingolipid function in animals.

EMBO J, 2004 Jan 28, 23(2), 302 - 11 Epub 2003 Dec 18.
Nbp2 targets the Ptc1-type 2C Ser/Thr phosphatase to the HOG MAPK pathway; Mapes J et al.; The yeast high osmolarity glycerol (HOG) pathway signals via the Pbs2 MEK and the Hog1 MAPK, whose activity requires phosphorylation of Thr and Tyr in the activation loop . The Ptc1-type 2C Ser/Thr phosphatase (PP2C) inactivates Hog1 by dephosphorylating phospho-Thr, while the Ptp2 and Ptp3 protein tyrosine phosphatases dephosphorylate phospho-Tyr . In this work, we show that the SH3 domain-containing protein Nbp2 negatively regulates Hog1 by recruiting Ptc1 to the Pbs2-Hog1 complex . Consistent with this role, NBP2 acted as a negative regulator similar to PTC1 in phenotypic assays . Biochemical analysis showed that Nbp2, like Ptc1, was required to inactivate Hog1 during adaptation . As predicted for an adapter, deletion of NBP2 disrupted Ptc1-Pbs2 complex formation . Furthermore, Nbp2 contained separate binding sites for Ptc1 and Pbs2: the novel N-terminal domain bound Ptc1, while the SH3 domain bound Pbs2 . In addition, the Pbs2 scaffold bound the Nbp2 SH3 via a Pro-rich motif distinct from that which binds the SH3 domain of the positive regulator Sho1 . Thus, Nbp2 recruits Ptc1 to Pbs2, a scaffold for both negative and positive regulators.

J Biol Chem, 2004 Mar 5, 279(10), 8867 - 72 Epub 2003 Dec 18.
Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70; Tsukahara F et al.; Heat shock cognate protein 70 (Hsc70) serves nuclear transport of several proteins as a molecular chaperone . We have recently identified a novel variant of human Hsc70, heat shock cognate protein 54 (Hsc54), that lacks amino acid residues 464-616 in the protein binding and variable domains of Hsc70 . In the present study, we examined nucleocytoplasmic localization of Hsc70 and Hsc54 by using green fluorescent protein (GFP) fusions . GFP-Hsc70 is localized in both the cytoplasm and the nucleus at 37 degrees C and accumulated into the nucleolus/nucleus after heat shock, whereas GFP-Hsc54 always remained exclusively in the cytoplasm under these conditions . Mutation studies indicated that 20 amino acid residues of nuclear localization-related signals, which are missing in Hsc54 but are retained in Hsc70, are required for proper nuclear localization of Hsc70 . We further found that Hsc54 contains a functional leucine-rich nuclear export signal (NES, (394)LDVTPLSL(401)) which is differently situated from the previously proposed NES in Saccharomyces cerevisiae Ssb1p . The cytoplasmic localization of Hsc54 was impaired by a mutation in NES as well as by a nuclear export inhibitor, leptomycin B, suggesting that Hsc54 is actively exported from the nucleus to the cytoplasm through a CRM1-dependent mechanism . In contrast, the nucleocytoplasmic localization of Hsc70 was not affected by the same mutation of NES or leptomycin B . These results suggest that the nuclear localization-related signal could functionally mask NES leading to prolonged retention of Hsc70 in the nucleus . An additional mechanism for unmasking the NES may regulate nucleocytoplasmic trafficking of Hsc70.

J Biol Chem, 2004 Apr 2, 279(14), 13889 - 95 Epub 2003 Dec 18.
The structure of the 1L-myo-inositol-1-phosphate synthase-NAD+-2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate complex demands a revision of the enzyme mechanism; Jin X et al.; 1l-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of d-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds . It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction . Here we present the structure of MIP synthase in complex with NAD(+) and a high-affinity inhibitor, 2-deoxy-d-glucitol 6-(E)-vinylhomophosphonate . This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-d-glucitol 6-phosphate in the previously published structure of MIP synthase-NAD(+)-2-deoxy-d-glucitol 6-phosphate . There are several other conformational changes in NAD(+) and the enzyme active site as well . Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.

J Biol Chem, 2004 Mar 12, 279(11), 9713 - 24 Epub 2003 Dec 18.
The type 1 phosphatase Reg1p-Glc7p is required for the glucose-induced degradation of fructose-1,6-bisphosphatase in the vacuole; Cui DY et al.; Protein phosphatases play an important role in vesicular trafficking and membrane fusion processes . The type 1 phosphatase Glc7p and its regulatory subunit Reg1p were identified as required components in the glucose-induced targeting of the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) to the vacuole for degradation . The interaction of Reg1p with Glc7p was important for the transport of FBPase from intermediate vacuole import and degradation (Vid) vesicles to vacuoles . The glc7-T152K mutant strain exhibited a reduced Reg1p binding along with defects in FBPase degradation and Vid vesicle trafficking to the vacuole . In this mutant, Vid vesicles were the most defective components, whereas the vacuole was also defective . Shp1p and Glc8p regulate Glc7p phosphatase activity and are required for FBPase degradation . In the Deltashp1 and Deltaglc8 strains, Reg1p-Glc7p interaction was not affected, suggesting that phosphatase activity is also necessary for FBPase degradation . Similar to those seen in the glc7-T152K mutant, the Deltashp1 and Deltaglc8 mutants exhibited severely defective Vid vesicles, but partially defective vacuoles . Taken together, our results suggest that Reg1p-Glc7p interaction and Glc7p phosphatase activity play a required role in the Vid vesicle to vacuole-trafficking step along the FBPase degradation pathway.

Plant Mol Biol, 2003 Aug, 52(6), 1191 - 202
Isolation and characterization of a novel rice Ca2+-regulated protein kinase gene involved in responses to diverse signals including cold, light, cytokinins, sugars and salts; Kim KN et al.; We have isolated a cold-inducible gene (designated OsCK1) from Oryza sativa by a differential cDNA screening technique . Sequence analysis indicated that the open reading frame of the OsCK1 gene consists of 1350 bp encoding 449 amino acid residues, which is very similar to a family of calcineurin B-like protein (CBL)-interacting protein kinases (CIPKs) or salt overly sensitive 2 (SOS2)-like protein kinases (PKS) in Arabidopsis . CIPKs/PKS are a group of Ser/Thr protein kinases associated with the AtCBL/SOS3-like calcium-binding proteins (SCaBP) . OsCK1 actually interacts with AtCBL3 through the C-terminal region in a yeast two-hybrid system, suggesting that OsCK1 is probably a rice orthologue of one of the CIPK/PKS members . Expression of the OsCK1 gene was detected mainly in the shoots and highly inducible by diverse signals such as cold, light, salt, sugar and cytokinins . In addition, calcium increased the OsCK1 transcript level, whereas a calcium ionophore, A23187, partially abolished stimulus-induced expressions . OsCK1 phosphorylated itself and a generic substrate, myelin basic protein, in the preference of Mn2+ . Deletion of the C-terminal region from OsCK1 significantly decreased autophosphorylation activity without affecting the ability for substrate phosphorylation . These findings suggest that the CBL/CIPK or SCaBP/PKS signaling pathways recently found in Arabidopsis may also exist in rice and function in cold response in which calcium signal serves as a second messenger.

Cell Mol Biol (Noisy-le-grand), 2003 Nov, 49(7), 1025 - 35
Overexpression of genes involved in vesicular trafficking to the vacuole defends against lethal effects of oxidative damage; Martinez M et al.; Anticancer bleomycins and structurally-related analogs are oxidative agents that mimic ionizing radiation in many of their cellular effects . The current study was designed to better understand this class of radiomimetic and oxidative drugs, and how cells defend against them to become resistant . Based on some of the properties conferred by the blm5-1 mutation of Saccharomyces cerevisiae, a multi-step cloning strategy was developed to search for genes that protect cells against oxidative damage and lethal effects of bleomycin treatments . The strategy employed blm5-1 mutant strains to search for genes that rescued the drug hypersensitivities conferred by the mutation, and utilized the inability of homozygous blm5-1 mutant diploid strains to grow at elevated temperatures . This approach identified the VPS3, VPS8 and PEP7 genes that function in vesicular trafficking between the endosome and the yeast vacuole via the carboxypeptidase Y (CpY) pathway . Mutant blm5-1 strains possess several phenotypic characteristics consistent with CpY mutants, including reduced mitotic growth rates and sporulative abilities . However, blm5-1 strains were not found to be defective in the transport of CpY into the vacuole . We suggest that the ability of the VPS3, VPS8 and PEP7 genes to rescue lethal effects of oxidative damage resulted from the overexpression of these genes.

Ukr Biokhim Zh, 2003 Jul-Aug, 75(4), 75 - 80
{Use of three-hybrid system to detect RNA-binding activity of alfalfa mosaic virus coat protein}; Spiridonov VG et al.; We used yeast three-hybrid system, for studying interaction of alfalfa mosaic virus coat protein AMVCP (AMVCP) with RNA4, which codes this protein . We have shown that AMVCP with high affinity is bound to plus-chain of RNA4 in vivo . The mutational analysis has shown, that the N-terminal part of AMVCP (aa 1 to 85) contains RNA-binding domain . C-terminal part of this protein (aa 86 to 221) does not participate in direct interaction with RNA4 . However activity of the reporter-gene LacZ, which codes beta-galactosidase, in case of interaction only N-terminal part of AMVCP is five times lower, in comparison with full-length hybrid protein, that confirms that the tertiary structure of full-length AMVCP is more favourable for interaction with RNA4.

Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D323 - 5
yMGV: a cross-species expression data mining tool; Lelandais G et al.; The yeast Microarray Global Viewer (yMGV @ was created 3 years ago as a database that houses a collection of Saccharomyces cerevisiae and Schizosaccharo myces pombe microarray data sets published in 82 different articles . yMGV couples data mining tools with a user-friendly web interface so that, with a few mouse clicks, one can identify the conditions that affect the expression of a gene or list of genes regulated in a set of experiments . One of the major new features we present here is a set of tools that allows for inter-organism comparisons . This should enable the fission yeast community to take advantage of the large amount of available information on budding yeast transcriptome . New tools and ongoing developments are also presented here.

Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 831 - 42
Mouse MafA, homologue of zebrafish somite Maf 1, contributes to the specific transcriptional activity through the insulin promoter; Kajihara M et al.; Large Maf transcription factors, which are members of the basic leucine zipper (b-Zip) superfamily, have been reported to be involved in embryonic development and cell differentiation . Previously, we isolated a novel zebrafish large Maf cDNA, somite Maf1 (SMaf1), which possesses transactivational activity within its N-terminus domain . To elucidate SMaf1 function in mammals, we tried to isolate the mouse homologue of zebrafish SMaf1 . We isolated the mouse homologue of zebrafish SMaf1, which is the same molecule as the recently reported MafA . MafA mRNA was detected in formed somites, head neural tube, and liver cells in the embryos . In the adult mouse, MafA transcript was amplified in the brain, lung, spleen, and kidney by RT-PCR . MafA mRNA was also detectable in beta-cell line . Next, we analyzed the transcriptional activity of MafA using rat insulin promoters I and II (RIPI and II), since a part of RIP sequence was similar to the Maf recognition element (MARE) and MafA was expressed in pancreatic beta cells . MafA was able to activate transcription from RIPII, but not RIPI, in a dose dependent manner and the activity was dependent on RIPE3b/C1 sequences . In addition, the amount of MafA protein was regulated by glucose concentration . These results indicate that MafA is the homologue of zebrafish SMaf1 and acts as a transcriptional activator of the insulin gene promoter through the RIPE3b element.

Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 623 - 8
TBBz but not TBBt discriminates between two molecular forms of CK2 in vivo and its implications; Zien P et al.; Two ATP-competitive inhibitors-4,5,6,7-tetrabromo-benzotriazole (TBBt) and 4,5,6,7-tetrabromo-benzimidazole (TBBz) have been shown to decrease activity of CK2 holoenzyme . Surprisingly it occurs that TBBz contrary to TBBt does not inhibit free catalytic subunit CK2 {Formula: see text} . Both inhibitors are virtually inactive against RAP protein kinase . The above-mentioned protein kinases phosphorylate in vitro a set of acidic ribosomal P-proteins of the 60S ribosomal subunit . Such a modification is one of the mechanisms regulating translational activity of ribosomes in vivo . Application of these two very selective inhibitors allows us to define the role of free catalytic {Formula: see text} subunit of CK2 in phosphorylation of ribosomal proteins . It occurs that CK2 {Formula: see text} but not CK2 holoenzyme is responsible for phosphorylation of P-proteins in vivo . Moreover, elimination of both forms of protein kinase CK2 (hCK2 and CK2 {Formula: see text} ) activity in living cells led to dramatic loss of the translational activity of the ribosome.

Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 555 - 61
A single point mutation resulting in an adversely reduced expression of DPM2 in the Lec15.1 cells; Pu L et al.; Mammalian dolichol-phosphate-mannose (DPM) synthase consists of three subunits, DPM1, DPM2, and DPM3 . Lec15.1 Chinese hamster ovary cells are deficient in DPM synthase activity . The present paper reports that DPM1 cDNA from wild type and Lec15.1 CHO cells were found to be identical, and transfection with CHO DPM1 cDNA did not reverse the Lec15.1 phenotype . Neither did a chimeric cDNA containing the complete hamster DPM1 open reading frame fused to the Saccharomyces cerevisiae DPM1 C-terminal transmembrane domain . In contrast, Lec15.1 cells were found to have a single point mutation G29A within the coding region of the DPM2 gene, resulting in a glycine to glutamic acid change in amino acid residue 10 of the peptide . Moreover, mutant DPM2 cDNA expressed a drastically reduced amount of DPM2 protein and poorly corrects the Lec15.1 cell phenotype when compared with wild type CHO DPM2 cDNA (G(29) form).

Curr Biol, 2003 Dec 16, 13(24), 2159 - 69
Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1; Balcer HI et al.; BACKGROUND: Dynamic remodeling of the actin cytoskeleton requires rapid turnover of actin filaments, which is regulated in part by the actin filament severing/depolymerization factor cofilin/ADF . Two factors that cooperate with cofilin are Srv2/CAP and Aip1 . Human CAP enhances cofilin-mediated actin turnover in vitro, but its biophysical properties have not been defined, and there has been no in vivo evidence reported for its role in turnover . Xenopus Aip1 forms a cofilin-dependent cap at filament barbed ends . It has been unclear how these diverse activities are coordinated in vivo . RESULTS: Purified native yeast Srv2/CAP forms a high molecular weight structure comprised solely of actin and Srv2 . The complex is linked to actin filaments via the SH3 domain of Abp1 . Srv2 complex catalytically accelerates cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhances the ability of profilin to stimulate nucleotide exchange on ADP-actin . Yeast Aip1 forms a cofilin-dependent filament barbed end cap, disrupted by the cof1-19 mutant . Genetic analyses show that specific combinations of activities mediated by cofilin, Srv2, Aip1, and capping protein are required in vivo . CONCLUSIONS: We define two genetically and biochemically separable functions for cofilin in actin turnover . One is formation of an Aip1-cofilin cap at filament barbed ends . The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex . We show that the Srv2 complex is a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization.

J Biol Chem, 2004 Mar 12, 279(11), 9987 - 96 Epub 2003 Dec 15.
Determination of four sequential stages during microautophagy in vitro; Kunz JB et al.; Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen . This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses . Vacuoles are lysosomal compartments of yeasts . Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle . Using this in vitro system, we defined different reaction states . We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis . This allowed us to characterize four biochemically distinguishable steps of the reaction . We propose that these correspond to sequential stages of vacuole invagination and vesicle scission . Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice . Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

J Biol Chem, 2004 Feb 27, 279(9), 7812 - 8 Epub 2003 Dec 16.
Merlin neutralizes the inhibitory effect of Mdm2 on p53; Kim H et al.; The stability of p53 tumor suppressor is regulated by Mdm2 via the ubiquitination and proteasome-mediated proteolysis pathway . The c-Abl and PTEN tumor suppressors are known to stabilize p53 by blocking the Mdm2-mediated p53 degradation . This study investigated the correlation between p53 and merlin, a neurofibromatosis 2 (NF2)-related tumor suppressor, in association with the Mdm2 function . The results showed that merlin increased the p53 stability by inhibiting the Mdm2-mediated degradation of p53, which accompanied the increase in the p53-dependent transcriptional activity . The stabilization of p53 by merlin appeared to be accomplished through Mdm2 degradation, and the N-terminal region of merlin was responsible for this novel activity . This study also showed that overexpression of merlin-induced apoptosis of cells depending preferentially on p53 in response to the serum starvation or a chemotherapeutic agent . These results suggest that merlin could be a positive regulator of p53 in terms of tumor suppressor activity, and provide the promising therapeutic means for treating tumors with non-functional merlin or Mdm2 overexpression.

J Biol Chem, 2004 Mar 5, 279(10), 9475 - 80 Epub 2003 Dec 15.
A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases; Brdlik CM et al.; TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins . Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1 . To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance . Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors . Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine . Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin . These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.

J Biol Chem, 2004 Feb 13, 279(7), 5049 - 52 Epub 2003 Dec 15.
Differential export requirements for shuttling serine/arginine-type mRNA-binding proteins; Hacker S et al.; Messenger RNAs are transported to the cytoplasm bound to several shuttling mRNA-binding proteins . Here, we present the characterization of Hrb1, a novel component of the transported ribonucleoprotein complex in Saccharomyces cerevisiae . The protein is similar to the other two serine/arginine (SR)-type proteins in yeast, Gbp2 and Npl3 . Hrb1 is nuclear at steady state and its import is mediated by the karyopherin Mtr10 . Hrb1 binds to poly(A)+ RNA in vivo and its binding is significantly increased in MTR10 mutants, suggesting a role for Mtr10 in dissociating Hrb1 from the mRNAs . Interestingly, by comparing the export requirements of all three SR proteins we find similarities but also striking differences . While the export of all three proteins is dependent on the export of mRNAs in general, as no transport is observed in mutants defective in transcription (rpb1-1) or mRNA export (mex67-5), we find specific requirements for components of the THO complex, involved in transcription elongation . While both Hrb1 and Gbp2 depend on Mft1 and Hpr1 for their nuclear export, Npl3 is exported independently of both proteins . These findings suggest that Hrb1 and Gbp2, but not Npl3, might be loaded onto the growing mRNA via the THO complex components Mtf1 and Hrp1.

FEBS Lett, 2003 Dec 18, 555(3), 511 - 5
Multiple functions of tail-anchor domains of mitochondrial outer membrane proteins; Habib SJ et al.; Tail-anchored proteins form a distinct class of membrane proteins that have a single membrane anchor sequence at their C-terminus, the tail-anchor . Their N-terminal portion is exposed to the cytosol . We have studied the roles of tail-anchor domains of proteins residing in the mitochondrial outer membrane . Four distinct functions of the tail-anchor domain were identified . First, the domain mediates the targeting to mitochondria in a process that probably requires a net positive charge at the C-terminally flanking segment . Second, tail-anchor domains facilitate the insertion into the mitochondrial outer membrane . Third, the tail-anchor is responsible for the assembly of the respective protein into functional multi-subunit complexes; and fourth, tail-anchor domains can stabilize such complexes.

Cell, 2003 Dec 12, 115(6), 739 - 50
Ribosome loading onto the mRNA cap is driven by conformational coupling between eIF4G and eIF4E; Gross JD et al.; The eukaryotic initiation factor 4G (eIF4G) is the core of a multicomponent switch controlling gene expression at the level of translation initiation . It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation . We describe the solution structure of the complex between yeast eIF4E/cap and eIF4G (393-490) . Binding triggers a coupled folding transition of eIF4G (393-490) and the eIF4E N terminus resulting in a molecular bracelet whereby eIF4G (393-490) forms a right-handed helical ring that wraps around the N terminus of eIF4E . Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo . Our data explain how mRNA, eIF4E, and eIF4G exists as a stable mRNP that may facilitate multiple rounds of ribosomal loading during translation initiation, a key determinant in the overall rate of protein synthesis.

Methods Enzymol, 2003, 366, 175 - 87
Using the Ras Recruitment System to identify PP2A-B55-interacting proteins; Barr HM et al.; The RRS system facilitated the discovery of hitherto unknown interactions with the PP2A-B55 subunit . The advantages of the system lie in its ability to identify interactions that may not be detected by traditional yeast two-hybrid systems . The RRS can thus provide a complementary genetic approach to the identification of protein-protein interactions.

Curr Opin Rheumatol, 2004 Jan, 16(1), 38 - 42
Behçet syndrome; Yurdakul S et al.; PURPOSE OF REVIEW: Interest in Behcet syndrome (BS), although the condition is rare in many parts of the world, is increasing as judged by the growing number of related publications and scientific meetings . RECENT FINDINGS: Recent work reconfirmed the following without further insight into their meaning in pathogenesis: males have a more severe course; one third of the patients have a thrombophilia; papulopustular lesions and arthritis go together; gammadelta T are increased; and HLA B51 is still the main genetic association . Presence of increased levels of antibodies to Saccharomyces cerevisiae and the proposal of alpha-enolase as the target antigen of antiendothelial cell antibodies, known to be present also in BS, are observations that might help in delineating the unknown pathogenesis . For management, preliminary yet promising experience with the use of anti-tumor necrosis factor agents is being reported . SUMMARY When compared with 2 decades ago, we know considerably more and can do more about BS.

Mol Cell Biol, 2004 Jan, 24(1), 352 - 61
Mutations in the gal83 glycogen-binding domain activate the snf1/gal83 kinase pathway by a glycogen-independent mechanism; Wiatrowski HA et al.; The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress . Previous studies identified a glycogen-binding domain in the AMPK beta1 subunit, and the sequence is conserved in the Snf1 kinase beta subunits Gal83 and Sip2 . Here we use genetic analysis to assess the role of this domain in vivo . Alteration of Gal83 at residues that are important for glycogen binding of AMPK beta1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo . Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element . Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase . Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15736 - 41 Epub 2003 Dec 12.
Activation of a LTR-retrotransposon by telomere erosion; Scholes DT et al.; Retrotransposons can facilitate repair of broken chromosomes, and therefore an important question is whether the host can activate retrotransposons in response to chromosomal lesions . Here we show that Ty1 elements, which are LTR-retrotransposons in Saccharomyces cerevisiae, are mobilized when DNA lesions are created by the loss of telomere function . Inactivation of telomerase in yeast results in progressive shortening of telomeric DNA, eventually triggering a DNA-damage checkpoint that arrests cells in G2/M . A fraction of cells, termed survivors, recover from arrest by forming alternative telomere structures . When telomerase is inactivated, Ty1 retrotransposition increases substantially in parallel with telomere erosion and then partially declines when survivors emerge . Retrotransposition is stimulated at the level of Ty1 cDNA synthesis, causing cDNA levels to increase 20-fold or more before survivors form . This response is elicited through a signaling pathway that includes Rad24, Rad17, and Rad9, three components of the DNA-damage checkpoint . Our findings indicate that Ty1 retrotransposons are activated as part of the cellular response to telomere dysfunction.

Genome Res, 2004 Jan, 14(1), 99 - 108 Epub 2003 Dec 12.
Whole-genome discovery of transcription factor binding sites by network-level conservation; Pritsker M et al.; Comprehensive identification of DNA cis-regulatory elements is crucial for a predictive understanding of transcriptional network dynamics . Strong evidence suggests that these DNA sequence motifs are highly conserved between related species, reflecting strong selection on the network of regulatory interactions that underlie common cellular behavior . Here, we exploit a systems-level aspect of this conservation-the network-level topology of these interactions-to map transcription factor (TF) binding sites on a genomic scale . Using network-level conservation as a constraint, our algorithm finds 71% of known TF binding sites in the yeast Saccharomyces cerevisiae, using only 12% of the sequence of a phylogenetic neighbor . Most of the novel predicted motifs show strong features of known TF binding sites, such as functional category and/or expression profile coherence of their corresponding genes . Network-level conservation should provide a powerful constraint for the systematic mapping of TF binding sites in the larger genomes of higher eukaryotes.

J Biol Chem, 2004 Mar 5, 279(10), 9625 - 33 Epub 2003 Dec 11.
The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells; Cook JG et al.; Chromosomal DNA replication requires the recruitment of the six-subunit minichromosome maintenance (Mcm) complex to chromatin through the action of Cdc6 and Cdt1 . Although considerable work has described the functions of Cdc6 and Cdt1 in yeast and biochemical systems, evidence that their mammalian counterparts are subject to distinct regulation suggests the need to further explore the molecular relationships involving Cdc6 and Cdt1 . Here we demonstrate that Cdc6 and Cdt1 are mutually dependent on one another for loading Mcm complexes onto chromatin in mammalian cells . The association of Cdt1 with Mcm2 is regulated by cell growth . Mcm2 prepared from quiescent cells associates very weakly with Cdt1, whereas Mcm2 from serum-stimulated cells associates with Cdt1 much more efficiently . Cdc6, which normally accumulates as cells progress from quiescence into G(1), is capable of inducing the binding of Mcm2 to Cdt1 when ectopically expressed in quiescent cells . We further show that Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region we had previously shown to be essential for Cdc6 function . Cdt1 activity is inhibited by the geminin protein, and we provide evidence that the mechanism of this inhibition involves blocking the binding of Cdt1 to both Mcm2 and Cdc6 . These results identify novel molecular functions for both Cdc6 and geminin in controlling the association of Cdt1 with other components of the replication apparatus and indicate that the association of Cdt1 with the Mcm complex is controlled as cells exit and reenter the cell cycle.

J Mol Biol, 2004 Jan 9, 335(2), 399 - 408
Recognition of 5'-YpG-3' sequences by coupled stacking/hydrogen bonding interactions with amino acid residues; Lamoureux JS et al.; The combined biochemical and structural study of hundreds of protein-DNA complexes has indicated that sequence-specific interactions are mediated by two mechanisms termed direct and indirect readout . Direct readout involves direct interactions between the protein and base-specific atoms exposed in the major and minor grooves of DNA . For indirect readout, the protein recognizes DNA by sensing conformational variations in the structure dependent on nucleotide sequence, typically through interactions with the phosphodiester backbone . Based on our recent structure of Ndt80 bound to DNA in conjunction with a search of the existing PDB database, we propose a new method of sequence-specific recognition that utilizes both direct and indirect readout . In this mode, a single amino acid side-chain recognizes two consecutive base-pairs . The 3'-base is recognized by canonical direct readout, while the 5'-base is recognized through a variation of indirect readout, whereby the conformational flexibility of the particular dinucleotide step, namely a 5'-pyrimidine-purine-3' step, facilitates its recognition by the amino acid via cation-pi interactions . In most cases, this mode of DNA recognition helps explain the sequence specificity of the protein for its target DNA.

BMC Genomics . 2003 Dec 12;4(1):50.
The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function; van Nocker S et al.; BACKGROUND: The WD motif (also known as the Trp-Asp or WD40 motif) is found in a multitude of eukaryotic proteins involved in a variety of cellular processes . Where studied, repeated WD motifs act as a site for protein-protein interaction, and proteins containing WD repeats (WDRs) are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins . In the model plant Arabidopsis thaliana, members of this superfamily are increasingly being recognized as key regulators of plant-specific developmental events . RESULTS: We analyzed the predicted complement of WDR proteins from Arabidopsis, and compared this to those from budding yeast, fruit fly and human to illustrate both conservation and divergence in structure and function . This analysis identified 237 potential Arabidopsis proteins containing four or more recognizable copies of the motif . These were classified into 143 distinct families, 49 of which contained more than one Arabidopsis member . Approximately 113 of these families or individual proteins showed clear homology with WDR proteins from the other eukaryotes analyzed . Where conservation was found, it often extended across all of these organisms, suggesting that many of these proteins are linked to basic cellular mechanisms . The functional characterization of conserved WDR proteins in Arabidopsis reveals that these proteins help adapt basic mechanisms for plant-specific processes . CONCLUSIONS: Our results show that most Arabidopsis WDR proteins are strongly conserved across eukaryotes, including those that have been found to play key roles in plant-specific processes, with diversity in function conferred at least in part by divergence in upstream signaling pathways, downstream regulatory targets and /or structure outside of the WDR regions.

J Biol Chem, 2004 Mar 5, 279(10), 8684 - 93 Epub 2003 Dec 10.
Kruppel-like factor 4 (KLF4) represses histidine decarboxylase gene expression through an upstream Sp1 site and downstream gastrin responsive elements; Ai W et al.; Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion . Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements . In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements . Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter . Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements . In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity . Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression . We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements . Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.

Anal Chem, 2003 Dec 15, 75(24), 6912 - 21
A correlation algorithm for the automated quantitative analysis of shotgun proteomics data; MacCoss MJ et al.; Quantitative shotgun proteomic analyses are facilitated using chemical tags such as ICAT and metabolic labeling strategies with stable isotopes . The rapid high-throughput production of quantitative "shotgun" proteomic data necessitates the development of software to automatically convert mass spectrometry-derived data of peptides into relative protein abundances . We describe a computer program called RelEx, which uses a least-squares regression for the calculation of the peptide ion current ratios from the mass spectrometry-derived ion chromatograms . RelEx is tolerant of poor signal-to-noise data and can automatically discard nonusable chromatograms and outlier ratios . We apply a simple correction for systematic errors that improves the accuracy of the quantitative measurement by 32 +/- 4% . Our automated approach was validated using labeled mixtures composed of known molar ratios and demonstrated in a real sample by measuring the effect of osmotic stress on protein expression in Saccharomyces cerevisiae.

Nature, 2003 Dec 11, 426(6967), 676 - 80
Optimization of specificity in a cellular protein interaction network by negative selection; Zarrinpar A et al.; Most proteins that participate in cellular signalling networks contain modular protein-interaction domains . Multiple versions of such domains are present within a given organism: the yeast proteome, for example, contains 27 different Src homology 3 (SH3) domains . This raises the potential problem of cross-reaction . It is generally thought that isolated domain-ligand pairs lack sufficient information to encode biologically unique interactions, and that specificity is instead encoded by the context in which the interaction pairs are presented . Here we show that an isolated peptide ligand from the yeast protein Pbs2 recognizes its biological partner, the SH3 domain from Sho1, with near-absolute specificity--no other SH3 domain present in the yeast genome cross-reacts with the Pbs2 peptide, in vivo or in vitro . Such high specificity, however, is not observed in a set of non-yeast SH3 domains, and Pbs2 motif variants that cross-react with other SH3 domains confer a fitness defect, indicating that the Pbs2 motif might have been optimized to minimize interaction with competing domains specifically found in yeast . System-wide negative selection is a subtle but powerful evolutionary mechanism to optimize specificity within an interaction network composed of overlapping recognition elements.

Mol Biol Cell, 2004 Mar, 15(3), 1445 - 58 Epub 2003 Dec 10.
Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes; Vasiljev A et al.; Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps . First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex . They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane . In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria . The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped . The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles . Thus, these components are sufficient for translocation of AAC precursor across the outer membrane . Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex . The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

Mol Biol Cell, 2004 Mar, 15(3), 1197 - 210 Epub 2003 Dec 10.
Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton; Richardson SC et al.; In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to "tether" vacuolar membranes before fusion . To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6 . We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells . Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events . Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro . Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis . Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments . These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function.

Mol Biol Cell, 2004 Mar, 15(3), 1470 - 8 Epub 2003 Dec 10.
Sec61p contributes to signal sequence orientation according to the positive-inside rule; Goder V et al.; Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences . They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated . Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the "positive-inside rule." We tested the role of conserved charged residues of Sec61p, the major component of the translocon in Saccharomyces cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis by using diagnostic model proteins . Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences . These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal . For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides . Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.

Genetics, 2003 Nov, 165(3), 1105 - 15
The PHOA and PHOB cyclin-dependent kinases perform an essential function in Aspergillus nidulans; Dou X et al.; Unlike Pho85 of Saccharomyces cerevisiae, the highly related PHOA cyclin-dependent kinase (CDK) of Aspergillus nidulans plays no role in regulation of enzymes involved in phosphorous acquisition but instead modulates differentiation in response to environmental conditions, including limited phosphorous . Like PHO85, Aspergillus phoA is a nonessential gene . However, we find that expression of dominant-negative PHOA inhibits growth, suggesting it may have an essential but redundant function . Supporting this we have identified another cyclin-dependent kinase, PHOB, which is 77% identical to PHOA . Deletion of phoB causes no phenotype, even under phosphorous-limited growth conditions . To investigate the function of phoA/phoB, double mutants were selected from a cross of strains containing null alleles and by generating a temperature-sensitive allele of phoA in a deltaphoB background . Double-deleted ascospores were able to germinate but had a limited capacity for nuclear division, suggesting a cell cycle defect . Longer germination revealed morphological defects . The temperature-sensitive phoA allele caused both nuclear division and polarity defects at restrictive temperature, which could be complemented by expression of mammalian CDK5 . Therefore, an essential function exists in A . nidulans for the Pho85-like kinase pair PHOA and PHOB, which may involve cell cycle control and morphogenesis.

J Biol Chem, 2004 Mar 5, 279(10), 9222 - 32 Epub 2003 Dec 10.
The Plasmodium falciparum PfGatp is an endoplasmic reticulum membrane protein important for the initial step of malarial glycerolipid synthesis; Santiago TC et al.; During its 48-h asexual life cycle within human erythrocytes, Plasmodium falciparum grows to many times its own size and divides to produce 16-32 new parasites . This rapid multiplication requires active synthesis of new membranes and is fueled by phospholipid precursors and fatty acids that are scavenged from the human host . Plasmodium membrane biogenesis relies heavily on the expression of parasite enzymes that incorporate these precursors into phospholipids . However, little is known about the genes involved in membrane biogenesis or where this process takes place within the parasite . Here, we describe the analysis in P . falciparum of the first step of phospholipid biosynthesis that controls acylation of glycerol 3-phosphate (GPAT) at the sn-1 position . We show that this activity is of parasite origin and is specific for glycerol 3-phosphate substrate . We have identified the gene, PfGAT, encoding this activity in P . falciparum and reconstituted its codon composition for optimal expression in the yeast Saccharomyces cerevisiae . PfGAT complements the lethality of a yeast double mutant gat1Deltagat2Delta, lacking GPAT activity . Biochemical analysis revealed that PfGatp is a low affinity GPAT enzyme with a high specificity for C16:0 and C16:1 substrates . PfGatp is an integral membrane protein of the endoplasmic reticulum expressed throughout the intraerythrocytic life cycle of the parasite but induced mainly at the trophozoite stage . This study, which describes the first protozoan GPAT gene, reveals an important role for the endoplasmic reticulum in the initial step of Plasmodium membrane biogenesis.

J Biol Chem, 2004 Feb 27, 279(9), 7378 - 83 Epub 2003 Dec 10.
The prion curing agent guanidinium chloride specifically inhibits ATP hydrolysis by Hsp104; Grimminger V et al.; The molecular chaperone Hsp104 from Saccharomyces cerevisiae dissolves protein aggregates in the cell and is thus of crucial importance for the thermotolerance of yeast . In addition to this disaggregase activity, Hsp104 has a key function in yeast prion propagation, as Hsp104 was found to be essential for the maintenance of the associated phenotypes . In vivo data suggest that Hsp104 function is affected by guanidinium chloride . Adding small amounts of this compound to yeast medium causes curing of the prions: cells lose their prion-related phenotype . Guanidinium chloride was also found to impair heat shock resistance . Here, we present a detailed in vitro analysis showing that guanidinium chloride is an uncompetitive inhibitor of Hsp104 . Micromolar concentrations of this agent reduce the ATPase activity of Hsp104 to approximately 35% of its normal activity . This inhibition is not related to the denaturing properties of this compound, because Hsp104 was not affected by urea . Guanidinium ions selectively bind to the nucleotide-bound, hexameric state of the molecular chaperone . Thus, they increase the affinity of Hsp104 for adenine nucleotides and promote the nucleotide-dependent oligomerization of the chaperone . Our findings strongly suggest that guanidinium chloride causes curing of yeast prions by perturbing the ATPase of Hsp104, which is essential for both prion propagation and thermotolerance.

J Biol Chem, 2004 Feb 27, 279(9), 7427 - 37 Epub 2003 Dec 10.
Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer; Li D et al.; Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR) . A permissive heterodimer (e.g . peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex . In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner . However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells . Here we extended our study of the interrelationship of TR and RXR . We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera . This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers . Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand.

Comput Biol Chem, 2003 Dec, 27(6), 565 - 74
Rival penalized competitive learning (RPCL): a topology-determining algorithm for analyzing gene expression data; Nair TM et al.; DNA arrays have become the immediate choice in the analysis of large-scale expression measurements . Understanding the expression pattern of genes provide functional information on newly identified genes by computational approaches . Gene expression pattern is an indicator of the state of the cell, and abnormal cellular states can be inferred by comparing expression profiles . Since co-regulated genes, and genes involved in a particular pathway, tend to show similar expression patterns, clustering expression patterns has become the natural method of choice to differentiate groups . However, most methods based on cluster analysis suffer from the usual problems (i) dead units, and (ii) the problem of determining the correct number of clusters (k) needed to classify the data . Selecting the k has been an open problem of pattern recognition and statistics for decades . Since clustering reveals similar patterns present in the data, fixing this number strongly influences the quality of the result . While there is no theoretical solution to this problem, the number of clusters can be decided by a heuristic clustering algorithm called rival penalized competitive learning (RPCL) . We present a novel implementation of RPCL that transforms the correct number of clusters problem to the tractable problem of clustering based on the degree of similarity . This is biologically significant since our implementation clusters functionally co-regulated genes and genes that present similar patterns of expression . This new approach reveals potential genes that are co-involved in a biological process . This implementation of the RPCL algorithm is useful in differentiating groups involved in concerted functional regulation and helps to progressively home into patterns, which are closely similar.

J Biol Chem, 2004 Feb 27, 279(9), 8018 - 28 Epub 2003 Dec 09.
Definition of the consensus motif recognized by gamma-adaptin ear domains; Mattera R et al.; The heterotetrameric adaptor complex 1 (AP-1) and the monomeric Golgi-localized, gamma ear-containing, Arf-binding (GGA) proteins are components of clathrin coats associated with the trans-Golgi network and endosomes . The carboxyl-terminal ear domains (or gamma-adaptin ear (GAE) domains) of two gamma-adaptin subunit isoforms of AP-1 and of the GGAs are structurally similar and bind to a common set of accessory proteins . In this study, we have systematically defined a core tetrapeptide motif PsiG(P/D/E)(Psi/L/M) (where Psi is an aromatic residue), which is responsible for the interactions of accessory proteins with GAE domains . The definition of this motif has allowed us to identify novel GAE-binding partners named NECAP and aftiphilin, which also contain clathrin-binding motifs . These findings shed light on the mechanism of accessory protein recruitment to trans-Golgi network and endosomal clathrin coats.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 16006 - 11 Epub 2003 Dec 08.
Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3; Flajolet M et al.; Cell surface receptor membrane localization is strongly dependent on protein-protein interactions often involving regulation by phosphorylation/dephosphorylation of the intracellular domains of membrane proteins . The present study was carried out to identify metabotropic glutamate receptor (mGluR) 3 regulatory binding proteins . Using the yeast two-hybrid technique, we found that the 50-aa C-terminal cytoplasmic tail of mGluR3 interacts specifically with protein phosphatase 2Calpha (PP2Calpha) . This interaction was confirmed by GST pull-down and coimmunoprecipitation assays . mGluR3 interacts with PP2Calpha, beta, gamma, and delta isoforms; however, among the mGluR family only mGluR3 interacted with PP2C . The minimal interacting domain of mGluR3 comprised residues 836-855 . Alignment between mGluR3 and mGluR2, a closely related group II receptor, indicated that this domain is not conserved between the two receptors . The mGluR3 cytoplasmic C-terminal tail contains one phosphorylation site for protein kinase A (Ser-845), but the phosphatase that dephosphorylates this site has not been previously identified . We find that PP2C, but not PP1, PP2A, or PP2B, dephosphorylates the mGluR3 cytoplasmic tail in vitro . The dephosphorylated form of the mGluR3 cytoplasmic tail, but not the equivalent region of mGluR2, inhibited PP2C assayed by using {32P}casein as a substrate . However, phosphorylation of the mGluR3 cytoplasmic tail at Ser-845 inhibits the interaction with PP2C . These results indicate distinct functions for mGluR2 and mGluR3 and suggest a dynamic regulation of mGluR3 by PP2C.

Microbiology, 2003 Dec, 149(Pt 12), 3519 - 30
Mitochondrial-type iron-sulfur cluster biosynthesis genes (IscS and IscU) in the apicomplexan Cryptosporidium parvum; LaGier MJ et al.; Several reports have indicated that the iron-sulfur cluster {Fe-S} assembly machinery in most eukaryotes is confined to the mitochondria and chloroplasts . The best-characterized and most highly conserved {Fe-S} assembly proteins are a pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of {Fe-S} prior to their incorporation into apoproteins . In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available . Primary sequence analysis (CpIscS and CpIscU) and phylogenetic studies (CpIscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms . Moreover, the N-terminal signal sequences of CpIscS and CpIscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae . Overall, these findings suggest that the previously identified mitochondrial relict of C . parvum may have been retained by the parasite as an intracellular site for {Fe-S} assembly.

J Biol Chem, 2004 Feb 27, 279(9), 8159 - 68 Epub 2003 Dec 08.
Ectopic expression of bovine type 5 phosphodiesterase confers a renal phenotype in Drosophila; Broderick KE et al.; cGMP signaling regulates epithelial fluid transport by Drosophila Malpighian (renal) tubules . In order to directly evaluate the importance of cGMP-degrading phosphodiesterases (PDEs) in epithelial transport, bovine PDE5 (a bona fide cGMP-PDE), was ectopically expressed in vivo . Transgenic UAS-PDE5 Drosophila were generated, and PDE5 expression was driven in specified tubule cells in vivo by cell-specific GAL4 drivers . Targeted expression was verified by PCR and Western blotting . Immunolocalization of PDE5 in tubule confirmed specificity of expression and demonstrated localization to the apical plasma membrane . GAL4/UAS-PDE5 tubules exhibit increased cG-PDE activity and reduced basal cGMP levels compared with control lines . We show that wild-type and control tubules are sensitive to the PDE5-specific inhibitor sildenafil and that GAL4/UAS-PDE5 tubules display enhanced sensitivity to sildenafil, compared with controls . cGMP content in GAL4/UAS-PDE5 tubules is restored to control levels by treatment with sildenafil . Thus bovine PDE5 retains cGMP-degrading activity and inhibitor sensitivity when expressed in Drosophila . Expression of PDE5 in tubule principal cells results in an epithelial phenotype, reducing rates of basal and cGMP-/Cardioaccelatory peptide(2b)(CAP(2b))-stimulated fluid transport . Furthermore, inhibition of PDE5 activity by sildenafil restores basal and cGMP-stimulated fluid transport rates to control levels . However, corticotrophin releasing factor-like-stimulated transport, which is activated by cAMP signaling, was unaffected, confirming that only cGMP-stimulated signaling events in tubule are compromised by overexpression of PDE5 . Successful ectopic expression of a vertebrate cG-PDE in Drosophila has shown that cG-PDE has a critical role in tubule function in vivo and that cG-PDE function is conserved across evolution . The transgene also provides a generic tool for the analysis of cGMP signaling in Drosophila.

J Cell Biol, 2003 Dec 8, 163(5), 1111 - 21
ARF6 controls post-endocytic recycling through its downstream exocyst complex effector; Prigent M et al.; The small guanosine triphosphate (GTP)-binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway . Here, we show that GTP-bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane . We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes . In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway . Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading . Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.

J Cell Biol, 2003 Dec 8, 163(5), 937 - 47
Meiotic condensin is required for proper chromosome compaction, SC assembly, and resolution of recombination-dependent chromosome linkages; Yu HG et al.; Condensin is an evolutionarily conserved protein complex that helps mediate chromosome condensation and segregation in mitotic cells . Here, we show that condensin has two activities that contribute to meiotic chromosome condensation in Saccharomyces cerevisiae . One activity, common to mitosis, helps mediate axial length compaction . A second activity promotes chromosome individualization with the help of Red1 and Hop1, two meiotic specific components of axial elements . Like Red1 and Hop1, condensin is also required for efficient homologue pairing and proper processing of double strand breaks . Consistent with these functional links condensin is necessary for proper chromosomal localization of Red1 and Hop1 and the subsequent assembly of the synaptonemal complex . Finally, condensin has a Red1/Hop1-independent role in the resolution of recombination-dependent linkages between homologues in meiosis I . The existence of distinct meiotic activities of condensin (axial compaction, individualization, and resolution of recombination-dependent links) provides an important framework to understand condensin's role in both meiotic and mitotic chromosome structure and function.

Biochemistry, 2003 Dec 16, 42(49), 14349 - 55
"In the beginning": initiation of minus strand DNA synthesis in retroviruses and LTR-containing retrotransposons; Le Grice SF; Sequestering a host-coded tRNA for initiation of minus (-) strand DNA synthesis is central to the reverse transcription cycle of a number of retroviruses and long terminal repeat (LTR) retrotransposons . However, "self-priming" from a hydrolysis product of the viral genome has been observed for the LTR retrotransposon Tf1 and most likely exists for related elements . Furthermore, in contrast to retroviruses, where DNA synthesis is initiated from the 3'-terminus of the cognate tRNA primer, examples are available where nucleotides of the tRNA anticodon domain are complementary to the viral primer binding site (PBS), necessitating internal cleavage of the primer to provide the appropriate 3'-OH for DNA synthesis . Thus, although the ensuing steps of reverse transcription are common to these elements, several variations in which the replication primer is used have been exploited . In addition, the PBS of the viral RNA genome can vary in size from an 11 nt sequence, through a bipartite cis-acting element, to 18 contiguous nucleotides complementary to the 3'-end of the replication primer . These diverse tRNA-viral RNA interactions, and their consequences for initiation of (-) strand DNA synthesis, are the subject of this review.

Nat Biotechnol, 2004 Jan, 22(1), 98 - 103 Epub 2003 Dec 07.
Unraveling protein interaction networks with near-optimal efficiency; Lappe M et al.; The functional characterization of genes and their gene products is the main challenge of the genomic era . Examining interaction information for every gene product is a direct way to assemble the jigsaw puzzle of proteins into a functional map . Here we demonstrate a method in which the information gained from pull-down experiments, in which single proteins act as baits to detect interactions with other proteins, is maximized by using a network-based strategy to select the baits . Because of the scale-free distribution of protein interaction networks, we were able to obtain fast coverage by focusing on highly connected nodes (hubs) first . Unfortunately, locating hubs requires prior global information about the network one is trying to unravel . Here, we present an optimized 'pay-as-you-go' strategy that identifies highly connected nodes using only local information that is collected as successive pull-down experiments are performed . Using this strategy, we estimate that 90% of the human interactome can be covered by 10,000 pull-down experiments, with 50% of the interactions confirmed by reciprocal pull-down experiments.

J Biol Chem, 2004 Feb 27, 279(9), 8343 - 50 Epub 2003 Dec 04.
A novel human nucleolar protein, Nop132, binds to the G proteins, RRAG A/C/D; Sekiguchi T et al.; RRAG A (Rag A)/Gtr1p is a member of the Ras-like small G protein family that genetically interacts with RCC1, a guanine nucleotide exchange factor for RanGTPase . RRAG A/Gtr1p forms a heterodimer with other G proteins, RRAG C and RRAG D/Gtr2p, in a nucleotide-independent manner . To further elucidate the function of RRAG A/Gtr1p, we isolated a protein that interacts with RRAG A . This protein is a novel nucleolar protein, Nop132 . Nop132 is associated with the GTP form, but not the GDP form, of RRAG A, suggesting that RRAG A might regulate Nop132 function . Nop132 is also associated with RRAG C and RRAG D . The Nop132 amino acid sequence is similar to the Saccharomyces cerevisiae nucleolar Nop8p, which is associated with Gtr1p, Gtr2p, and Nip7p . Nop132 also interacts with human Nip7 and is colocalized with RRAG A, RRAG C, and Nip7 . RNA interference knockdown of Nop132 inhibited cell growth of HeLa cells.

J Biol Chem, 2004 Feb 20, 279(8), 6553 - 9 Epub 2003 Dec 02.
Evidence of a transcriptional co-activator function of cohesin STAG/SA/Scc3; Lara-Pezzi E et al.; Cohesins hold sister chromatids together from DNA replication until they are segregated . Although cohesins Smc1, Smc3, and Scc1/Rad21 are involved in chromatid cohesion and other cellular processes, little is known about the other mitotic cohesin subunit, Scc3/STAG . Here we describe STAG/Scc3, which may act as a transcriptional co-activator . STAG2 is able to enhance the activity of the tumor necrosis factor alpha, the CD69, and the human immunodeficiency virus long terminal repeat promoters in a NF-kappaB-dependent manner . In addition, STAG2 interacts with the viral transactivator Tat and enhances the Tat-mediated activation of the human immunodeficiency virus long terminal repeat promoter . Moreover, STAG2 co-activates a multimeric NF-kappaB reporter construct and enhances the activity of the transactivation domain of p65/RelA in a Gal4 system . This function is dependent on one of the LXXLL co-activation motives present in this cohesin and is substantiated by the interaction of STAG2 with the p65 subunit of NF-kappaB . These results describe a novel activity for cohesins, suggesting a role for STAG/Scc3 in transcriptional regulation.

J Biol Chem, 2004 Feb 27, 279(9), 8351 - 8 Epub 2003 Dec 04.
Structure of protein phosphatase methyltransferase 1 (PPM1), a leucine carboxyl methyltransferase involved in the regulation of protein phosphatase 2A activity; Leulliot N et al.; The important role of the serine/threonine protein phosphatase 2A (PP2A) in various cellular processes requires a precise and dynamic regulation of PP2A activity, localization, and substrate specificity . The regulation of the function of PP2A involves the reversible methylation of the COOH group of the C-terminal leucine of the catalytic subunit, which, in turn, controls the enzyme's heteromultimeric composition and confers different protein recognition and substrate specificity . We have determined the structure of PPM1, the yeast methyltransferase responsible for methylation of PP2A . The structure of PPM1 reveals a common S-adenosyl-l-methionine-dependent methyltransferase fold, with several insertions conferring the specific function and substrate recognition . The complexes with the S-adenosyl-l-methionine methyl donor and the S-adenosyl-l-homocysteine product and inhibitor unambiguously revealed the co-substrate binding site and provided a convincing hypothesis for the PP2A C-terminal peptide binding site . The structure of PPM1 in a second crystal form provides clues to the dynamic nature of the PPM1/PP2A interaction.

Appl Environ Microbiol, 2003 Dec, 69(12), 6979 - 86
Improvement of foreign-protein production in Aspergillus niger var . awamori by constitutive induction of the unfolded-protein response; Valkonen M et al.; Unfolded-protein response (UPR) denotes the upregulation of endoplasmic reticulum (ER)-resident chaperone and foldase genes and numerous other genes involved in secretory functions during the accumulation of unfolded proteins into the ER . Overexpression of individual foldases and chaperones has been used in attempts to improve protein production in different production systems . We describe here a novel strategy to improve foreign-protein production . We show that the constitutive induction of the UPR pathway in Aspergillus niger var . awamori can be achieved by expressing the activated form of the transcription factor hacA . This induction enhances the production of Trametes versicolor laccase by up to sevenfold and of bovine preprochymosin by up to 2.8-fold in this biotechnically important fungus . The regulatory range of UPR was studied by analyzing the mRNA levels of novel A . niger var . awamori genes involved in different secretory functions . This revealed both similarities and differences to corresponding studies in Saccharomyces cerevisiae.

Gene, 2003 Dec 24, 323, 133 - 40
The mouse zinc-fingers and homeoboxes (ZHX) family; ZHX2 forms a heterodimer with ZHX3; Kawata H et al.; Human zinc-fingers and homeoboxes (ZHX) 1, ZHX2 and ZHX3, members of the ZHX family, contain two Cys(2)-His(2)-type zinc-finger motifs and five homeodomains (HDs) . These proteins not only form homodimers but heterodimers with ZHX1 as well and act as ubiquitous transcriptional repressors . The cloning of mouse ZHX2 and ZHX3 cDNAs and the corresponding genes from a 129 mouse genomic library are reported, along with an analysis of the heterodimerization of ZHX2 with ZHX3 . The mouse ZHX2 and ZHX3 proteins consist of 836 and 951 amino acid residues, respectively . The similarity of amino acid sequences of each protein with those of human orthologue is 87.0% and 85.2%, respectively . An analysis of genomic clones revealed that an entire coding sequence and a portion of the 5'- and 3'-noncoding sequence of mouse ZHX2 cDNA are encoded by a single exon of the mouse ZHX2 gene as well as the mouse ZHX1 gene . In contrast, in the case of the mouse ZHX3 gene, the coding sequences of ZHX3 cDNA are separated by an intron . A 4.5-kb ZHX2 transcript, and three ZHX3 transcripts, 9.5-, 6.5- and 4.4-kb, are ubiquitously expressed, although their levels vary . Lastly, in vitro and in vivo protein-protein interaction assays revealed that ZHX2 is able to form a heterodimer with ZHX3 via a region containing each HD1.

Gene, 2003 Dec 24, 323, 1 - 10
The telomeric protein Rap1 is conserved in vertebrates and is expressed from a bidirectional promoter positioned between the Rap1 and KARS genes; Tan M et al.; We have identified the chicken homolog of the mammalian telomere protein repression and activation protein 1 (Rap1) . Although cRap1 has only 36% sequence identity to hRap1, it contains the same conserved BRCA1 C-terminal (BRCT), Myb and Rap C-terminus (RCT) domains . Two-hybrid analysis and immunolocalization experiments revealed that cRap1 interacts with the telomere-binding protein telomeric repeat binding factor (TRF)2 and localizes to telomeres . Thus, despite considerable sequence divergence, the identity and overall domain structure of telomere-associated proteins is conserved in vertebrates . Analysis of the cRap1 genomic locus revealed that the cRap1 gene lies immediately adjacent to the cKARS (lysyl-tRNA synthetase) gene with the two genes in a head-to-head orientation separated by only 57 nt . This same organization is conserved at the human Rap1-KARS locus . When 5' regions of the cRap1 and cKARS genes were tested for promoter activity, the promoters of both genes were found to lie in or near the intergenic spacer . The two promoters lack TATA boxes but appear to have downstream promoter elements (DPEs) . Analysis of human Rap1 and KARS expressed sequence tags (ESTs) indicated that this localization of TATA-less promoters to the intergenic spacer is a conserved feature of the Rap1-KARS locus.

J Mol Biol, 2004 Jan 2, 335(1), 57 - 70
A distal, high-affinity binding site on the cyclin-CDK substrate Pho4 is important for its phosphorylation and regulation; Byrne M et al.; Cyclins and cyclin-dependent kinases (CDKs) are key components of signaling pathways essential for cell growth and survival . The cyclin-CDK Pho80-Pho85 inactivates the transcription factor Pho4 in budding yeast by phosphorylating it on five sites . We isolated seven single amino acid substitutions outside of the phosphorylation sites that cause Pho4 to be constitutively active . The substitutions decrease the amount of Pho4 phosphorylation in vivo, and they increase the apparent K(M) of the in vitro phosphorylation reaction by an order of magnitude but do not alter k(cat) substantially . These data suggest that the substituted residues are part of a cyclin-CDK-binding site that is distal to the phosphorylation sites . Further analysis revealed that all of Pho4 variants were phosphorylated by Pho80-Pho85 in a more distributive manner than the wild-type protein, further supporting the idea that binding at a distal, high-affinity binding site is important in determining the processivity of Pho4 phosphorylation . In addition, computational modeling of the Pho4 phosphorylation reactions shows that the K(D) of binding between the Pho4 mutants and Pho80-Pho85 increases, confirming that the mutations are located in a relatively high-affinity "docking site" for the kinase . Interestingly, the K(D) derived from the in vitro data correlates well with the strength of the in vivo phenotypes, demonstrating that the in vitro data are relevant to the in vivo regulation of Pho4.

Trends Biochem Sci, 2003 Dec, 28(12), 655 - 62
TPR proteins: the versatile helix; D'Andrea LD et al.; Tetratrico peptide repeat (TPR) proteins have several interesting properties, including their folding characteristics, modular architecture and range of binding specificities . In the past five years, many 3D structures of TPR domains have been solved, revealing at a molecular level the versatility of this basic fold . Here, we discuss the structure of TPRs and highlight the diversity of arrangements and functions that are associated with these ubiquitous domains . Genomic analyses of the distribution of TPR domains are presented along with implications for protein engineering.

Carbohydr Res, 2004 Jan 2, 339(1), 29 - 35
Synthesis of two oligosaccharides, the GPI anchor glycans from S . cerevesiae and A . fumigatus; Ma Z et al.; Two oligosaccharides, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)-alpha-D-Manp-(1-->4)-alpha-D-GlcpNAc (I) and alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)-alpha-D-Manp-(1-->4)-alpha-D-GlcpNAc (II), the glycosylphosphatidylinositol (GPI) anchor glycans from S . cerevesiae and A . fumigatus were synthesized as their methyl glycosides in a regio- and stereoselective manner . The pentasaccharide I was obtained from 6-O-selective glycosylation of methyl 2,3-di-O-benzoyl-alpha-D-mannopyranosyl-(1-->4)-2-acetamido-3,6-di-O-benzoyl-2-deoxy-alpha-D-glucopyranoside (8) with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (9), followed by benzoylation, deacetylation, and mannosylation, and then by deprotection . The hexasaccharide (II) was obtained via condensation of allyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (17) with 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (16), followed by deallylation, trichloroacetimidation, and coupling with acceptor (8), and finally by deprotection.

Genome Biol . 2003;4(12):240 . Epub 2003 Nov 18.
Localizing the proteome; Simpson JC et al.; The subcellular localization of the entire proteome of an organism, the yeast Saccharomyces cerevisiae, has been revealed for the first time . Comparison with less comprehensive studies of mammalian cells provides insights into the localization of the mammalian proteome.

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