J Mol Biol, 2000 Mar 3, 296(4), 1105 - 16 Preformed secondary structure drives the association reaction of GCN4-p1, a model coiled-coil system; Zitzewitz JA et al.; The structure of the transition state for the rate-limiting step in the folding and association of the homodimeric coiled-coil peptide GCN4-p1, was probed by mutational analysis . A series of quadruple amino acid replacements that spanned the helix propensity scale were made at the four external f positions in the heptad repeat . Equilibrium and kinetic circular dichroism studies demonstrate that both the stability and the unfolding and refolding rate constants vary with helix propensity but also reflect interactions of the altered side-chains with their local environments . Pairwise replacements and fragment studies show that the two C-terminal heptads are the likely source of the nucleating helices . Helix-helix recognition between preformed elements of secondary structure plays an important role in this fundamental folding reaction . Z Naturforsch {C}, 1999 Dec, 54(12), 1084 - 8 Carboxymethylated glucan inhibits lipid peroxidation in liposomes; Babincova M et al.; Protective capabilities were studied of carboxymethylated (1-->3)-beta-D-glucan from Saccharomyces cerevisiae cell wall against lipid peroxidation in phosphatidylcholine liposomes induced by OH radicals produced with Fenton's reagent (H2O2/Fe2+) and also by microwave radiation using absorption UV-VIS spectrophotometry . A significant decrease in the conjugated diene production, quantified as Klein oxidation index, was observed in the presence of a moderate amount of added glucan . Increase of the oxidation index was accompanied with enhanced carboxyfluorescein leakage as a result of liposome membrane destabilization . This process was markedly suppressed with glucan present in the liposome suspension . Therefore, glucan may be considered as a potent protector against microwave radiation-induced cell damage. Biochemistry, 2000 Feb 15, 39(6), 1234 - 42 Structural and mechanistic basis for the activation of a low-molecular weight protein tyrosine phosphatase by adenine; Wang S et al.; Although the activation of low-molecular weight protein tyrosine phosphatases by certain purines and purine derivatives was first described three decades ago, the mechanism of this rate enhancement was unknown . As an example, adenine activates the yeast low-molecular weight protein tyrosine phosphatase LTP1 more than 30-fold . To examine the structural and mechanistic basis of this phenomenon, we have determined the crystal structure of yeast LTP1 complexed with adenine . In the crystal structure, an adenine molecule is found bound in the active site cavity, sandwiched between the side chains of two large hydrophobic residues at the active site . Hydrogen bonding to the side chains of other active site residues, as well as some water-mediated hydrogen bonds, also helps to fix the position of the bound adenine molecule . An ordered water was found in proximity to the bound phosphate ion present in the active site, held by hydrogen bonding to N3 of adenine and Odelta1 of Asp-132 . On the basis of the crystal structure, we propose that this water molecule is the nucleophile that participates in the dephosphorylation of the phosphoenzyme intermediate . Solvent isotope effect studies show that there is no rate-determining transfer of a solvent-derived proton in the transition state for the dephosphorylation of the phosphoenzyme intermediate . Such an absence of general base catalysis of water attack is consistent with the stability of the leaving group, namely, the thiolate anion of Cys-13 . Consequently, adenine activates the enzyme by binding and orienting a water nucleophile in proximity to the phosphoryl group of the phosphoenzyme intermediate, thus increasing the rate of the dephosphorylation step, a step that is normally the rate-limiting step of this enzymatic reaction. Biochemistry, 2000 Feb 15, 39(6), 1223 - 33 Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage; West KL et al.; A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex . The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD . We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium . In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis . It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations . The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s) . These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue . A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction. Biopolymers, 2000 Apr 5, 53(4), 293 - 307 Osmolyte-induced changes in protein conformational equilibria; Saunders AJ et al.; Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability . Interpreting the molecular details governing the solute-induced effects, however, remains controversial . We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c . Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein . The stabilities of the A and N states increase linearly with osmolyte concentration . Interestingly, osmolytes stabilize the A state to a greater degree than the N state . To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions . We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data . We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state . The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria . Nucleic Acids Res, 2000 Mar 15, 28(6), 1407 - 17 A new double-stranded RNA-binding protein that interacts with PKR; Coolidge CJ et al.; We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein . p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA . Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members . Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas hetero-dimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity . We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R) . While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression . These transformants could be rescued by overexpression of the alpha-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2alpha), one of the known substrates for PKR . We hypothesize that competing heterodimers between p74-K296R PKR and eIF2alpha-K296R PKR may control cell growth such that stabilization of the p74-K296R PKR heterodimer induces abnormal morphology and cell death. Nucleic Acids Res, 2000 Mar 15, 28(6), 1365 - 73 Assembly of archaeal signal recognition particle from recombinant components; Bhuiyan SH et al.; Signal recognition particle (SRP) takes part in protein targeting and secretion in all organisms . Searches for components of archaeal SRP in primary databases and completed genomes indicated that archaea possess only homologs of SRP RNA, and proteins SRP19 and SRP54 . A recombinant SRP was assembled from cloned, expressed and purified components of the hyperthermophilic archaeon Archaeoglobus fulgidus . Recombinant Af-SRP54 associated with the signal peptide of bovine pre-prolactin translated in vitro . As in mammalian SRP, Af-SRP54 binding to Af-SRP RNA required protein Af-SRP19, although notable amounts bound in absence of Af-SRP19 . Archaeoglobus fulgidus SRP proteins also bound to full-length SRP RNA of the archaeon Methanococcus jannaschii, to eukaryotic human SRP RNA, and to truncated versions which corresponded to the large domain of SRP . Dependence on SRP19 was most pronounced with components from the same species . Reconstitutions with heterologous components revealed a significant potential of human SRP proteins to bind to archaeal SRP RNAs . Surprisingly, M.jannaschii SRP RNA bound to human SRP54M quantitatively in the absence of SRP19 . This is the first report of reconstitution of an archaeal SRP from recombinantly expressed purified components . The results highlight structural and functional conservation of SRP assembly between archaea and eucarya. IUBMB Life, 1999 Dec, 48(6), 593 - 9 5' to 3' single strand DNA exonuclease activity in a preparation of human Ku protein; Morozov VE et al.; We describe a novel 5' to 3' single-strand exonuclease activity exhibited by a Ku preparation purified from a human cell line . The enzyme removes 5' single-strand extensions from duplex DNA molecules . The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase . The exonuclease activity does not require divalent cations . The potential implications of the exonuclease activity findings for repair of double-strand breaks and recombination processes are discussed. FEBS Lett, 2000 Feb 18, 468(1), 101 - 4 Structure of the cytosolic domain of TOM5, a mitochondrial import protein; Hammen PK et al.; TOM5 is a small outer mitochondrial membrane protein in Saccharomyces cerevisiae and is part of a multi-protein translocator complex, which mediates protein import into mitochondria . Presently, nothing is known about the conformational preferences of TOM5 or other mitochondrial import proteins . In this report, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy are used to determine the conformational preferences of the cytosolic domain of TOM5 . The CD spectra show evidence of a helical structure that is invariant with pH . NOESY data revealed that TOM5 forms a stable helical core between E11 and R15 with a less structurally rigid helix extending to the C-terminus. Arch Biochem Biophys, 2000 Mar 1, 375(1), 138 - 44 Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein; Shingledecker K et al.; Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein . The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions . Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration . This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups . The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates . Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions . The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8 . This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group . Comparison of the rates of cleavage of the linear ester intermediates derived from the M . tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins . FEBS Lett, 2000 Jan 28, 466(2-3), 239 - 43 cDNA-derived amino acid sequence of acetoacetyl-CoA synthetase from rat liver; Iwahori A et al.; In order to examine the primary structure of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 6.2.1.16; AA-CoA synthetase), the cDNA clone encoding this enzyme has been isolated from the cDNA library which was prepared from the liver of rat fed a diet supplemented with 4% cholestyramine and 0.4% pravastatin for 4 days . Nucleotide sequence analysis of cloned cDNA revealed that AA-CoA synthetase of rat liver contains an open reading frame of 2019 nucleotides, and the deduced amino acid sequence (672 amino acid residues) bears 25.0 and 38.9% homologies with acetyl-CoA synthetases of Saccharomyces cerevisiae and Archaeoglobus fulgidus, respectively. J Biol Chem, 2000 Feb 25, 275(8), 6038 - 44 Metalloproteolytic release of endothelial cell protein C receptor; Xu J et al.; Previous studies observed that there is about 100 ng/ml soluble endothelial cell protein C receptor (EPCR) in human plasma and that the levels increase in inflammatory diseases . In this study we examine the potential mechanisms involved in release of EPCR from cells . We find that EPCR is released from the surface of endothelium and transfected 293 cells by a metalloprotease in a constitutive fashion . The mass of soluble EPCR is 4 kDa less than intact EPCR . Release is blocked by either the hydroxamic acid based inhibitor, KD-IX-73-4 or by 1,10-phenanthroline, but not by matrix metalloprotease inhibitors . Release is stimulated by phorbol 12-myristate 13-acetate, thrombin, interleukin-1beta, and hydrogen peroxide . Stimulation with these agents reduces EPCR expression levels sufficiently to decrease the rate of protein C activation to a limited extent . The influence of phorbol 12-myristate 13-acetate on both EPCR release and inhibition of protein C activation are enhanced by microtubule disruption with nocodazole . EPCR release is augmented by transfection of EPCR expressing 293 cells with caveolin, suggesting that release is caveolae dependent . These studies indicate that metalloproteolytic release of EPCR is a highly regulated process that is sensitive to both coagulation factors and inflammatory mediators. J Biol Chem, 2000 Feb 25, 275(8), 5904 - 10 Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures; Tatsumi Y et al.; An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae . Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells . It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle . A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei . Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA . Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase . Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes. J Biol Chem, 2000 Feb 25, 275(8), 5874 - 9 Dissecting the interactions between NTF2, RanGDP, and the nucleoporin XFXFG repeats; Chaillan-Huntington C et al.; We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial for nuclear trafficking . Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the interaction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 microM range . Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP . These results indicate that, instead, Ran increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nuclear pore-associated Ran. J Biol Chem, 2000 Feb 25, 275(8), 5767 - 72 The assembly factor Atp11p binds to the beta-subunit of the mitochondrial F(1)-ATPase; Wang ZG et al.; Atp11p is a protein of Saccharomyces cerevisiae required for the assembly of the F(1) component of the mitochondrial F(1)F(0)-ATP synthase . This study presents evidence that Atp11p binds selectively to the beta-subunit of F(1) . Under conditions in which avidin-Sepharose beads specifically adsorbed biotinylated Atp11p from yeast mitochondrial extracts, the F(1) beta-subunit coprecipitated with the tagged Atp11p protein . Binding interactions between Atp11p and the entire beta-subunit of F(1) or fragments of the beta-subunit were also revealed by a yeast two-hybrid screen: Atp11p bound to a region of the nucleotide-binding domain of the beta-subunit located between Gly(114) and Leu(318) . Certain elements of this sequence that would be accessible to Atp11p in the free beta-subunit make contact with adjacent alpha-subunits in the assembled enzyme . This observation suggests that the alpha-subunits may exchange for bound Atp11p during the process of F(1) assembly. Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 1988 - 92 An artificial transcriptional activating region with unusual properties; Lu X et al.; We describe a series of transcriptional activators generated by adding amino acids (eight in one case, six in another) to fragments of the yeast Saccharomyces cerevisiae activator Gal4 that dimerize and bind DNA . One of the novel activating regions identified by this procedure is unusual, compared with previously characterized yeast activating regions, in the following ways: it works more strongly than does Gal4's natural activating region as assayed in yeast; it is devoid of acidic residues; and several lines of evidence suggest that it sees targets in the yeast transcriptional machinery at least partially distinct from those seen by Gal4's activating region. Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2011 - 6 Anatomy of a proficient enzyme: the structure of orotidine 5'-monophosphate decarboxylase in the presence and absence of a potential transition state analog; Miller BG et al.; Orotidine 5'-phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme . The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5'-phosphate, at a resolution of 2.1 A and 2.4 A, respectively . Orotidine 5'-phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel . The binding of 6-hydroxyuridine 5'-phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring . Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product . In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state . Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation. J Mass Spectrom, 2000 Feb, 35(2), 258 - 64 Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry; Koomen JM et al.; Laser desorption/ionization mass spectrometry (LDI-MS) has been used to assess the potential of using surrogate markers, bound to cellular structures containing nucleic acids, to image or map the position of these structures within biological samples . In this study, organic dyes were used as markers because of their established use in the histochemical marking of nucleic acids, and also because they are amenable to LDI-MS . Eight cationic dyes were tested and all could be desorbed from nucleic acid samples without additional matrix after specifically binding to these molecules . Methylene Blue was the best of these based on its sensitivity to detection by LDI-MS and the fact that it can be washed from the tissue in areas where it was not specifically bound to provide low-intensity background signals . Experiments are reported which characterize the M(+) ion signal obtained from Methylene Blue with regard to sensitivity, reproducibility and possible use for quantitation . This dye was used to map (with a lateral resolution of 25 microm) several nucleic acid-containing samples spotted on prepared surfaces, and to image the location of nucleic acids in two model tissues, retinal vertical sections and thyroid whole mount sections . Curr Biol, 2000 Feb 10, 10(3), 165 - 8 Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks; Teo SH et al.; DNA ligases catalyse the joining of DNA single- and double-strand breaks . Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair . The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair {1} {2} {3} {4} . Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 {5} . This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability . We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p . Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation . Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed . We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks . Furthermore, this targeting requires another key NHEJ protein, Ku. Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 704 - 10 The human PEX3 gene encoding a peroxisomal assembly protein: genomic organization, positional mapping, and mutation analysis in candidate phenotypes; Muntau AC et al.; In yeasts, the peroxin Pex3p was identified as a peroxisomal integral membrane protein that presumably plays a role in the early steps of peroxisomal assembly . In humans, defects of peroxins cause peroxisomal biogenesis disorders such as Zellweger syndrome . We previously reported data on the human PEX3 cDNA and its protein, which in addition to the peroxisomal targeting sequence contains a putative endoplasmic reticulum targeting signal . Here we report the genomic organization, sequencing of the putative promoter region, chromosomal localization, and physical mapping of the human PEX3 gene . The gene is composed of 12 exons and 11 introns spanning a region of approximately 40 kb . The highly conserved putative promoter region is very GC rich, lacks typical TATA and CCAAT boxes, and contains potential Sp1, AP1, and AP2 binding sites . The gene was localized to chromosome 6q23-24 and D6S279 was identified to be the closest positional marker . As yeast mutants deficient in PEX3 have been shown to lack peroxisomes as well as any peroxisomal remnant structures, human PEX3 is a candidate gene for peroxisomal assembly disorders . Mutation analysis of the human PEX3 gene was therefore performed in fibroblasts from patients suffering from peroxisome biogenesis disorders . Complementation groups 1, 4, 7, 8, and 9 according to the numbering system of Kennedy Krieger Institute were analyzed but no difference to the wild-type sequence was detected . PEX3 mutations were therefore excluded as the molecular basis of the peroxisomal defect in these complementation groups . Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 530 - 4 Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated by the cyclin A-Cdk2 complex; Ohtoshi A et al.; The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase . The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C . By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC) . Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells . The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A . In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase . These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A . Curr Opin Genet Dev, 2000 Feb, 10(1), 26 - 31 Complexity in the spindle checkpoint; Burke DJ; Cell viability requires accurate chromosome segregation at mitosis . The spindle checkpoint ensures that anaphase is not attempted until the sister chromatids of each chromosome are attached to spindle microtubules from opposite poles . The checkpoint mechanism involves a signal transduction cascade that is more complex than was originally envisioned. Cell, 2000 Feb 4, 100(3), 333 - 43 Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel; Song W et al.; Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR) . Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex . We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis . In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked . Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate . These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54. EMBO J, 2000 Feb 15, 19(4), 683 - 90 The novel coactivator C1 (HCF) coordinates multiprotein enhancer formation and mediates transcription activation by GABP; Vogel JL et al.; Transcription of the herpes simplex virus 1 (HSV-1) immediate early (IE) genes is determined by multiprotein enhancer complexes . The core enhancer assembly requires the interactions of the POU-homeodomain protein Oct-1, the viral transactivator alphaTIF and the cellular factor C1 (HCF) . In this context, the C1 factor interacts with each protein to assemble the stable enhancer complex . In addition, the IE enhancer cores contain adjacent binding sites for other cellular transcription factors such as Sp1 and GA-binding protein (GABP) . In this study, a direct interaction of the C1 factor with GABP is demonstrated, defining the C1 factor as the critical coordinator of the enhancer complex assembly . In addition, mutations that reduce the GABP transactivation potential also impair the C1-GABP interaction, indicating that the C1 factor functions as a novel coactivator of GABP-mediated transcription . The interaction and coordinated assembly of the enhancer proteins by the C1 factor may be critical for the regulation of the HSV lytic-latent cycle. EMBO J, 2000 Feb 15, 19(4), 672 - 82 A single point mutation in TFIIA suppresses NC2 requirement in vivo; Xie J et al.; Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB . Here we study molecular mechanisms of repression by human NC2 in vivo in yeast . Yeast NC2 genes are essential and can be exchanged with human NC2 . The physiologically relevant regions of NC2 have been determined and shown to match the histone-fold dimerization motif . A suppressor screen based upon limiting concentrations of NC2beta yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1 . The single point mutation in Toa1 alleviates the requirement for both subunits of NC2 . Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP-TFIIA-DNA complexes . Wild-type but not the mt-Toa1 can relieve NC2 effects in purified transcription systems . These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes. EMBO J, 2000 Feb 15, 19(4), 581 - 8 Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan processing and endoplasmic reticulum quality control; Vallee F et al.; Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins . The crystal structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion . The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases. FEBS Lett, 2000 Feb 11, 467(2-3), 359 - 64 The polyomavirus major capsid protein VP1 interacts with the nuclear matrix regulatory protein YY1; Palkova Z et al.; Polyomavirus reaches the nucleus in a still encapsidated form, and the viral genome is readily found in association with the nuclear matrix . This association is thought to be essential for viral replication . In order to identify the protein(s) involved in the virus-nuclear matrix interaction, we focused on the possible roles exerted by the multifunctional cellular nuclear matrix protein Yin Yang 1 (YY1) and by the viral major capsid protein VP1 . In the present work we report on the in vivo association between YY1 and VP1 . Using the yeast two-hybrid system we demonstrate that the VP1 and YY1 proteins physically interact through the D-E region of VP1 and the activation domain of YY1. FEBS Lett, 2000 Feb 11, 467(2-3), 348 - 55 A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch; Kaye FJ et al.; We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein . Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Delta rad23Delta Saccharomyces cerevisiae mutants . Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts . While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop . These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events. FEBS Lett, 2000 Feb 11, 467(2-3), 263 - 7 Expression of fatty acid-CoA ligase 4 during development and in brain; Cao Y et al.; Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters . We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production . We report here the expression of this gene in tissues, particularly in different parts of the brain . We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain . FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo . In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum. J Pept Sci, 2000 Jan, 6(1), 36 - 46 Synthesis of bivalent inhibitors of eucaryotic proteasomes; Loidl G et al.; Based on the peculiar spatial array of the active sites in the internal chamber of the multicatalytic proteasome, as derived from the X-ray structure of yeast proteasome, homo- and heterobivalent inhibitors were designed and synthesized to exploit the principle of multivalency for enhancing inhibition potency . Peptidic bis-aldehyde compounds of the octapeptide size were synthesized to address adjacent active sites, whilst a PEG spacer with a statistical length distribution of 19-25 monomers was used to link two identical or different tripeptide aldehydes as binding heads . These bis-aldehyde compounds were synthesized applying both methods in solution and solid phase peptide synthesis . Bivalent binding was observed only for the PEG-spaced inhibitors suggesting that binding from the primed side prevents hemiacetal formation with the active site threonine residue. Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 692 - 6 Interferon-alpha signaling promotes nucleus-to-cytoplasmic redistribution of p95Vav, and formation of a multisubunit complex involving Vav, Ku80, and Tyk2; Adam L et al.; Interferons (IFNs) are a family of hormone-like secretory proteins with multiple phenotypical changes, including gene expression and morphological alterations . Earlier studies have shown that IFN-activated Tyk2 kinase physical associates with p95Vav (Vav), a proto-oncogene gene product expressed in hematopoietic cells . Since Tyk2 is a cytoplasmic kinase and Vav is believed to be localized in the nuclear compartment, here we explored the possibility of Vav redistribution in IFN-alpha-activated cells, using the U266 human myeloma cell line as a model system . Using biochemical assays and in situ confocal microscopy, we demonstrate that IFN-alpha treatment triggers a rapid (10 min) translocation of Vav from the nuclear compartment to the cytoplasm . In addition, we also show the existence of IFN-alpha-induced physical interaction between Vav and Ku80, Ku80, and Tyk2, and among Vav, Ku80, and Tyk2 in the cytoplasmic compartment of IFN-stimulated cells . The observed IFN-alpha-induced association among Vav, Ku80, and Tyk2 was dependent on cellular tyrosine kinase activity . Since recently Vav has been shown to promote the GDP/GTP exchange activity of the cytoskeleton signaling molecule small GTPase Rac1 and activates its downstream signaling, our present findings raise the possibility of involvement of the small GTPase in IFN signaling leading to its biological effects, including cytoskeleton reorganization . Exp Parasitol, 2000 Feb, 94(2), 75 - 83 Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme; Brady CP et al.; Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae . The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases . However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2 . The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6 . 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35 . Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes . Additionally, both enzymes were observed in the excretory/secretory products of adult worms . The RT-PCR analysis indicated that neither enzyme is expressed in S . mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s) . Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form . Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles . Int Rev Immunol, 1999, 18(5-6), 429 - 48 Ribosomal proteins in cell proliferation and apoptosis; Chen FW et al.; Ribosomal proteins have the complex task of coordinating protein biosynthesis to maintain cell homeostasis and survival . Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis . A number of these proteins function as cell proliferation regulators and in some instances as inducers of cell death . Specifically, expression of human ribosomal protein L13a has been shown to induce apoptosis, presumably by arresting cell growth in the G2/M phase of the cell cycle . In addition, inhibition of expression of L13a induces apoptosis in target cells, suggesting that this protein is necessary for cell survival . Similar results have been obtained in the yeast Saccharomyces cerevisiae, where inactivation of the yeast homologues of L13a, rp22 and rp23, by homologous recombination results in severe growth retardation and death . In addition, a closely related ribosomal protein, L7, arrests cells in G1 and also induces apoptosis . Thus, it appears that a group of ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators. Acta Virol, 1999 Feb, 43(1), 5 - 18 The "small" polydisperse cytoplasmic extrachromosomal DNA of chicken leukaemic myeloblasts and the avian myeloblastosis virus core-bound DNA seem to descend from origin regions of chromosomal DNA replication; Pajer P et al.; Nucleotide sequences are presented for 12, 7 and 12 cloned extrachromosomal DNAs by nature harbored in nucleoprotein (NP) complexes forming chicken leukaemic myeloblast (CHLM) post microsomal sediment (POMS) components A, B and C, respectively, and for 11 cloned avian myeloblastosis virus (AMV) DNAs . Analysis of the abundance of sequence motifs significant for eukaryotic chromosomal DNA replication origin (ori) regions (and their initiation zones) has shown that these DNAs are reminiscent of cell DNA fragments enriched in ori sequences (Rao et al., 1990) and/or sequence features of several eukaryotic chromosomal oris containing clusters of modular sequence elements (Dobbs et al., 1994) . Accordingly, these DNAs, with an (A + T) content prevalently higher than that of the total cell DNA, revealed the presence of asymmetrically distributed (A + T)-rich stretches, scaffold attachment region (SAR) T consensuses, polypyrimidine nucleotide (poly(Py)) tracts and minimal Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, in abundance comparable with that of these sequences of DNA fragments enriched in oris . All these DNAs were found to be enriched also in sequence elements held as primase (Pr) attachment sites . Moreover, DNAs of POMS component B and those of AMV DNA were found to be enriched in the asymmetric pyrimidine (Py) heptanucleotide motif of Waltz et al . (1996) occurring in the initiation zones of ori region . Consequently, these extrachromosomal DNAs, portion of which represents a precursor of AMV DNA, seem to descent from initiation zones of various ori regions of an early replicating chromosomal myeloblast DNA . In addition, a possible explanation of the inclination of these DNAs to form multimers is presented. Eur J Biochem, 2000 Feb, 267(4), 1008 - 18 High resolution solution structure of the protein part of Cu7 metallothionein; Bertini I et al.; The three-dimensional solution structure of the protein part of Cu7 metallothionein (Cu7MT) of Saccharomyces cerevisiae has been attempted by 1H two-dimensional NMR spectroscopy at 800 MHz . The protein part constitutes 53 amino acids . A total of 1192 NOEs, of which 1048 are meaningful, were used to determine the solution structure of the first 40 residues, the last 13 residues being disordered . A family of 30 structures was generated . Root-mean-square deviation (rmsd) values from the average structure of 0.32 +/- 0.13 A and 0.61 +/- 0.15 A for backbone and all heavy atoms, respectively, were obtained for the residues 2-40 . The ten copper-coordinating cysteine sulfurs and the empty spaces around them are well defined . The structure of the protein part is similar but not identical to the available ones of the same holoprotein and of the Ag7 metallothionein, and is qualitatively superior . If the same metal-sulfur connectivities reported in the literature from 1H-109Ag heteronuclear multiple quantum coherence spectroscopy are assumed to hold for the present copper derivative, a peptide structure is obtained which is again similar, but still not identical, within indetermination, to that available . The structure of the copper polymetallic center may well be different from that proposed for the silver derivative, and indeed a number of different arrangements of the seven copper ions are consistent with the present highly refined structure of the protein part. J Biol Chem, 2000 Feb 18, 275(7), 4897 - 905 Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha; Yuasa K et al.; cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways . To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Ialpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein) . Although the N-terminal region (amino acids 1-66) of cGK-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, cGK-II, or cAMP-dependent protein kinase . GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids . Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42 . Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42 . Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo . Finally, we demonstrated that the kinase-deficient mutant of cGK-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42 . These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42. J Biol Chem, 2000 Feb 18, 275(7), 4827 - 33 ERO1-L, a human protein that favors disulfide bond formation in the endoplasmic reticulum; Cabibbo A et al.; Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins . Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding . When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans . In isolated microsomes, ERO1-L behaves as a type II integral membrane protein . ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y . ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated . These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells. J Biol Chem, 2000 Feb 18, 275(7), 4571 - 8 Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2; Hell K et al.; We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex . Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein . The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase . Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase . All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p . Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex . We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process. Mol Cell Biol, 2000 Mar, 20(5), 1772 - 83 Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics; Vartiainen M et al.; In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin . Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase . In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein . However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro . A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen . In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures . A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively . Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm . Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1. Mol Cell Biol, 2000 Mar, 20(5), 1537 - 45 Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation; Becker E et al.; The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module . NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck . To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR) . EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems . In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2 . EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1 . Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein . Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck . We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1 . Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. J Mol Biol, 2000 Feb 18, 296(2), 421 - 33 Extensive central disruption of a four-way junction on binding CCE1 resolving enzyme; Declais AC et al.; Junction-resolving enzymes are nucleases that are selective for the structure of the four-way DNA junction that is important in genetic recombination . They exhibit selectivity for the structure of the junction, but they also manipulate the structure . Local disruption of DNA structure around the centre of the junction by CCE1 of Saccharomyces cerevisiae has been investigated using 2-aminopurine fluorescence . On binding CCE1, 2-aminopurine bases located at the point of strand exchange exhibit a large increase in fluorescence intensity (up to 39-fold enhancement), consistent with complete unstacking . This was observed for all positions around the centre of the junction, both 5' and 3' to the point of strand exchange . Thymine bases complementary to the modified adenine bases adjacent to the junction centre were strongly reactive to potassium permanganate . The results indicate that binding of CCE1 results in a complete unpairing of the four central base-pairs of the junction, with a lesser disruption of the next base-pairs . DNA Cell Biol, 2000 Jan, 19(1), 9 - 18 C/EBPalpha is required to maintain postmitotic growth arrest in adipocytes; Tao H et al.; Terminal differentiation is often coupled with irreversible loss of proliferative potential . The CCAAT enhancer binding protein alpha (C/EBPalpha) preferentially accumulates in postmitotic, differentiated 3T3-L1 adipocytes but declines during tumor necrosis factor alpha (TNFalpha)-induced dedifferentiation . We have discovered that this decline in C/EBPalpha correlates with an increased mitotic growth potential . In order to further investigate the antimitotic activity of C/EBPalpha, we introduced antisense C/EBPalpha RNA into 3T3-L1 cells to block endogenous C/EBPalpha expression . When treated according to the standard differentiation protocol, stable cells lines harboring antisense C/EBPalpha RNA did not differentiate into fat-laden adipocytes, consistent with previous findings (Lin F, Lane MD, Genes Dev 1992;6:533-544) . We found that these undifferentiated cells expressing antisense-C/EBPalpha can reenter the cell cycle after mitogenic stimulation at a time in development when parental 3T3-L1 cells cannot . Moreover, the expression profiles of the growth-arrest-associated genes gas1 and gas2 revealed that the antisense C/EBPalpha-expressing cells withdrew from the cell cycle after the period of clonal expansion but failed to progress to the state of least proliferative potential characteristic of terminally differentiated adipocytes. Nature, 2000 Jan 27, 403(6768), 451 - 6 DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination {corrected}; Mol CD et al.; Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases . The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis . Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand . APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites . Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism . Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product . These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair. Cancer Res, 2000 Jan 15, 60(2), 238 - 41 Nickel compounds are novel inhibitors of histone H4 acetylation; Broday L et al.; Environmental factors influence carcinogenesis by interfering with a variety of cellular targets . Carcinogenic nickel compounds, although generally inactive in most gene mutation assays, induce chromosomal damage in heterochromatic regions and cause silencing of reporter genes when they are located near telomere or heterochromatin in either yeast or mammalian cells . We studied the effects of nickel on the lysine acetylation status of the NH2-terminal region of histone H4 . At nontoxic levels, nickel decreased the levels of histone H4 acetylation in vivo in both yeast and mammalian cells, affecting only lysine 12 in mammalian cells and all of the four lysine residues in yeast . In yeast, lysine 12 and 16 were more greatly affected than lysine 5 and 8 . Interestingly, a histidine Ni2+ anchoring site is found at position 18 from the NH2-terminal tail of H4 . Nickel was also found to inhibit the acetylation of H4 in vitro using purified recombinant histone acetyltransferase . To our knowledge, this is the first agent shown to decrease histone H4 acetylation at nontoxic levels. J Theor Biol, 2000 Feb 21, 202(4), 273 - 82 Distributions of dimeric tandem repeats in non-coding and coding DNA sequences; Dokholyan NV et al.; We study the length distribution functions for the 16 possible distinct dimeric tandem repeats in DNA sequences of diverse taxonomic partitions of GenBank (known human and mouse genomes, and complete genomes of Caenorhabditis elegans and yeast) . For coding DNA, we find that all 16 distribution functions are exponential . For non-coding DNA, the distribution functions for most of the dimeric repeats have surprisingly long tails, that fit a power-law function . We hypothesize that: (i) the exponential distributions of dimeric repeats in protein coding sequences indicate strong evolutionary pressure against tandem repeat expansion in coding DNA sequences; and (ii) long tails in the distributions of dimers in non-coding DNA may be a result of various mutational mechanisms . These long, non-exponential tails in the distribution of dimeric repeats in non-coding DNA are hypothesized to be due to the higher tolerance of non-coding DNA to mutations . By comparing genomes of various phylogenetic types of organisms, we find that the shapes of the distributions are not universal, but rather depend on the specific class of species and the type of a dimer . Arch Biochem Biophys, 2000 Feb 15, 374(2), 181 - 8 Isolation and characterization of a novel 530-kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes; Tanaka E et al.; A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns . The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration . Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome . The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa . The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits . These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition . Nucleic Acids Res, 2000 Mar 1, 28(5), 1211 - 20 Poly(A)-binding protein I of Leishmania: functional analysis and localisation in trypanosomatid parasites; Bates EJ et al.; Regulation of gene expression in trypanosomatid parasites is predominantly post-transcriptional . Primary transcripts are trans-spliced and polyadenylated to generate mature mRNAs and transcript stability is a major factor controlling stage-specific gene expression . Degenerate PCR has been used to clone the gene encoding the Leishmania homologue of poly(A)-binding protein (Lm PAB1), as an approach to the identification of trans-acting factors involved in this atypical mode of eukaryotic gene expression . lmpab1 is a single copy gene encoding a 63 kDa protein which shares major structural features but only 35-40% amino acid identity with other PAB1 sequences, including those of other trypanosomatids . Lm PAB1 is expressed at constant levels during parasite differentiation and is phosphorylated in vivo . It is localised predominantly in the cytoplasm but inhibition of transcription with actinomycin D also reveals diffuse localisation in the nucleus . Lm PAB1 binds poly(A) with high specificity and affinity but fails to complement a null mutation in Saccharomyces cerevisiae . These properties are indicative of functional divergence in vivo. J Virol, 2000 Mar, 74(5), 2372 - 82 A chimeric protein containing the N terminus of the adeno-associated virus Rep protein recognizes its target site in an in vivo assay; Cathomen T et al.; The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration . They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19 . Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization . In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo . Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression . Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast genome . Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers . These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications. Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2082 - 90 Encoding of a cytochrome P450-dependent lauric acid monooxygenase by CYP703A1 specifically expressed in the floral buds of petunia hybrida; Imaishi H et al.; The cDNA clone of novel cytochrome P450 CYP703A1 from petunia floral buds was isolated by RT-PCR . The nucleotide sequences of this cDNA clone contained the open reading frame that has been predicted to encode polypeptides consisting of 539 amino acid residues . A significantly high level of the transcript of the cyp703A1 gene was found in the early stage of petunia flower buds, but not in the leaves, stems and roots . The 1041bp 5'-flanking sequences of the cyp703A1 gene contained the conserved motifs of ATHB-1, AGAMOUS, MYB.Ph3, P and SBF-1 binding boxes . CYP703A1 cDNA was expressed in yeast Saccharomyces cerevisiae AH22 cells under the control of an alcohol dehydrogenase I promoter and terminator . The recombinant yeast microsomes containing the CYP703A1 hemoprotein were found to metabolize lauric acid . Based on these results, CYP703A1 was specifically expressed in the early stage of flower development and appeared to participate in the monooxygenation of fatty acids. FEBS Lett, 2000 Feb 4, 467(1), 111 - 6 The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest; Abbas-Terki T et al.; The molecular chaperone Cdc37 is thought to act in part as a targeting subunit of the heat-shock protein 90 (Hsp90) chaperone complex . We demonstrate here that Cdc37 is required for activity of the kinase Ste11 in budding yeast . A cdc37 mutant strain is defective in Ste11-mediated pheromone signaling and in accumulation and functional maturation of the constitutively active Ste11 version Ste11DeltaN . Moreover, Cdc37, Ste11DeltaN and Hsp90 coprecipitate pairwise . Thus, Hsp90 and Cdc37 may transiently associate with Ste11 to promote proper folding and/or association with additional regulatory factors . Our results establish Ste11 as the first endogenous Cdc37 client protein in yeast. Plant Cell, 2000 Feb, 12(2), 279 - 90 The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors; Despres C et al.; The Arabidopsis NPR1 gene is essential in activating systemic, inducible plant defense responses . To gain a better understanding of NPR1 function, we conducted a yeast two-hybrid screening procedure and identified a differential interaction between NPR1 and all known members of the Arabidopsis TGA family of basic leucine zipper transcription factors . In the electrophoretic mobility shift assay, NPR1 substantially increased the binding of TGA2 to its cognate promoter element (as-1) as well as to a positive salicylic acid-inducible element (LS7) and a negative element (LS5) in the promoter of the pathogenesis-related PR-1 gene . Proteins encoded by npr1 mutants interacted poorly with TGA2 and did not substantially increase TGA2 binding to the as-1, LS5, or LS7 elements, thus establishing a link between the loss of disease resistance and the loss of TGA2 interaction and NPR1-enhanced DNA binding . Coupled with observations that the DNA binding activity of TGA factors is deregulated in npr1 plants, the results suggest that NPR1-mediated DNA binding of TGA2 is critical for activation of defense genes. Plant Cell, 2000 Feb, 12(2), 237 - 48 The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize; Tonukari NJ et al.; The production of cell wall-degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glucose) repression . In Saccharomyces cerevisiae, the SNF1 gene is required for expression of catabolite-repressed genes when glucose is limiting . An ortholog of SNF1, ccSNF1, was isolated from the maize pathogen Cochliobolus carbonum, and ccsnf1 mutants of HC toxin-producing (Tox2(+)) and HC toxin-nonproducing (Tox2(-)) strains were created by targeted gene replacement . Growth in vitro of the ccsnf1 mutants was reduced by 50 to 95% on complex carbon sources such as xylan, pectin, or purified maize cell walls . Growth on simple sugars was affected, depending on the sugar . Whereas growth on glucose, fructose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced . Production of HC toxin was normal in the Tox2(+) ccsnf1 mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation . Activities of secreted beta-1,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2(+) ccsnf1 mutant were reduced by 53, 24, and 65%, respectively . mRNA expression was downregulated under conditions that induced the following genes encoding secreted wall-degrading enzymes: XYL1, XYL2, XYL3, XYL4, XYP1, ARF1, MLG1, EXG1, PGN1, and PGX1 . The Tox2(+) ccsnf1 mutant was much less virulent on susceptible maize, forming fewer spreading lesions; however, the morphology of the lesions was unchanged . The Tox2(-) ccsnf1 mutant also formed fewer nonspreading lesions, which also retained their normal morphology . The results indicate that ccSNF1 is required for biochemical processes important in pathogenesis by C . carbonum and suggest that penetration is the single most important step at which ccSNF1 is required . The specific biochemical processes controlled by ccSNF1 probably include, but are not necessarily restricted to, the ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars produced. Virology, 2000 Feb 15, 267(2), 185 - 98 Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus; Choi IR et al.; The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins . To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system . Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast . Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins . The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI) . Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another . The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected . In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3 . The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells . The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes . Biotechnol Prog, 2000 Jan-Feb, 16(1), 44 - 51 Macro approach and fuzzy modeling of entrapped biocatalyst; Ruggeri B et al.; The interactions between a strain of Saccharomyces cerevisiae and an alginate matrix are investigated to ascertain the main factors affecting the bioreaction evolution . During the tests several parameters (glucose, ethanol, calcium ion and biomass concentration, pH, and alginate bed diameter) were evaluated, coupled with microscopic investigation inside the beads to determine the spatial biomass distribution . A detailed analysis of macro parameters and a correlation among them are proposed using a fuzzy algorithm . A global two-step fuzzy model results in which biomass distribution inside the beads is represented as a hidden parameter. J Cell Biol, 2000 Feb 7, 148(3), 465 - 80 Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways; Noda T et al.; In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle . When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway . Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues . The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol . Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents . Key questions remain regarding the origin and formation of the transport vesicle . In this study, we have cloned the APG9/CVT7 gene and characterized the gene product . Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport . Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins . Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles . From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways. J Biol Chem, 2000 Feb 11, 275(6), 4311 - 22 PSK, a novel STE20-like kinase derived from prostatic carcinoma that activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates actin cytoskeletal organization; Moore TM et al.; Degenerate polymerase chain reaction against conserved kinase catalytic subdomains identified 15 tyrosine and serine-threonine kinases expressed in surgically removed prostatic carcinoma tissues, including six receptor kinases (PDGFBR, IGF1-R, VEGFR2, MET, RYK, and EPH-A1), six non-receptor kinases (ABL, JAK1, JAK2, TYK2, PLK-1, and EMK), and three novel kinases . Several of these kinases are oncogenic, and may function in the development of prostate cancer . One of the novel kinases is a new member of the sterile 20 (STE20) family of serine-threonine kinases which we have called prostate-derived STE20-like kinase (PSK) and characterized functionally . PSK encodes an open reading frame of 3705 nucleotides and contains an N-terminal kinase domain . Immunoprecipitated PSK phosphorylates myelin basic protein and transfected PSK stimulates MKK4 and MKK7 and activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway . Microinjection of PSK into cells results in localization of PSK to a vesicular compartment and causes a marked reduction in actin stress fibers . In contrast, C-terminally truncated PSK (1-349) did not localize to this compartment or induce a decrease in stress fibers demonstrating a requirement for the C terminus . Kinase-defective PSK (K57A) was unable to reduce stress fibers . PSK is the first member of the STE20 family lacking a Cdc42/Rac binding domain that has been shown to regulate both the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and the actin cytoskeleton. J Biol Chem, 2000 Feb 11, 275(6), 4081 - 91 Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1; Petersen C et al.; Ran GTPase is required for nucleocytoplasmic transport of many types of cargo . Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD) . Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins) . In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells . We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo . In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs . However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1 . These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells . Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro . However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP . A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation . When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP . The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex . Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes. J Biol Chem, 2000 Feb 11, 275(6), 3810 - 8 Identification of the transactivation domain of the transcription factor Sox-2 and an associated co-activator; Nowling TK et al.; The importance of interactions between Sox and POU transcription factors in the regulation of gene expression is becoming increasingly apparent . Recently, many examples of the involvement of Sox-POU partnerships in transcription have been discovered, including a partnership between Sox-2 and Oct-3 . Little is known about the mechanisms by which these factors modulate transcription . To better understand the molecular interactions involved, we mapped the location of the transactivation domain of Sox-2 . This was done in the context of its interaction with Oct-3, as well as its ability to transactivate as a fusion protein linked to the DNA-binding domain of Gal4 . Both approaches demonstrated that Sox-2 contains a transactivation domain in its C-terminal half, containing a serine-rich region and the C terminus . We also determined that the viral oncoprotein E1a inhibits the ability of the Gal4/Sox-2 fusion protein to transactivate, as well as the transcriptional activation mediated by the combined action of Sox-2 and Oct-3 . In contrast, a mutant form of E1a, unable to bind p300, lacks both of these effects . Importantly, we determined that p300 overcomes the inhibitory effects of E1a in both assays . Together, these findings suggest that Sox-2 mediates its effects, at least in part, through the co-activator p300. Curr Biol, 2000 Jan 13, 10(1), 39 - 42 A novel Rad24 checkpoint protein complex closely related to replication factor C; Green CM et al.; Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae . Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex . The Rad24 complex was purified to homogeneity . In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa . The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair {3} . We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex . Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins . These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not . We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway. Mol Gen Genet, 2000 Jan, 262(6), 1147 - 56 SLG1 plays a role during G1 in the decision to enter or exit the cell cycle; Ivanovska I et al.; Saccharomyces cerevisiae cells decide to divide during G1 . If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication . Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events . At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate . Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate . The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1 . This is consistent with a role of SLG1 in G1 regulation . Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell . We propose that SLG1 plays a regulatory role in bud emergence or stationary phase. Mol Gen Genet, 2000 Jan, 262(6), 1001 - 11 Developmental and metabolic regulation of the phosphoglucomutase-encoding gene, pgmB, of Aspergillus nidulans; Hoffmann B et al.; We have isolated the pgmB gene from Aspergillus nidulans, which encodes a phosphoglucomutase, one of the key enzymes in carbohydrate metabolism . The pgmB gene is located on chromosome VII and its ORF encodes 557 amino acids . Mutant phenotypes were analysed by expression of high levels of pgmB antisense RNA, which lead to a loss of detectable levels of sense RNA . Under conditions of antisense RNA expression, a 30% reduction in the growth rate was observed in comparison to wild-type . On the enzyme level, expression of pgmB antisense RNA resulted in a 35% reduction in total phosphoglucomutase activity . Two pgmB mRNAs were observed under all conditions tested and differ with respect to the location of the poly(A) site . Expression of pgmB driven by the GAL1 promoter in Saccharomyces cerevisiae complemented the growth phenotype of a pgm2delta mutant strain and suppressed the sensitivity of a gcn4delta mutant strain to amino acid starvation in the presence of galactose . Cultivation of A . nidulans in the presence of glucose or galactose as carbon source did not affect transcription of pgmB . However, amino acid starvation conditions resulted in a six-fold reduction in the level of pgmB mRNA, while genes for amino acid biosynthesis showed increased transcription . Transcription of pgmB was low during hyphal growth and in the sexual phase of development, but was significantly increased during the asexual stage of the A . nidulans life cycle. Genome, 1999 Dec, 42(6), 1077 - 87 Sequence and phylogenetic analysis of the SNF4/AMPK gamma subunit gene from Drosophila melanogaster; Yoshida EN et al.; To optimize gene expression under different environmental conditions, many organisms have evolved systems which can quickly up- and down-regulate the activity of other genes . Recently, the SNF1 kinase complex from yeast and the AMP-activated protein kinase complex from mammals have been shown to represent homologous metabolic sensors that are key to regulating energy levels under times of metabolic stress . Using heterologous probing, we have cloned the Drosophila melanogaster homologue of SNF4, the noncatalytic effector subunit from this kinase complex . A sequence corresponding to the partial genomic sequence as well as the full-length cDNA was obtained, and shows that the D . melanogaster SNF4 is encoded in a 1944-bp cDNA representing a protein of 648 amino acids (aa) . Southern analysis of Drosophila genomic DNA in concert with a survey of mammalian SNF4 ESTs indicates that in metazoans, SNF4 is a duplicated gene, and possibly even a larger gene family . We propose that one gene copy codes for a short (330 aa) protein, whereas the second locus codes for a longer version (<410 aa) that is extended at the carboxy terminus, as typified by the Drosophila homologue presented here . Phylogenetic analysis of yeast, invertebrate, and multiple mammalian isoforms of SNF4 shows that the gene duplication likely occurred early in the metazoan lineage, as the protein products of the different loci are relatively divergent . When the phylogeny was extended beyond the SNF4 gene family, SNF4 shares sequence similarity with other cystathionine-beta-synthase domain-containing proteins, including IMP dehydrogenase and a variety of uncharacterized Methanococcus proteins. J Steroid Biochem Mol Biol, 1999 Dec 15, 71(3-4), 93 - 102 The nuclear-receptor interacting protein (RIP) 140 binds to the human glucocorticoid receptor and modulates hormone-dependent transactivation; Windahl SH et al.; The glucocorticoid receptor (GR) regulates target gene expression in response to corticosteroid hormones . We have investigated the mechanism of transcriptional activation by the GR by studying the role of the receptor interacting protein RIP140 . Both in vivo and in vitro protein-protein interaction assays revealed a ligand-dependent interaction between the GR and RIP140 . The ligand binding domain of the GR was sufficient for this interaction, while both the N- and C-terminal regions of RIP140 bound to the receptor . In a yeast transactivation assay RIP140 and SRC-1, a member of the steroid receptor coactivator family of proteins, both enhanced the transactivation activity of a GR protein (GRA-1) in which the potent N-terminal tau1 transactivation domain has been deleted . In contrast, in COS-7 cells increasing amounts of RIP140 significantly inhibited GRdeltatau1 function . In cotransfection studies in COS-7 cells, RIP140 also inhibited receptor activity in presence of both SRC-1 and the coactivator protein CBP together . Thus, in yeast cells a stimulation of receptor activity was observed, while in mammalian cells RIP140 repressed GR function . Taken together, these data suggest that, (1) RIP140 is a target protein for the GR and (2) RIP140 can modulate the transactivation activity of the receptor. Anal Chem, 2000 Jan 15, 72(2), 267 - 75 A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry; Rappsilber J et al.; Most cellular functions are performed by multi-protein complexes . The identity of the members of such complexes can now be determined by mass spectrometry . Here we show that mass spectrometry can also be used in order to define the spatial organization of these complexes . In this approach, components of a protein complex are purified via molecular interactions using an affinity tagged member and the purified complex is then partially cross-linked . The products are separated by gel electrophoresis and their constituent components identified by mass spectrometry yielding nearest-neighbor relationships . In this study, a member of the yeast nuclear pore complex (Nup85p) was tagged and a six-member sub-complex of the pore was cross-linked and analyzed by 1D SDS-PAGE . Cross-linking reactions were optimized for yield and number of products . Analysis by MALDI mass spectrometry resulted in the identification of protein constituents in the cross-linked bands even at a level of a few hundred femtomoles . Based on these results, a model of the spatial organization of the complex was derived that was later supported by biological experiments . This work demonstrates, that the use of mass spectrometry is the method of choice for analyzing cross-linking experiments aiming on nearest neighbor relationships. J Neurosci Res, 2000 Jan 1, 59(1), 94 - 9 GAL4/UAS-WGA system as a powerful tool for tracing Drosophila transsynaptic neural pathways; Tabuchi K et al.; Visualization of specific transsynaptic neural pathways is an indispensable technique for understanding the relationship between structure and function in the nervous system . Here, we demonstrate the application of the wheat germ agglutinin (WGA) transgene technique for tracing transsynaptic neural pathways in Drosophila . The intracellular localization of WGA was examined by immunoelectron microscopy . WGA signals were detected in granule-like structures in both the outer photoreceptor cells expressing WGA and the second-order laminar neurons . Misexpression of tetanus toxin (TNT), which inactivates N-synaptobrevin, in the outer photoreceptor cells resulted in the elimination of on/off transients in electroretinogram (ERG) recordings and in a great reduction in WGA transfer into laminar neurons, suggesting that anterograde WGA transsynaptic transfer is dependent mainly on synaptic transmission . Retrograde WGA transfer was also detected upon its forced expression in muscle cells . WGA primarily expressed in muscle cells was taken up by motoneuron axons and transported to their cell bodies in the ventral nerve cord, suggesting that WGA can trace motoneuronal pathways in combination with the muscle-specific GAL4 driver . Thus, the GAL4/UAS-WGA system should facilitate the dissection of the Drosophila neural circuit formation and/or synaptic activity in various regions and at various developmental stages. J Neurosci Res, 2000 Jan 1, 59(1), 19 - 23 Localization and processing of CLN3, the protein associated to Batten disease: where is it and what does it do? Pearce DA. Although the CLN3 gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive . A key to understanding the pathology of this devastating disease will be to elucidate the function of CLN3 at the molecular level . CLN3 has proven difficult to study, as it is predicted to be a membrane protein, and is of apparently low abundance in cells . Different groups have reported differing subcellular localization of CLN3 . The purpose of this review is to critically examine the various cell biological approaches undertaken to localize CLN3 and to piece together a potential function for CLN3 in neuronal cells . The most likely conclusion of this is that CLN3 is a lysosomal/endosomal protein that is trafficked through the endoplasmic reticulum (ER) and Golgi . Furthermore, studies are required to confirm whether CLN3 has a potential role in the recycling of synaptic vesicles through the endosome/lysosome. Science, 2000 Feb 4, 287(5454), 873 - 80 Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles; Roberts CJ et al.; Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response . Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis . A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions . Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways . Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses . Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways. Biochem J, 2000 Feb 15, 346 Pt 1, 63 - 70 Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis; Way G et al.; The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcepsilonR1, with antigen . We demonstrate a novel function for ADP-ribosylation factor 1 (ARF1) in the regulation of antigen-stimulated secretion using cytosol-depleted RBL-2H3 mast cells for reconstitution of secretory responses . When antigen is used as the stimulus, ARF1 also reconstitutes phospholipase D activation . Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to phosphatidylethanol causes inhibition of ARF1-reconstituted secretion . In addition . ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels at the expense of phosphatidylinositol 4-monophosphate . The requirement for PIP(2) in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPalpha) to increase PIP(2) levels . Exocytosis, restored by either ARF1 or PITPalpha, was inhibited when PIP(2) levels were depleted by phospholipase Cdelta1 . We conclude that the function of ARF1 and PITPalpha is to increase the local synthesis of PIP(2), the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby recruitment of key components of the exocytotic machinery are targeted to the appropriate membrane compartment. Biochim Biophys Acta, 2000 Feb 14, 1470(1), R21 - 8 Keystone Symposia on Molecular and Cellular Biology: 'The molecular basis of cancer' . Organizers: Carol L . Prives and George F . Vande Woude, Taos, NM, 15-21 March 1999; Wang JY et al.; The Keystone Symposium on the Molecular Basis of Cancer was an excellent meeting, which stimulated the exchange of a great deal of information . This report was prepared to organize some of the results that provided new insights into the regulation of cell proliferation and apoptosis . We were unable to report on all of the talks and posters due mostly to our limited capacity to absorb and digest the large amount of results presented at the meeting . We apologize to those whose results were not covered in this report. J Mol Biol, 2000 Feb 11, 296(1), 217 - 28 Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c; Godbole S et al.; The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride . The thermodynamics of unfolding by guanidine hydrochloride is also reported . All variants have decreased stability relative to the wild-type protein . The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme . The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions . Significant deviations from random coil behavior are observed . Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied . The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein . The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding . The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c . J Mol Biol, 2000 Jan 28, 295(4), 791 - 801 DNA polymerase switching: I . Replication factor C displaces DNA polymerase alpha prior to PCNA loading; Maga G et al.; An important not yet fully understood event in DNA replication is the DNA polymerase (pol) switch from pol alpha to pol delta . Indirect evidence suggested that the clamp loader replication factor C (RF-C) plays an important role, since a replication competent protein complex containing pol alpha, pol delta and RF-C could perform pol switching in the presence of proliferating cell nuclear antigen (PCNA) . By using purified pol alpha/primase, pol delta, RF-C, PCNA and RP-A we show that: (i) RF-C can inhibit pol alpha in the presence of ATP prior to PCNA loading, (ii) RF-C decreases the affinity of pol alpha for the 3'OH primer ends, (iii) the inhibition of pol alpha by RF-C is released upon PCNA loading, (iv) ATP hydrolysis is required for PCNA loading and subsequent release of inhibition of pol alpha, (v) under these conditions a switching from pol alpha/primase to pol delta is evident . Thus, RF-C appears to be critical for the pol alpha to pol delta switching . Based on these results, a model is proposed in which RF-C induces the pol switching by sequestering the 3'-OH end from pol alpha and subsequently recruiting PCNA to DNA . Nat Struct Biol, 2000 Feb, 7(2), 161 - 6 Asparagine-mediated self-association of a model transmembrane helix; Choma C et al.; In membrane proteins, the extent to which polarity, hydrogen bonding, and van der Waals packing interactions of the buried, internal residues direct protein folding and association of transmembrane segments is poorly understood . The energetics associated with these various interactions should differ substantially between membrane versus water-soluble proteins . To help evaluate these energetics, we have altered a water-soluble, two-stranded coiled-coil peptide to render its sequence soluble in membranes . The membrane-soluble peptide associates in a monomer-dimer-trimer equilibrium, in which the trimer predominates at the highest peptide/detergent ratios . The oligomers are stabilized by a buried Asn side chain . Mutation of this Asn to Val essentially eliminates oligomerization of the membrane-soluble peptide . Thus, within a membrane-like environment, interactions involving a polar Asn side chain provide a strong thermodynamic driving force for membrane helix association. Nat Struct Biol, 2000 Feb, 7(2), 154 - 60 Interhelical hydrogen bonding drives strong interactions in membrane proteins; Zhou FX et al.; Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins . To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper . We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence . Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur . Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces. Nat Struct Biol, 2000 Feb, 7(2), 97 - 9 Are DEAD-box proteins becoming respectable helicases? Linder P, Daugeron MC. The vaccinia NPH-II RNA helicase, a member of the DEAD/DExH-box protein family, has been shown to be a processive, unidirectional RNA helicase with a step size of about one half turn of a helix . This finding demonstrates that RNA helicases can function as molecular motors. Nat Struct Biol, 2000 Feb, 7(2), 91 - 4 Understanding membrane protein structure by design; Bowie JU; In contrast to soluble proteins, the primary interactions that specify and stabilize membrane protein structures are still largely a matter of speculation . Although van der Waals interactions have been gaining increasing favor as the dominant player, new results demonstrate the strength of hydrogen bonding in a membrane environment. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 978 - 83 A relation between the principal axes of inertia and ligand binding; Foote J et al.; The principal axes of inertia are eigenvectors that can be calculated for any rigid body . We report studies of the position of the principal axes in crystallographically solved protein molecules . We find with high frequency that at least one principal axis penetrates the surface of the respective protein in a region used for ligand binding . In antibody variable regions, an axis goes through the third hypervariable loop of the heavy chain . In major histocompatibility complex proteins, an axis goes through the peptide-binding groove . In protein-protein heterodimers, a principal axis of one subunit will often penetrate the interface formed with the other subunit . In many of these protein-protein complexes, the axis specifically intersects residues known to be critical for molecular recognition. Oncogene, 2000 Jan 20, 19(3), 444 - 51 c-Myb acetylation at the carboxyl-terminal conserved domain by transcriptional co-activator p300; Tomita A et al.; Transcription factor c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells . Here we demonstrate that c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo . The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine . Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity . Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb . Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb, and hGCN5 is revealed to be a factor acetyl-transferase for c-Myb in vitro . We present a new manner of post-translational modification of the c-Myb protein and the potential significance of the acetylation in c-Myb. EMBO J, 2000 Feb 1, 19(3), 463 - 71 The controlling role of ATM in homologous recombinational repair of DNA damage; Morrison C et al.; The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation . DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear . To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells . ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation . Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway . These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation. EMBO J, 2000 Feb 1, 19(3), 421 - 33 The Bbp1p-Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication; Schramm C et al.; In budding yeast, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope via its central plaque structure . Here, we describe the identification of BBP1 in a suppressor screen with a conditional lethal allele of SPC29 . Bbp1p was detected at the central plaque periphery of the SPB and bbp1-1 cells were found to be defective in SPB duplication . bbp1-1 cells extend their satellite into a duplication plaque like wild-type cells; however, this duplication plaque then fails to insert properly into the nuclear envelope and does not assemble a functional inner plaque . This function in SPB duplication is probably fulfilled by a stable complex of Bbp1p and Mps2p, a nuclear envelope protein that is also essential for duplication plaque insertion . In addition, we found that Bbp1p interacts with Spc29p and the half-bridge component Kar1p . These interactions are likely to play a role in connecting the SPB with the nuclear envelope and the central plaque with the half-bridge. EMBO J, 2000 Feb 1, 19(3), 317 - 23 Crystal structure of a fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution; Wang H et al.; Fibrillarin is a phylogenetically conserved protein essential for efficient processing of pre-rRNA through its association with a class of small nucleolar RNAs during ribosomal biogenesis . The protein is the antigen for the autoimmune disease scleroderma . Here we report the crystal structure of the fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution . The structure consists of two domains, with a novel fold in the N-terminal region and a methyltransferase-like domain in the C-terminal region . Mapping temperature-sensitive mutations found in yeast fibrillarin Nop1 to the Methanococcus homologue structure reveals that many of the mutations cluster in the core of the methyltransferase-like domain. J Exp Zool, 2000 Feb 15, 286(3), 231 - 7 Enzyme activities involved in lipid metabolism during embryonic development of Macrobrachium borellii; Gonzalez-Baro MR et al.; The activities of the enzymatic systems involved in the activation and degradation of fatty acids, and in the synthesis of triacylglycerols and phospholipids were studied in vitro using total cellular homogenate and subcellular fractions of eggs of the shrimp Macrobrachium borellii at different developing stages . Egg development was divided into seven stages based on morphological features of the embryo . Palmitoyl-CoA ligase activity increased as the embryo developed and showed its maximum at stage V . An increase in the synthesis of triacylglycerols and diacylglycerols was also observed at this stage . Diacylglycerylethers were synthesized more actively during the first stages of development . The higher specific activity observed in total homogenate than in microsomal fraction suggested that their synthesis was not exclusively microsomal . Phospholipid synthesis was very active all along development, reflecting active membrane biosynthesis . The highest activity of the cytosolic triacylglycerol lipase was observed at stage V . Fatty acid degradation, measured as mitochondrial beta-oxidation activity, did not vary significantly during development . We conclude that both the anabolic and catabolic processes concerning lipid metabolism are very active, with values similar to those described for adult hepatopancreas, revealing the major role of lipids during shrimp embryogenesis energetics, and that the highest activities of lipid synthesis-hydrolysis take place at stage V when embryos are under active organogenesis . J . Exp . Zool . 286:231-237, 2000 . J Mol Biol, 2000 Feb 4, 295(5), 1139 - 62 Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions; Peng H et al.; The KRAB domain is a 75 amino acid residue transcriptional repression module commonly found in eukaryotic zinc-finger proteins . KRAB-mediated gene silencing requires binding to the corepressor KAP-1 . The KRAB:KAP-1 interaction requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a widely distributed motif, hypothesized to be a protein-protein interface . Little is known about RBCC-mediated ligand binding and the role of the individual sub-domains in recognition and specificity . We have addressed these issues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction using purified components . Our results show that KRAB binding to KAP-1 is direct and specific, as the related RBCC domains from TIF1alpha and MID1 do not bind the KRAB domain . A combination of gel filtration, analytical ultracentrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC must oligomerize likely as a homo-trimer in order to bind the KRAB domain . The RING finger, B2 box, and coiled-coil region are required for oligomerization of KAP-1-RBCC and KRAB binding, as mutations in these domains concomitantly abolished these functions . KRAB domain binding stabilized the homo-oligomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies . Mutant KAP-1-RBCC molecules hetero-oligomerize with the wild-type KAP-1, but these complexes were inactive for KRAB binding, suggesting a potential dominant negative activity . Substitution of the coiled-coil region with heterologous dimerization, trimerization, or tetramerization domains failed to recapitulate KRAB domain binding . Chimeric KAP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil regions from MID1 also failed to bind the KRAB domain . The KAP-1-RBCC mediates a highly specific, direct interaction with the KRAB domain, and it appears to function as an integrated, possibly cooperative structural unit wherein each sub-domain contributes to oligomerization and/or ligand recognition . These observations provide the first principles for RBCC domain-mediated protein-protein interaction and have implications for identifying new ligands for RBCC domain proteins . Pflugers Arch, 2000, 439(3 Suppl), R94 - 6 Interaction trap experiment with CDC6; Zavec PB et al.; CDC6 is an essential gene of yeast Saccharomyces cerevisiae . Although DNA sequence of the gene is available for a long time, biochemical function of Cdc6 protein in the cell cycle remains unclear . Using the interaction trap experiment we were looking for proteins interacting specifically with Cdc6 . Four gene products interacting with Cdc6 were detected . By sequence analysis we found that ECM11 codes for the protein involved in the cell wall synthesis, YNL201 codes for the protein of unknown function, probably involved in the carbon metabolism, YOR279 codes for protein of completely unknown function with no significant similarity with any known protein, and the interaction with Ty1 retrotransposition element was also found . The strongest interaction with Cdc6 bait measured as beta-galactosidase activity was observed with ECM11 and YNL201; YOR279 interacts slightly weaker . The weakest beta-galactosidase activity was obtained by Ty1A element . The strongest suppression of cdc6-1 mutation was observed by Ty1A element, the slight one with ECM11 and YNL201 but no suppression of thermosensitive mutation was detected for YOR279. Trends Cell Biol, 2000 Feb, 10(2), 60 - 7 Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors; Jackson CL et al.; ARF proteins are important regulators of membrane dynamics and protein transport within the eukaryotic cell . The Sec7 domain is approximately 200 amino acids in size and stimulates guanine-nucleotide exchange on members of the ARF class of small GTPases . The members of one subclass of Sec7-domain proteins are direct targets of the secretion-inhibiting drug brefeldin A, which blocks the exchange reaction by trapping a reaction intermediate in an inactive, abortive complex . A separate subclass of Sec7-domain proteins is involved in signal transduction and possess a domain that mediates membrane binding in response to extracellular signals. J Biol Chem, 2000 Feb 4, 275(5), 3377 - 81 The transcriptional co-activator ADA5 is required for HAC1 mRNA processing in vivo; Welihinda AA et al.; Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates signaling pathways to induce transcription of a number of genes encoding ER protein chaperones and-folding catalysts . In Saccharomyces cerevisiae this transcriptional induction is mediated by an increase in the synthesis of the transcription factor Hac1p . The transmembrane receptor Ire1p/Ern1p containing a Ser/Thr protein kinase and endoribonuclease activity transmits the unfolded protein response (UPR) from the ER to the nucleus . Activation of Ire1p kinase induces its endoribonuclease activity to cleave unspliced HAC1 mRNA and generate exon fragments that are subsequently ligated by tRNA ligase (RLG1) . Whereas unspliced HAC1 mRNA is poorly translated, spliced HAC1 mRNA is efficiently translated . Subunits of the yeast transcriptional co-activator complex SAGA also play a role in the UPR . Deletion of GCN5, ADA2, or ADA3 reduces, and deletion of ADA5 completely abolishes, the UPR . Although HAC1 mRNA requires only Ire1p and Rlg1p in vitro, we demonstrate that ADA5 is required for the IRE1/RLG1-dependent splicing reaction of HAC1 mRNA in vivo . In addition, Ada5p interacts with Ire1p . These results suggest that subcomponents of transcriptional co-activator complexes may be involved in RNA processing events. J Biol Chem, 2000 Feb 4, 275(5), 3051 - 6 Molecular